Monday, May 31, 2010

Grow Taller by Cycling with Ankle Weights?

As many of you know Shinbone Routine 2010 involves cycling with ankle weights.  I've always thought ankle weights were rather cumbersome as they always keep bouncing around on your ankle.  Maybe someone knows a way to keep them tight?  Anyways, the method involves putting an ankle weight on the upper part of the diaphysis of your tibia and another ankle weight on the lower part of your diaphysis of the tibia.  Sky doesn't have before and after pictures yet but he claims that it's similar to Ginza Kogima's method.  I went to Ginza Kojima's page but I couldn't find any before and after pictures(although I have seen some on another one Kojima's pages albeit in Japanese). 

Kojima's method involves some sort of green light(something to decrease Bone Mineral Content I believe) and a rotary device.  Now what does a rotary device do but place spiral forces on the bone?  So Kojima's method works by two things: decreasing Bone Mineral Content and causing spiral forces.  I don't like however that the method is intentionally vague given that distraction osteogenesis is so incredibly transparent in regards to the research involved and it works.

So I can't trust Ginza Kojima's "success" as proof of Shinbone Routine 2010 given that we don't know about the green light nor how exactly the rotary machine works.  I do however think that cycling with ankle weights could work if you manage to get these ankle weights to stay still. 

The cycling motion in combination with the force of the ankle weights puts a twisting and pulling force on the bone.  Pulling plus microfractures in the cortical bone equals height gain.  Now is the pulling and twisting force of cycling with ankle weights sufficient?  Are the ankle weights too cumbersome?

That's what we need the before and after pictures for.

Saturday, May 29, 2010

Grow taller with Spiral Fractures?

As many of you may know, Sky's limbcenter is based on non-surgical bone stretching.  His system involves stretching bone to lengthen it.  Now he states that this is due to Wolf's law but Wolf's law is incredibly outdated and only states that bone adapts to the demands placed on it(but not always in the ways we want).  Now before I unearthed lateral synovial joint loading my research for height increase dealt with growing taller by a combination of microstrain and microfractures.

My hypothesis for how Sky's limbcenter works is as follows:  Sky has a system of pulleys that stretches the bone.  The bone is elastic due to the collagen content so it is capable of stretching.  Now imagine you are pulling a stick apart, when you pull the stick apart tiny cracks form within the stick.  The cracks help the stick remain longer than before.  Except in the case of bone the microfractures can heal so now the bone is longer than before.

Now, the problem is that a pulley system is cumbersome and there are a lot of tendons and ligaments in the way.  So, the alternative: spiral forces.  Imagine you're twisting a stick.  The stick is being pulled apart and cracks in the stick structure will be induced.  But unlike a stick, the bone can heal the cracks and still be as long as before.

The easiest way to apply spiral forces on a bone would be to surgical insert an apparatus that enables you to turn and twist the bone.  But, you can apply spiral forces on the bone by hand by just twisting your appendages like giving yourself an Indian burn but on your bones.  Rotate your foot in such a way as to apply a twisting force on the bone.

The advantages are over pulling your bones apart is that you can contort yourself in such a way as to apply a twisting force on any long bone on your body including your clavicle, toes, and fingers.  You can do LSJL on your fingers and toes too but I'm working on my legs and arms first because it will take forever to do LSJL on all your fingers and toes(especially since four out of five of them have three long bones).  On the other hand you don't have to use dumbells or anything on your fingers and toes as you can generate a large amount of pressure yourself relative to the size of your finger bones.

The disadvantage is that it's a pain to take the time to twist all your long bones in such a way as to cause sufficient spiral forces on the bone to both lengthen and microfracture.  However, you will gain results faster if you do in conjunction with LSJL as it works by a different mechanism.

I am personally not doing this method(if I plateaued on LSJL then I would apply LSJL to my fingers first to get proof before I would consider doing this)  but it is something to think about trying if you're considering doing a Sky-esque bone stretching method.  And this is why I believe pitchers throwing arms are longer than their sedentary arm(I could never find any official evidence though, only anecdotal but scientific evidence is lacking in many areas) due to the spiral forces put on a pitchers arm when they throw a fast ball.

Friday, May 28, 2010

Grow Taller with Jumping

There are three ways to increase your torso height: increase the width of the periosteum(since the vertebrae are irregular bones), cause your osteoblasts to deposit more bone underneath the periosteum, and to increase your intervertebral disc height.

We want trabecular bone microfractures in the vertabrae to release the stem cells which differentiate into osteoblasts which then deposit new bone beneath the periosteum.  What's the best way to do that given that your spine is hard to access(you can only get the spinous process given the fact that the ribs block everything)?  Jumping(or rather landing with straight legs).  Jumping is the exercise where you can generate the most force.  Other exercises with potential include squats, deadlifts, power cleans, etc but jumping is the simplest exercise and those exercises mostly causing shearing forces(periosteal width) it may take a lot of weight to cause trabecular microfractures.  Whereas jumping is an easy exercise to perform.

Upregulation of osteogenic factors induced by high-impact jumping suppresses adipogenesis in marrow but not adipogenic transcription factors in rat tibiae.

"Jump training is a high-impact training regimen that increases bone volume in young bones[Volume includes height]. The aim of our study was to determine whether downregulation of adipogenesis that is associated with upregulation of osteogenesis is detected after jump training in growing rat tibiae. Four-week-old rats were jump trained for 1, 2, or 4 weeks for 5 days/week, and the height of jumping progressively increased to 35 cm. We performed morphometry to directly quantitate changes in bone volume and marrow adipocyte distribution in tibiae after the jump training. We also examined changes in the expression of osteogenic and adipogenic transcription factor proteins and mRNAs after the jump training. Four weeks of jump training induced an increase in trabecular bone volume, which was associated with the recruitment of runt-related transcription factor 2 expressing cells, as well as a decrease in marrow fat volume. However, peroxisome proliferator-activated receptor-gamma2 protein and mRNA expression levels did not change after high-impact jump training. The mRNA expression levels of the adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBPs)alpha, C/EBPbeta, and C/EBPdelta also showed no change during the training period in jump-trained rats. We suggest that the levels of osteogenic factors that were upregulated by mechanical loading from high-impact jumping suppress adipogenesis in marrow rather than adipogenic transcription factors."

Remember stem cells can differentiate into adipocytes, chondrocytes, or osteoblasts.  If stem cells aren't differentiating into adipocytes they are more likely to be differentiating into chondrocyte(which is what increases height).  Further, more trabecular bone colume means more red bone marrow means more stem cells.  This is the tibia but still an increase in trabecular bone in the epiphysis in that bone would be good for height and the rats may have gained the same benefit in the trabecular bone in their vertebrae.

4 week old Fischer strain female rats were used.

"Bone volume and marrow fat volume are inversely proportional"

"Mechanical loading of the mesenchymal stem cell line C3H10T1/2 increases Runx2 expression and decreases PPARγ2 expression"

"we were able to study only the influence of jumping and not of landing."

"There were no differences in the tibia length (Cont 30.8 ± 0.4 mm; Jump 30.6 ± 0.4 mm) or the periosteal perimeter of the tibia–fibula junction (Cont 7.3 ± 0.3 mm; Jump 7.4 ± 0.2 mm) between the Jump and Cont groups."

" BMP-2 mRNA expression levels did not significantly change during the training period. BMP-4 mRNA expression levels were significantly higher after 1 and 2 weeks of jump training than in each age-matched control group. Runx2 mRNA expression levels were significantly higher following 4 weeks of jump training than in the age-matched control group, whereas osterix mRNA expression levels were significantly higher following 2 weeks of jump training. No significant changes in PPARγ2 or C/EBPs mRNA expression levels were observed between the Cont and Jump groups throughout the training period."

Cross-sectional geometry of weight-bearing tibia in female athletes subjected to different exercise loadings

"The association of long-term sport-specific exercise loading with cross-sectional geometry of the weight-bearing tibia was evaluated among 204 female athletes representing five different exercise loadings and 50 referents. All exercises involving ground impacts (e.g., endurance running, ball games, jumping) were associated with thicker cortex at the distal and diaphyseal sites of the tibia and also with large diaphyseal cross-section, whereas the high-magnitude (powerlifting) and non-impact (swimming) exercises were not.
Bones adapt to the specific loading to which they are habitually subjected. In this cross-sectional study, the association of long-term sport-specific exercise loading with the geometry of the weight-bearing tibia was evaluated among premenopausal female athletes representing 11 different sports. METHODS: A total of 204 athletes were divided into five exercise loading groups, and the respective peripheral quantitative computed tomographic data were compared to data obtained from 50 physically active, non-athletic referents. Analysis of covariance was used to estimate the between-group differences.
At the distal tibia, the high-impact, odd-impact, and repetitive low-impact exercise loading groups had ~30% to 50% greater cortical area (CoA) than the referents. At the tibial shaft, these three impact groups had ~15% to 20%  greater total area (ToA) and ~15% to 30% greater CoA. By contrast, both the high-magnitude and repetitive non-impact groups had similar ToA and CoA values to the reference group at both tibial sites.
High-impact, odd-impact, and repetitive low-impact exercise loadings were associated with thicker cortex at the distal tibia. At the tibial shaft, impact loading was not only associated with thicker cortex, but also a larger cross-sectional area. High-magnitude exercise loading did not show such associations at either site but was comparable to repetitive non-impact loading and reference data. Collectively, the relevance of high strain rate together with moderate-to-high strain magnitude as major determinants of osteogenic loading of the weight-bearing tibia is implicated."

Now, we've already established that the irregular bones(like the spine) grow in the same way as appositional growth.  The tibia grew appositionally by 15 to 20%!  That would be an insane increase in height in the spine(15to20%).  You could also increase height this way in the pelvis, top bone of the skull, and the calcaneus heel bone(although those you can tap wholly or at least partially).

The high impact group had the largest height whereas the high magnitude group had the lowest height.

Jumping improves hip and lumbar spine bone mass in prepubescent children: a randomized controlled trial.

"Physical activity during childhood is advocated as one strategy for enhancing peak bone mass (bone mineral content [BMC]) as a means to reduce osteoporosis-related fractures. Thus, we investigated the effects of high-intensity jumping on hip and lumbar spine bone mass in children. Eighty-nine prepubescent children between the ages of 5.9 and 9.8 years were randomized into a jumping (n = 25 boys and n = 20 girls) or control group (n = 26 boys and n = 18 girls). Both groups participated in the 7-month exercise intervention during the school day three times per week. The jumping group performed 100, two-footed jumps off 61-cm boxes each session, while the control group performed nonimpact stretching exercises. BMC (g), bone area (BA; cm2), and bone mineral density (BMD; g/cm2) of the left proximal femoral neck and lumbar spine (L1-L4) were assessed by dual-energy X-ray absorptiometry (DXA; Hologic QDR/4500-A). Peak ground reaction forces were calculated across 100, two-footed jumps from a 61-cm box. In addition, anthropometric characteristics (height, weight, and body fat), physical activity, and dietary calcium intake were assessed. At baseline there were no differences between groups for anthropometric characteristics, dietary calcium intake, or bone variables. After 7 months, jumpers and controls had similar increases in height, weight, and body fat. Using repeated measures analysis of covariance (ANCOVA; covariates, initial age and bone values, and changes in height and weight) for BMC, the primary outcome variable, jumpers had significantly greater 7-month changes at the femoral neck and lumbar spine than controls (4.5% and 3.1%, respectively). In repeated measures ANCOVA of secondary outcomes (BMD and BA), BMD at the lumbar spine was significantly greater in jumpers than in controls (2.0%) and approached statistical significance at the femoral neck (1.4%; p = 0.085). For BA, jumpers had significantly greater increases at the femoral neck area than controls (2.9%) but were not different at the spine. Our data indicate that jumping at ground reaction forces of eight times body weight is a safe, effective, and simple method of improving bone mass at the hip and spine in children. This program could be easily incorporated into physical education classes."

So, the reason that the children didn't gain height faster was that their method of jumping did not cause microfractures in the spine(the height difference due to the hip would be a lot smaller and possibly insignificant). I don't know if jumping off a box is a better, I've found the most impact just by jumping a little bit off the ground and then landing as hard as possible.  If the scientists had experimented in such a way as to cause lumbar microfractures there would've been height increase.

There seemed to be no apparent correlation between jumping and height.

Thursday, May 27, 2010

1/4" of height gain with Lateral Synovial Joint Loading

Here's last weeks pictures: Bone Length Increase | Two Months One Week In.  Length of lateral ankle to tibial tuberosity stayed the same at 16" while the length of the medial ankle to the medial epicondyle was 15 3/4".  My height increase from 5'9 1/4" to 5'9 1/2".  I measured up to my nose which was about 5'4" and is now 5'4 1/4".

Wednesday, May 26, 2010

Increase your Height with Sox9

In the study where the recreated the growth plate in hyaline cartilage using mesenchymal chondrosarcoma, they used Sox9 as a key marker for chondrogenesis.  If there's no Sox9 expression in the growth plate, then delivering mesenchymal stem cells there by trabecular microfracture followed by an increase in interstitial fluid flow won't recreate the growth plate(the chondrocytes won't proliferate and differentiate).  Of course the cells have to die too or otherwise the osteoblasts won't invade the dead chondrocytes.  How can we control Sox9 genetic expression in the growth plate and can that expression be increased by foods or exercise?

Anterior cruciate ligament-derived cells have high chondrogenic potential. 

"Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis[Scx] did not parallel the increase of Col2a1 expression along with chondrogenic induction." 

"ACL includes more chondrocytic cells, identified as fusiform, ovoid, and spheroid cells, compared with MCL. Fusiform and spheroid cells packaged in the ligament-to-bone interface can produce a cartilage specific extracellular matrix (ECM), type II collagen"

"Repair-associated molecules including transforming growth factor (TGF)-β and type III collagen do not increase in rabbit ACL injury, unlike MCL injury"

"TGF-β signal Smad3 promotes the early chondrogenesis through the activation of Sox9"<-Smad3 was not upregulated by LSJL.

"TGF-β-regulated Smad3 also activates the Sox9-dependent transcription on chromatin without influencing Sox9 expression itself"

"Scx expression is transiently increased in the early stage of embryonic stem cell-derived chondrogenesis. We have previously demonstrated that Scx stimulates the Sox9-dependent transcription by forming transcriptional complex with E47 and p300 in early chondrogenesis"

So maybe it is important to load the ligaments in Lateral Synovial Joint Loading.  Anterior is the outer part of the knee so perhaps outer knee loading is better for LSJL.  

Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures. 

"Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-beta (TGF-beta). Hypoxia, like aggregation and TGF-beta delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (approximately 170 cells/micropellet) as well as conventional pellet cultures (approximately 2 x 10(5) cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O(2) showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O(2) relative to 20% O(2) pellets but 2% O(2) micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size." 

Hypoxia is something we can induce by exercise.  Just run until you're out of breath.  Something to experiment with LSJL if results slow.  Do jumping jacks until you are out of breath and then apply the dumbell to the epiphysis of the bone.  Hypoxia enhances stem cell proliferation too. 

Sox9 family members negatively regulate maturation and calcification of chondrocytes through up-regulation of parathyroid hormone-related protein. 

"Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone-related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling." 

Parathyroid Hormone is pretty important for growth.  Not only does it increase osteoblast activity(which increases height in the spine) but it is involved in chondrocyte differentiation as well. 

p38 MAPK mediated in compressive stress-induced chondrogenesis of rat bone marrow MSCs in 3D alginate scaffolds. 

"Mesenchymal stem cells (MSCs) are well known to have the capability to form bone and cartilage, and chondrogenesis derived from MSCs is reported to be affected by mechanical stimuli. This research was aimed to study the effects of cyclic compressive stress on the chondrogenic differentiation of rat bone marrow-derived MSCs (BMSCs) which were encapsulated in alginate scaffolds and cultured with or without chondrogenic medium, and to investigate the role of p38 MAPK phospho-relay cascade in this process. The results show that the gene expression of chondrocyte-specific markers of Col2alpha1, aggrecan, Sox9, Runx2, and Ihh was upregulated by dynamic compressive stress introduced at the 8th day of chondrogenic differentiation in vitro. The p38 MAPK was activated by chondrogenic cytokines in a slow and lagged way, but activated by cyclic compressive stimulation in a rapid and transient manner. And inhibition of p38 activity with SB203580 suppressed gene expression of chondrocyte-specific genes stimulated by chondrogenic medium and (or) cyclic compressive stress. These findings suggest that p38 MAPK signal acts as an essential mediator in the mechano-biochemical transduction and subsequent transcriptional regulation in the process of chondrogenesis." 

MAPK's can be affected by the hormone leptin(positively).  Cyclic compressive stress is something we can induct with exercise in methods like you guess it LSJL.  A foam roller could induce compressive stress too(it's pretty awkward to use on your limbs though when you're turned on your side) . 

This research seems to suggest that both aerobic(induces hypoxia) and heavy compressive lifting(cyclic compressive stimulation) would be good for increasing height growth during development.  They could also help the factors involved in Lateral Synovial Joint Loading as well. 

Here's a study involving R. Bollock on Sox9:

Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes.

"The transcription factor Sox9 is necessary for early chondrogenesis. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and β-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1."

"Typical markers include Col2a1 (collagen 2) and Acan (aggrecan) for early chondrocytes; Fgfr3 (fibroblast growth factor receptor 3) for columnar cells; Ppr (parathyroid hormone-related protein receptor), Ihh (Indian hedgehog), and Col10a1 (collagen 10) for prehypertrophic cells; and Col10a1 only for hypertrophic cells. Terminal chondrocytes express Mmp13 (matrix metalloproteinase 13) and Bsp (bone sialoprotein), and mineralize the extracellular matrix, as do mature osteoblasts, whereas early osteoblasts express Osx (Osterix) and Col1a1 (collagen 1)"

"The three Sox proteins[Sox5, 6, and 9] are needed and sufficient for early chondrogenesis, and thus referred to as the chondrogenic trio"

"We previously showed that an Acan (aggrecan) upstream enhancer was sufficient to activate the Col2a1 promoter in differentiated chondrocytes in transgenic mice"

"Sox9 may delay prehypertrophy by downregulating Runx2 and Mef2c, but must control hypertrophy differently"

"Sox9 protein is present in cells activating Col10a1."

"Sox9 and Mef2c transactivate Col10a1, and act additively."

"The Sox trio is required for Col2a1 transcription"

MLTK may be involved with the Sox trio.

The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.

"Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrogenesis. The protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 expression by Sox9. We find that knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest development. Sox6 is specifically induced during the onset of chondrogenesis, and Sox6 induction is inhibited by MLTK knockdown. Sox6 knockdown phenocopies MLTK knockdown. Ectopic expression of MLTK induces Sox6 expression in a Sox9-dependent manner. p38 and JNK pathways function downstream of MLTK during chondrogenesis."

"Sox5 and Sox6 bind to the enhancers of cartilage matrix genes and enhance the activity of Sox9"

"MLTK-alpha and MLTK-beta form homo- and heterodimers, and can be activated by osmotic stress"<-LSJL can cause osmotic stress. This may be part of the mechanism as to how hydrostatic pressure induces chondrogenic differentiation.

"Activated MLTK-alpha and MLTK-beta activate the p38 and JNK pathways through MKK3/6 and MKK4/7 phosphorylation"

"overexpression of xSox9 alone did not induce a substantial increase in xSox6 expression, co-expression of xMLTK-alpha and xMLTK-beta with xSox9 induced marked increases in xSox6 expression in a dose-dependent manner"

"p38 signaling can increase the transcriptional activity of Sox9, and transgenic mice in which p38 signaling is constitutively activated in chondrocytes, show phenotypes similar to those of mice that overexpress Sox9 in chondrocytes"<-increasing p38 signaling might be a way to grow taller.

Disruption of a Sox9 - β-catenin circuit by mutant Fgfr3 in Thanatophoric Dysplasia Type II.

"Using a mouse model of Thanatophoric Dysplasia Type II (TDII) in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9[so maybe FGFR3 can increase height in the right circumstances] and downregulation of β-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease β-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of β-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and β-catenin protein stability with it promoting Sox9 stabilization and β-catenin degradation. Both Sox9 overexpression and β-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3."

"Sox9 is expressed in progenitor or “resting” chondrocytes, proliferating chondrocytes and chondrocytes in the initial stage of differentiation (prehypertrophy), but is then abruptly downregulated as these cells differentiate into non-proliferative and terminally differentiated hypertrophic chondrocytes"

"Sox9 downregulation in hypertrophic chondrocytes is thought to play a critical function in endochondral growth and ossification since its forced expression in growth plate chondrocytes suppressed the production of hypertrophic cells and Vegfa expression, resulting in compromised vascularization at the ossification front and a severe chondrodysplasia"

"Sox9 can also suppress β-catenin transcriptional activity independent of β-catenin degradation, possibly by interfering with β-catenin - Tcf/Lef interactions"

"Sox9 protein levels are negatively regulated by Beta-catenin"

"strong correlation between mutant Fgfr3 expression, decreased Beta-catenin expression and activity and a failure to downregulate Sox9."

"Addition of FGF2 repressed β-catenin activity alone and augmented Fgfr3K650E driven repression"

"deletion of Sox9 in growth plate chondrocyte was recently found to induce apoptosis, suppress
Col10a1 expression and prevent proper hypertrophic differentiation"

"Beta-catenin stability is regulated by a multiprotein complex that includes of Beta-catenin, Axin, APC, and Gsk-3Beta. According to canonical regulation of Beta-catenin degradation, Gsk-3Beta phosphorylates primed Beta-catenin within this complex, which marks Beta-catenin for ubiquitylation and subsequent proteasome degradation in the cytoplas. Sox9 promotes nuclear translocation of Beta-catenin together with components of the Beta-catenin destruction complex, which leads to enhanced phosphorylation and degradation of Beta-catenin in the nucleus. Fgfr3K650E promoted both upregulation of Sox9 and Beta-catenin phosphorylation at Gsk-3Beta sites"

Unraveling the transcriptional regulatory machinery in chondrogenesis.

"The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) and the Rho-associated coiled coil-forming kinase (ROCK) interacted with Sox9 and directly phosphorylate it at serine 181. This modification caused nuclear accumulation of Sox9, increased the efficiency of Sox9 binding to DNA, and/or increased transcriptional activity. It occurred principally in the prehypertrophic zone of the growth plate, which is the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor, suggesting that PTHrP may inhibit conversion of proliferating cells into prehypertrophic cells, at least in part, through activating PKA and thereby the activity of Sox9. Histone acetyl transferase Tip60 increased Sox9/Sox5-dependent transcription in association with acetylation of Sox9 at lysine 61, 253, and 398. Tip60, together with Sox9 and Sox5, was also present in the chromatin of the Col2a1 enhancer. The Sox9 protein is also modified by protein inhibitor of activated STAT (PIAS)1-mediated SUMOylation. PIAS1 [may repress] or [stimulate] Sox9 activity. "

"P54nrb, 54-kDa nuclear RNA-binding protein, which physically interacted with Sox9 to increase transactivation of Col2a1 and promoted splicing of the Col2a1 mRNA"<-Grow taller with P54nrb?

"Trap230 (Med12) [is] a coactivator of Sox9"

Chondrogenesis enhanced by overexpression of sox9 gene in mouse bone marrow-derived mesenchymal stem cells.

"a high density micromass culture may compensate for the necessity of co-factors, L-sox5 and sox6."

The study states that overexpression of Sox9 enhanced chondrogenesis.

The postnatal role of Sox9 in cartilage

"The postnatal inactivation of Sox9 led to stunted growth characterized by decreased proliferation, increased cell death, and de‐differentiation of growth plate chondrocytes."  Loss of Sox9 resulted in compression and degeneration in other cartilagenous areas too such as the discs.  So ensuring an equilibrium quantity of Sox9 in those areas may help increase height.

"Upon postnatal Sox9 inactivation in the articular cartilage, the sulfated proteoglycan and aggrecan content of the uncalcified cartilage were rapidly depleted and the degradation of aggrecan was accompanied by higher ADAMTS5 immunostaining and increased detection of the aggrecan neoepitope, NITEGE."

"the loss of Sox9 in the IVD decreased the expression of cytokines, cell surface receptors, and ion channels"

Genes downregulated by loss of Sox9:
MATN3(up in LSJL)
UCMA
Col2a1(up in LSJL)
Sfrp5
Grem1
Rspo4
Cytl1
Clec3a
Fxyd2(up in LSJL)
Cilp2
Col27a1
THBS1
CHAD
HAPLN1(up)
COL11A2
COMP
VCAN(up)
CILP
ACAN(up)
BGN(up)
Lect1
Frzb
IGFBP5
LOXL2,3,4
PAPSS2
FAM38B
PIEZO2
PKD1
FGFR3
TRPV4
GPR126

Upregulated genes:
Dbh
Ddc
Syt1
Syt4
Ucp1
Mpo
CXCR2

"Sox9 is almost exclusively localized to the articular chondrocytes of the uncalcified cartilage, with very few Sox9 positive cells in the region of calcified cartilage"

"the polycystin gene (pkd1) that encodes for an integral membrane protein that has been shown to be a mechanical flow “sensor” in the renal epithelium was downregulated. The homozygous pkd1-null mice display skeletal abnormalities, including stunted growth and kyphosis of the spine"

Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways.

"During endochondral bone formation, Sox9 expression starts in mesenchymal progenitors, continues in the round and flat chondrocyte stages at high levels, and ceases just prior to the hypertrophic chondrocyte stage. Sox9 is important in mesenchymal progenitors for their differentiation into chondrocytes, but its functions post-differentiation have not been determined. To investigate Sox9 function in chondrocytes, we deleted mouse Sox9 at two different steps after chondrocyte differentiation. Sox9 inactivation in round chondrocytes resulted in a loss of Col2a1 expression and in apoptosis. Sox9 inactivation in flat chondrocytes caused immediate terminal maturation without hypertrophy and with excessive apoptosis. Inactivation of Sox9 in the last few cell layers resulted in the absence of Col10a1 expression, suggesting that continued expression of Sox9 just prior to hypertrophy is necessary for chondrocyte hypertrophy. SOX9 knockdown also caused apoptosis of human chondrosarcoma SW1353 cells. These phenotypes were associated with reduced Akt phosphorylation {LSJL upregulates Sox9 and increases p-Akt}. Forced phosphorylation of Akt by Pten inactivation partially restored Col10a1 expression and cell survival in Sox9(floxdel/floxdel) mouse chondrocytes, suggesting that phosphorylated Akt mediates chondrocyte survival and hypertrophy induced by Sox9. When the molecular mechanism of Sox9-induced Akt phosphorylation was examined, we found that expression of the PI3K subunit Pik3ca (p110α) was decreased in Sox9(floxdel/floxdel) mouse chondrocytes. Sox9 binds to the promoter and enhances the transcriptional activities of Pik3ca. Thus, continued expression of Sox9 in differentiated chondrocytes is essential for subsequent hypertrophy and sustains chondrocyte-specific survival mechanisms by binding to the Pik3ca promoter, inducing Akt phosphorylation."

"mesenchymal condensation and subsequent cartilage formation are absent in the limbs of Prx1-Cre"

"misexpression of Sox9 in hypertrophic chondrocytes results in a lack of bone marrow, and that Sox9 is a major negative regulator of cartilage vascularization"

" In 11Enh-Cre transgenic mice, Cre recombinase activities are controlled by the Col11a2 promoter and enhancer and begin during the round chondrocyte stage. 11Enh-Cre directs recombination at a later stage (both developmental and within the chondrocyte differentiation pathway) than Col2a1-Cre"

"11Enh-Cre initiated the direct recombination of floxed Sox9 genes in round chondrocytes"

"chondrocytes lacking Sox9 enter into terminal maturation without hypertrophy"

" the deletion of Pten results in the accumulation of PtdIns(3,4,5)P3, leading to forced activation of Akt."

"most differentiated cell types, including osteoblasts and adipocytes, do not express Sox9 and survive."<-thus if osteoblasts were expressing Sox9 in LSJL then there should have been apoptosis markers whereas in LSJL many apoptosis markers were downregulated.


Regulation of bone and cartilage development by network between BMP signalling and transcription factors

"Sox9 has been shown to directly regulate chondrogenic genes, such as Col2a1, Col11a2 and aggrecan"

"BMP2 markedly stimulated Sox9 expression in mouse limb bud cells"

"p54nrb, a component of para-speckled body, associates with Sox9 and stimulates its transcriptional activity"

"Znf219 and Arid5a, which physically interact with each other, also associate and co-localize with Sox9"<-These two genes are involved in histone related alterations in chondrogenesis.

"Sox9 associates with Gli2, a mediator of Ihh, and Sox9 and Gli2 co-operatively stimulate PTHrP expression and PTHrP gene promoter activity"

"Overexpression of Msx2 also consistently stimulates maturation of chondrocytes. In addition, cartilage development seems to be reduced in Msx2 knockout mice"

"Runx2 stimulates and Sox9 inhibits maturation of chondrocytes."

"Osterix is expressed in the prehypertrophic zones of growth plates and that, in global and conditional Osterix knockout mice, chondrogenesis was totally blocked at the hypertrophic stage and there was no evidence of calcification of chondrogenic matrices or formation of matrix vesicles"

"When cells experience ER stress, several ER sensors, including IRE1, ATF6, Perk, Oasis{up in LSJL as CREB3l1} and BBF2H7, all of which are anchored in the membrane of the ER, are released by membrane truncation, and subsequently respond to the ER stress. Interestingly, truncated forms of Oasis and BBF2H7 translocate to nuclei and function as transcription factors."

"Sec23 is a major target of BBF2H7 in chondrocytes. As expected, introduction of Sec23, an important chaperon protein for protein folding, rescued chondrocyte differentiation in BBF2H7-deficient cells"


"All-trans retinoic acid (RA) and other active retinoids are generated from vitamin A (retinol), but key aspects of the signaling pathways required to produce active retinoids remain unclear. Retinoids generated by one cell type can affect nearby cells, so retinoids also function in intercellular communication. RA induces differentiation primarily by binding to RARs, transcription factors that associate with RXRs and bind RAREs in the nucleus. Binding of RA: (1) initiates changes in interactions of RAR/RXRs with co-repressor and co-activator proteins, activating transcription of primary target genes; (2) alters interactions with proteins that induce epigenetic changes; (3) induces transcription of genes encoding transcription factors and signaling proteins that further modify gene expression (e.g., FOX03A, Hoxa1, Sox9, TRAIL, UBE2D3); and (4) results in alterations in estrogen receptor α signaling. Proteins that bind at or near RAREs include Sin3a, N-CoR1, PRAME, Trim24, NRIP1, Ajuba, Zfp423, and MN1/TEL. Interactions among retinoids, RARs/RXRs, and these proteins explain in part the powerful effects of retinoids on stem cell differentiation."

"high Stra6 expression is suggestive of a requirement for the actions of retinol."

"Inside the cell, RA is transported to the nucleus bound to CRABP2{up in LSJL}"

"Once in the nucleus, RA binds to RARα, β, or γ. These RARs can bind to one of the RXRs (RXRα, β, or γ)."<-RXRb is downregulated in LSJL.

"The Rex1 (Zfp42) gene is expressed at high levels in embryonic stem cells, and is transcriptionally inhibited by RA. The mechanism of inhibition involves loss of binding of the positively acting transcription factor Oct4 to an Oct4 site in the Rex1 promoter in response to RA. Interestingly, transcriptional repression by RA in embryonic stem cells is often mediated by a different mechanism, an increase in the expression of the orphan nuclear receptor GCNF (germ cell nuclear receptor) (Nr6a1), which then represses pluripotency genes such as Sox2, Nanog, or Oct4"

"Sox9 was shown to inhibit proliferation by activating the transcription factor Hes1{up in LSJL}. Sox9 [is transcriptionally activated] by RA"

Control of chondrogenesis by the transcription factor Sox9

"In chondrocytes, Sox9 binds as a homodimer to a pair of the consensus sequences of Col2a1{up}, Col9a1{up}, Col27a1, or Matrilin-1, and this binding is mediated by a dimerization domain located closer to the N-terminus than the HMG domain"

"Sox9-null cells do not express chondrogenic marker genes, such as Col2a1, Col9a2, Col11a2, and aggrecan{up}."

"fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), human cartilage glycoprotein 39, transient receptor potential vanilloid 4 (TRPV4), RAR agonists, and Src inhibitor increase Sox9 expression"

"CCAAT-binding factor, Sp1, CREB, Sonic hedgehog, and hypoxia-inducible factor 1α directly transactivate the Sox9 proximal promoter"

"transcription factors including Pax1{up}, Pax9, Nkx3.1, Nkx3.2, and Barx2{up} control the level of Sox9"

"Sox9 [inhibits] hypertrophic conversion by phosphorylation of S64 and S211 of Sox9 by protein kinase A (PKA), a downstream intracellular signaling molecule of parathyroid hormone-related peptide (PTHrP)/PTHrP receptor"

"Translocation of Sox9 into the nucleus is controlled by Sox9-calmodulin interaction through the nuclear localization signal of the HMG domain within a consensus calmodulin-binding region"

"cGMP-dependent protein kinase type II{PRKG2 is up in LSJL} reduces the activity of Sox9 by inhibiting the nuclear import of Sox9, a process that is mediated by phosphorylation of S181. Sox9 activity is also regulated by protein inhibitor of activated STAT1 (PIAS1)-mediated sumoylation. PIAS1 enhances the sumoylation at K398 and represses Sox9 activity. The stability and degradation of Sox9 protein are determined by the ubiquitin-proteosome pathway"

"Sox9 uses a cAMP response element-binding protein-binding protein (CBP)/p300 to exert its effects, which is mediated by histone acetylation"

"Smad3, a signaling molecule of transforming growth factor-β,(TGF-β) stimulates the Sox9-dependent transcriptional activation by modulating the interaction between Sox9 and CBP/p300"

"peroxisome proliferation-activated receptor-gamma co-activator 1-α (PGC1-α) is a coactivator of Sox9 during chondrogenesis"

"Sox9 interacts with a component of the mediator complex, the thyroid hormone receptor-associated protein (Trap) 230/Med12"

Analysis of post transcriptional regulation of SOX9 mRNA during in vitro chondrogenesis.

"SOX9 mRNA half life exhibited an inverse correlation with total SOX9 mRNA levels in both dedifferentiating human articular chondrocytes and chondrogenic pellet cultures."

"SOX9 mRNA exhibits a moderately short half life in human articular chondrocytes (HACs) which can be extended if the cells are exposed to environmental stresses such as cycloheximide treatment or hyperosmolarity."

"as levels of the SOX9 mRNA were falling, its half life was increased"

"The BMSCs exhibit a rapid turnover of SOX9 in both their undifferentiated state in monolayer culture and as they differentiate into chondrocytic cells in pellet culture"

"fine tuning of SOX9 mRNA decay rates, through some form of feedback mechanism, keeps overall levels of SOX9 mRNA within a certain threshold, even as transcriptional rates change."

Trans-activation of the mouse cartilage-derived retinoic acid-sensitive protein gene by Sox9.

"To determine whether Sox9 is an inducer of the chondrocyte phenotype, we investigated the role of Sox9 in transcription of another cartilage gene encoding the cartilage-derived retinoic acid-sensitive protein (CD-RAP)[also known as MIA]. CD-RAP is specifically expressed during chondrogenesis. We show here that Sox9 protein is able to bind to a SOX consensus sequence in the CD-RAP promoter. Mutation of the SOX motif led to decreased transcription of a CD-RAP promoter construct in chondrocytes. Overexpression of SOX9 resulted in a dose-dependent increased activity of CD-RAP promoter-driven reporter gene in both chondrocytes and nonchondrogenic cells. A truncated SOX9, which contains a binding domain but no trans-activation function, inhibited CD-RAP promoter activity. Overexpression of SOX9 increased the level of endogenous CD-RAP mRNA in chondrocytes, but was unable to induce endogenous gene expression in 10T1/2 mesenchymal cells or BALB/c-3T3 fibroblasts. These results suggest that Sox9 is a general transcriptional regulator of cartilage-specific genes. However, Sox9 does not appear to be able to induce the chondrocyte phenotype in nonchondrogenic cells{but TGFB1 and BMP-2 which are involved in LSJL may be able to}, implying that other factors are involved in chondrogenesis."

"CD-RAP/MIA is down-regulated coordinately by retinoic acid with type II collagen, the most abundant extracellular protein made by chondrocytes and generally considered to be characteristic of cartilage."

"SOX9 enhanced the CD-RAP promoter activity in both chondrocytes and nonchondrogenic cells, including BALB/3T3, MC3T3, and 10T1/2 cells"

"The CD-RAP promoter activity was modulated by bone morphogenetic proteins, parathyroid hormone-related peptide, transforming growth factor β, fibroblast growth factor, interleukin-1 and interleukin-8, epidermal growth factor, and retinoic acid"

"Sox9 is unable to activate endogenous Col2a1 in nonchondrogenic cells."

"a 48-bp enhancer element in the first intron of type II collagen (which includes a Sox9 motif) is able to generate chondrocyte-specific expression in vivo. However, mutation in each of three HMG-like domains in this fragment abolished the cartilage-specific expression in vitro and in vivo, suggesting that additional HMG-like DNA domains other than the Sox9 binding site are also critical for the cartilage-specific expression."

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

"Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification."

Sox9 is good for height growth in all stages aside from the hypertrophic stage.

"Differentiation of proliferating cells into prehypertrophic and hypertrophic chondrocytes is marked by an up to ten-fold increase in cell volume and development of a granular cell surface with numerous microvilli, which release matrix vesicles required for cartilage mineralization. This step is associated with substantial matrix remodelling: the hyaline cartilage matrix, comprising aggrecan, type II, VI and XI collagen made by resting and proliferating chondrocytes, is substituted by a calcifiying matrix deposited by hypertrophic chondrocytes that produce type X collagen and alkaline phosphatase"

"Maturation of chondrocytes in the lower hypertrophic zone to terminally differentiated chondrocytes is marked by upregulation of osteopontin, Vegfa, and Mmp13"

"RUNX2 plays a key role in endochondral ossification as it promotes not only expression of type X collagen[and MMP13] and maturation to hypertrophic chondrocytes, but also induces Vegfa in hypertrophic chondrocytes"

"Sox9 is expressed in all chondroprogenitor cells; it is essential for the formation of cartilage blastema of the limb mesenchyme, for proliferation and differentiation of chondrocytes in the foetal growth plate and for regulation of cartilage-specific genes including Col2a1, Col9a1, Col11a1, aggrecan and others. In the foetal and juvenile growth plates, Sox9 is expressed in resting and proliferating chondrocytes, with a maximum of expression in prehypertropic chondrocytes, but disappears completely from the hypertrophic zone"

"misexpression of Sox9 in hypertrophic chondrocytes significantly impaired postnatal skeletal growth and bone length[20% length decrease]. "


"To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis."

"[SW1353] cells showed increased gene expression of SOX6 (up to 14-fold) and COL2A1 (up to 13-fold), but aggrecan expression was low and was unchanged by SOX9. The SW1353 cells expressed high levels of COL1A1 and this was reduced 6-fold by SOX9 transduction."

Genes upregulated by Sox9 also upregulated by LSJL:
Apod
Crlf1

Genes downregulated:
Spint1

Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes.

Bapx1 is also known as Nkx-3.2

"The suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation.  The transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes.  Sox9 physically bound to [a] region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. "

"Paired box gene 1 (Pax1){up}, Pax9, and mesenchyme homeobox 1 (Meox1) proteins regulate Bapx1 expression by directly activating its promoter"

"Pax1/Pax9 and Meox1 expression is maintained in chondrogenic mesenchymal cells until they reach the pre-chondrocyte stage"

"Pax1/9 and Meox1 may regulate initial and/or early induction of Bapx1, and Sox9 contributes to maintain Bapx1 expression during subsequent chondrogenic differentiation. "

"S100A1 and S100B were direct Sox9 targets, and that they suppress hypertrophic chondrocyte differentiation"

Sox9 modulates cell survival and adipogenic differentiation of multipotent adult rat mesenchymal stem cells.

"Sox9 activity level affect the expression of the key transcription factor in adipogenic differentiation, C/EBPß, and moreover, cyclin D1 mediated, the expression of osteogenic marker osteocalcin in undifferentiated adult bone marrow derived rat mesenchymal stem cells (rMSC).  Introducing a stable Sox9 knockdown in undifferentiated rMSC resulted in a marked decrease in proliferation rate and an increase in apoptotic activity. This was linked to a profound upregulation of p21 and cyclin D1 gene and protein expression accompanied with an induction of caspase 3/7 activity and an inhibition of Bcl-2.   Sox9 silencing provoked a delayed S-phase progression and an increased nuclear localization of p21. The protein stability of cyclin D1 was induced in the absence of Sox9 presumably as a function of altered p38 signalling.In addition, the major transcription factor for adipogenic differentiation, C/EBPß, was repressed after silencing Sox9. The nearly complete absence of C/EBPß protein due to increased destabilization of the C/EBPß mRNA and the impact on osteocalcin gene expression and protein synthesis, suggests that a delicate balance of Sox9 level is not only imperative for proper chondrogenic differentiation of progenitor cells, but also affects the adipogenic and most likely osteogenic differentiation pathways of MSC."

"In a rat chondrocytic cell line (CFK2), overexpression of Sox9 induces alteration of cell cycle progression. Stable Sox9 transfected cells accumulate an increased proportion of the cells in the G0/G1 and S-phase of the cell cycle with a reduced number of cells in the G2/M phase"

"As a consequence of using a specific inhibitor for p38 (SB203580), Sox9 expression was repressed in rMSC during in vitro chondrogenesis"

"On average, after knockdown Sox9 specific mRNA copies were reduced 10-fold compared to control cells. Overexpression of Sox9 resulted in an average 100-fold increase in gene expression compared to controls"

"The number of control cells increased 8-fold during 7 days (doubling time2.3days), -SOX9 cells increased 4-fold (doubling time 3.5days) whereas +SOX9 cells showed a 15-fold (doubling time 1.8days) increase in cell number."<-So Sox9 transgenic had the greatest number of cells and the shortest doubling time.

"10% increased S-phase population after overexpressing Sox9 (+SOX9) compared to control cells"

"Overexpression of Sox9 resulted in decreased expression of p21 and cyclin D1 on mRNA and
on protein level"

"reduced activity of Sox9 induces apoptosis in rMSC (increased caspase3/7activity) and reduces expression of the pro-survival protein Bcl-2 while overexpression of Sox9 induces expression of Bcl-2 and reduces activity of caspase3/7 resulting in less apoptosis. In this line, it was reported that inhibition of Bcl-2 resulted in reduction of Sox9 gene expression and consequently in Sox9-dependent chondrogenic gene expression"

Monday, May 24, 2010

Grow Taller with Rolfing?

I wrote an earlier article about the various ways of maintaining proper spinal alignment which included chiropractic, massage, and rolfing.  Rolfing is different from massage even though it will have the same positive effects on your posture(and therefore your height).  Rolfing is a form of deep tissue massage.  Deep tissue massage targets your connective tissue of which articular cartilage is a part of.  We know that dynamic loading of articular cartilage enhances gene expression of anabolic activities for cartilage and chondrocytes.  Deep tissue massage is dynamic loading of articular cartilage.  If your growth plates are still open, deep tissue massage may enhance chondrocyte proliferation and differentiation in your growth plate resulting in increased height.  If you have no active chondrocytes then dynamic loading(deep tissue massage) may give you some height in your cartilage.  If you do re-activate chondrocyte proliferation with a mechanism like lateral synovial joint loading then deep tissue massage will have the same effect on enhancing chondrocyte proliferation and differentiation.

The thing is of course that lateral synovial joint loading is also a mechanism for dynamic loading of the cartilage.  It just also increases interstitial fluid flow in the bone and causes trabecular bone microfractures which releases mesenchymal stem cells.  But, it's very hard to dynamically load the cartilage in the spine(maybe you can use the cobra stretch or side bends preferably finding a way to increase the load over time).  Someone else may be able to force your spinal muscles to the side and get their hands deep in there to provide dynamic load to those intervertebral discs.

We also know that muscle and bone growth tend to be linked.  So, stretching the fascia of muscles could initiate signaling pathways that aid in bone growth.  Proper skeletal alignment and improved nervous system function could help as well.  If you want to try fascia massage, you can try one of these:
It can also provide dynamic loading on your cartilage as well.  Just roll your entire body over it.  Your back, your sides, and your appendages.  Still can't get the spine though cause those pesky ribs are in the way.

Thai traditional massage increases biochemical markers of bone formation in postmenopausal women: a randomized crossover trial.

"Forty-eight postmenopausal women participated in the study. All volunteers were randomized to a 2-hour session of Thai traditional massage twice a week for 4 weeks and a 4-week control period after a 2-week washout, or vice versa. Twenty-one subjects were allocated to receiving Thai traditional massage first, followed by the control period, while 27 were initially allocated to the control period.
Serum P1NP increased significantly after Thai traditional massage, while there was no change in serum osteocalcin or CTX. During the control period, there was no significant change in P1NP, osteocalcin or CTX compared to baseline. When age and height were taken into account, P1NP in postmenopausal women whose ages were in the middle and higher tertiles and whose heights were in the lower and middle tertiles had a 14.8 +/- 3.3% increase in P1NP after massage, while no change in P1NP was found in the rest of the women.
Thai traditional massage results in an increase in bone formation as assessed by serum P1NP, particularly in postmenopausal women who are older and have a smaller body build."

Why would a smaller height be beneficial to increased bone formation in response to rolfing?  Smaller body size may mean that the massage may be more centralized over a smaller area increasing the frequency that an individual part is exposed to load.  "tall postmenopausal women received less repetitive cyclic numbers of massage per body surface area than shorter women during the same period"

"Thai Traditional massage exerts pressure on the body in a rhythmic fashion. The massage
performer uses the outstretched heels of both hands to exert pressure on the body of the
subject approximately once every 1–2 seconds for 2 hours."

No chondrogenic related genes were measured.

Effects of Meridian Massage on physical growth and infants' health as perceived by mothers.

"Meridian Massage is a traditional practice that manually stimulates the body's meridian system - the same network of vital energy channels used in acupuncture. The present study was to assess the effect of Meridian Massage on physical growth and infants' health as perceived by mothers.
A study was conducted in a community health center in Korea. A total of 169 healthy infant-mother dyads were assigned to the Meridian Massage group or the gentle touch massage group, based on the mother's preference. All massages were conducted by the mothers for 15 min per session, one time daily over a course of 6 weeks. In each group, the infant's weights, heights, and the number of days with illness as perceived by mothers and related clinic visits were measured.
Significant differences were observed in weight and height after 6 weeks between the Meridian group and the gentle touch massage group. Infants in the Meridian Massage group showed a significantly different number of days with perceived clinic visits compared to those in the control group.
Meridian Massage may facilitate physical growth and improve infants' health outcome as perceived by mothers. A randomized controlled trial is required to further explore the effects of Meridian Massage in early infancy."

"After completion of the 6-week intervention, the average height growth of infants in the Meridian Massage group was 4.5 ± 0.2 mm, higher than infants in the control group (3.2 ± 0.1 mm)."

The effects of infant massage on weight, height, and mother-infant interaction., found no significant effect of massage on baby height after four weeks. The study was in a foreign language so I could not get it.

Sunday, May 23, 2010

Smurf2

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β-Catenin

"Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β-catenin in Col2a1-Smurf2 embryos."

"Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β-catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1-Smurf2 limbs."

"Smurf2 directly interacts with Smad1 and Smad2 and indirectly interacts with the type I receptor of TGFβ and β-catenin through an adaptor Smad7 for ubiquitination and proteasome-dependent degradation.math image"

"we observed that the number of prehypertrophic and hypertrophic chondrocytes in a defined area was increased in the transgenic growth plates versus that in the wildtype counterparts"<-Endochondral ossification was also accelerated but sometimes this can result in increased height.

"the entire length of the transgenic limb was shorter"<-so Too much Smurf2 can decrease height.

"upregulation of β-catenin in Col2a1-Smurf2 transgenic limbs is caused by a decrease in GSK-3β by Smurf2."

According to Smurf2 regulates the senescence response and suppresses tumorigenesis in mice., Smurf2 is involved in cellular senescence and that may be the primary reason for decreasing height regardless of anything else.

Grow Taller Pills

We know that mechanical loading of cartilage and bone can help increase height.  We know that exercise can help to lower myostatin levels and increase insulin sensitivity and sensitivity to IGF-1.  We know that an increase in fluid flow in the bone can also help increase height.  We don't know exactly what the best exercises are increasing all those factors or what the best intervals/intensity levels to perform them are but we have some good ideas.

We also have a lot of potential tools to increase height but they are only available to people in the medical profession.  What are some pills to increase height that you can find at your local store or pharmacy?

We already know about Lithium.  Lithium increases stem cell proliferation and differentiation which is very, very good.  Lithium is available by prescription if you happen to suffer from depression or mood swings(like being prone to aggression).  It is toxic to the kidneys in one out of twenty individuals.  Lithium is also used in some batteries but I would not recommend sucking on batteries.

Dexamethasone is also something that could help...

Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells.

"To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype[This study was done in vitro but one can assume that fibroblast growth factor-2 would be available in humans]. MSCs [were] obtained from 5 young horses. First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10(7)M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1alpha. Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2-dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2-dexamethasone remained significantly increased. Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage."

Again more stem cells = good.  Dexamethasone had positive benefits even in the absence of FGF-2. Dexamethasone is unfortunately a steroid.  It is available by prescription though.

Phosphate is needed for optimal mesenchymal stem cell differentiation but I'm not sure excess phosphate will take MSCs to the next level.

Promotion of stem cell proliferation by vegetable peptone.
"The study described here was conducted to investigate the proliferative effect of vegetable peptone[Peptones are small polypeptides that are intermediate products in hydrolysis of proteins.] on adult stem cells in the absence of serum, and its possible mechanisms of action.  Cell viability and proliferation were determined using the MTT assay and Click-iT EdU flow cytometry, respectively. In addition, changes in expression of cytokine genes were analysed. Viability of cord blood-derived mesenchymal stem cells (CB-MSC) and adipose tissue-derived stem cells (ADSC) increased significantly when treated with the peptone. In addition, median value of the group treated with peptone shifted to the right when compared to the untreated control group. Furthermore, quantitative analysis of the cytokines revealed that production of vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) increased significantly in response to treatment with our vegetable peptone in both CB-MSCs and ADSCs. Vegetable peptone promotes proliferation of CB-MSCs and ADSCs. Induction of stem cell proliferation by vegetable peptone is likely to be related to its induction of VEGF, TGF-beta1, and IL-6 expression."

The peptones were characterized mainly by their amino acid contents.

"Treatment with pea peptone induced production of 678 pg/ml (increase of 8.83 ± 1.689 fold compared to untreated controls), 485 pg/ml (an increase of 3.79 ± 0.195 fold), and 332 pg/ml (an increase of 2.55 ± 0.217 fold) of VEGF, TGF-β1, and IL-6, respectively, wheat peptone induced production of 1019 pg/ml (increase of 13.27 ± 1.447 fold) and 387 pg/ml (increase of 3.02 ± 0.234 fold) of VEGF and TGF-β1, respectively, in CB-MSCs"<-Wheat peptone did not induce production of IL-6.  Pea Peptone had 5 times as much Arginine as Wheat peptone and that could be the cause of the IL-6.

"Pea peptone induced production of 122 pg/ml (increase of 6.70 ± 0.280 fold), 194 pg/ml (increase of 5.78 ± 0.445 fold), and 166 pg/ml (increase of 1.11 ± 0.080 fold) of VEGF, TGF-β1, and IL-6, respectively, whereas wheat peptone induced production of 172 pg/ml (increase of 9.49 ± 0.896 fold), 170 pg/ml (increase of 5.07 ± 0.356 fold), and 192 pg/ml (increase of 1.28 ± 0.324 fold), of VEGF, TGF-β1, and IL-6, respectively, in ADSCs"

"VEGF is known to exert its proliferation-promoting functions via two tyrosine kinase receptors: VEGF-1 (Flt-1) and VEGF-2 (Flk-1/KDR)".  VEGF is also involved in stem cell survival.  IL-6 activates STAT3 and MAPK.  TGF-Beta induces IL-6 and VEGF.

Vegetable peptone is available but I'm not sure if it's safe for human ingestion. 

Hexadecanoic Acid from Buzhong Yiqi Decoction Induced Proliferation of Bone Marrow Mesenchymal Stem Cells.

"Buzhong Yiqi decoction (BYD) is a well-known ancient tonic prescription in traditional Chinese medicine (TCM). The purpose of this study is to identify active components of BYD involved in promoting proliferation of mesenchymal stem cells (MSCs) and to investigate its mechanism. BYD was extracted with petroleum ether, ethanol, and water. Evidence provided by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, proliferation cell nuclear antigen immunoreactivity, cell cycle analysis, and gas chromatography-mass spectrometry indicated that hexadecanoic acid (HA) in BYD extracted with petroleum ether is the active compound responsible for increasing proliferation of MSCs. Western blot analysis show that HA significantly increase retinoic acid receptor (RAR) levels of MSCs, but not estrogen receptor, thyroid hormone receptor, vitamin D receptor, glucocorticoid receptor, and peroxisome proliferator-activated receptor. Reverse transcription-polymerase chain reaction revealed that HA significantly increased RAR mRNA levels. Furthermore, the mechanism of HA action depends on RAR pathway and up-regulates expression of mRNA for insulin-like growth factor-I, the target gene of RAR. Our findings have now allowed for a refinement in our understanding of TCM with respect to pharmacological regulation of stem cells and may be useful to stem cell biology and therapy."

"Buzhong Yiqi decoction (BYD), a well-known ancient tonic prescription in TCM, comprises eight herbs: Radix atragali, Radix codonopsis, Radix et rhizoma glycyrrhizae, Radix angelicae sinensis, Pericarpium citri reticulatae, Rhizoma cimicifugae, Radix bupleuri, and Rhizoma atractylodis macrocephalae."

"HA occurs naturally in many Chinese herbal medicines such as E. prostrata, Reishi Houshi, and Astragalus."

BYD is only available by prescription and only in China. 

Effects of immobilized glycosaminoglycans on the proliferation and differentiation of mesenchymal stem cells. 

"MSC proliferation and differentiation was studied on GAG-derivatized chitosan membranes. The GAGs[glycosaminoglycans] included heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid. The covalent GAG immobilization method and amount of immobilized GAG were varied. It was found that MSC growth increased as much as fivefold on GAG-immobilized surfaces compared to tissue culture plastic and chitosan-only controls. The MSC growth rates increased significantly with increasing GAG density on the culture surfaces. The MSC proliferation rates on heparin, heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate exhibited nonlinear increases with the level of fibronectin binding on these surfaces. In contrast, MSC proliferation on hyaluronic acid and chondroitin 4-sulfate was found to be independent of fibronectin or vitronectin binding on the surfaces, suggesting that these GAGs influenced MSC proliferation through different mechanisms. In conclusion, the results indicate that GAG immobilization on chitosan scaffolds provides an effective means of manipulating MSC proliferation." 

We already know about chondroitin sulfate.  Heparin is only available in clinical trials.  Hyaluronic acid is available in restylane which is used in cosmetics.

Saturday, May 22, 2010

Inhibiting Myostatin to increase your Height

We know it's possible to grow taller by inhibiting myostatin.  But how do we inhibit myostatin?  Myostatin helps regulate stem cell proliferation and differentiation(it inhibits it).  If we could inhibit myostatin we would benefit from enhanced stem cell proliferation and differentiation.  If you are doing Lateral Synovial Joint Loading, you want as much MSC proliferation and differentiation as possible.  If you are still naturally developing then inhibiting myostatin and nothing else may help you increase your stature. There are no products out right now that inhibit myostatin.  Are there natural ways to inhibit myostatin?  Can we design exercises? 

Caveolin-3 regulates myostatin signaling. Mini-review.

"Caveolins, components of the uncoated invaginations of plasma membrane, regulate signal transduction and vesicular trafflicking. Loss of caveolin-3, resulting from dominant negative mutations of caveolin-3 causes autosomal dominant limb-girdle muscular dystrophy (LGMD) 1C and autosomal dominant rippling muscle disease (AD-RMD). Myostatin, a member of the muscle-specific transforming growth factor (TGF)-beta superfamily, negatively regulates skeletal muscle volume[It negatively regulates bone volume too]. Herein we review caveolin-3 suppressing of activation of type I myostatin receptor, thereby inhibiting subsequent intracellular signaling. In addition, a mouse model of LGMD1C has shown atrophic myopathy with enhanced myostatin signaling. Myostatin inhibition ameliorates muscular phenotype in the model mouse, accompanied by normalized myostatin signaling. Enhanced myostatin signaling by caveolin-3 mutation in human may contribute to the pathogenesis of LGMD1C. Therefore, myostatin inhibition therapy may be a promising treatment for patients with LGMD1C. More recent studies concerning regulation of TGF-beta superfamily signaling by caveolins have provided new insights into the pathogenesis of several human diseases." 

Now remember that TGF-beta is important in causing stem cells to differentiate into chondrocytes.  This means that caveolins are very important.  Caveolin-3 actually supresses myostatin reception.  Extraneous injection of Caveolin-3 may inhibit myostatin and increase stem cell differentiation.   

Here's a way to inhibit myostatin if you haven't been born yet... 

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos. 

"Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals." 

Now it's possible to alter gene expression in fully developed adults too.  So perhaps this lentiviral vector can be used to alter stature in people already born.  

We can also block myostatin signaling... 

SB431542 treatment promotes the hypertrophy of skeletal muscle fibers but decreases specific force. 

"The small molecule inhibitor SB431542 inhibits activin type I receptors. The muscle growth-inhibitor myostatin binds to and signals via these receptors. The aim of this study was to test the hypothesis that SB431542 can inhibit myostatin-related Smad signaling and induce muscle growth in cultured C2C12 myotubes and increase growth and specific force in cultured Xenopus muscle fibers. The effect of SB431542 was assessed in vitro on C2C12 myotubes and ex vivo using mature Xenopus muscle fibers. SB431542 treatment reduced myostatin-induced C-terminal Smad2 phosphorylation and resulted in the formation of enlarged myotubes. However myogenin expression was unchanged, while p70 S6k phosphorylation at Thr389, total myosin heavy chain, and the rate of protein synthesis were all reduced. Mature Xenopus muscle fibers that were treated with SB431542 had a higher fiber cross-sectional area but decreased specific force production than control. SB431542 can initially antagonize myostatin signaling, but long-term unexpected signaling effects occur. Muscle fibers hypertrophy, but their specific force decreases compared to control." 

Two problems:  SB431542 did not have long-term benefits and we need to inhibit myostatin's effects in the bone not the muscle.  

Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway is involved in myostatin-regulated differentiation repression. 

"The cytokines of transforming growth factor beta (TGF-beta) and its superfamily members are potent regulators of tumorigenesis and multiple cellular events. Myostatin is a member of TGF-beta superfamily and plays a negative role in the control of cell proliferation and differentiation. We now show that myostatin rapidly activated the extracellular signal-regulated kinase 1/2 (Erk1/2) cascade in C2C12 myoblasts. A more remarkable Erk1/2 activation stimulated by myostatin was observed in differentiating cells than proliferating cells. The results also showed that Ras was the upstream regulator and participated in myostatin-induced Erk1/2 activation because the expression of a dominant-negative Ras prevented myostatin-mediated inhibition of Erk1/2 activation and proliferation. Importantly, the myostatin-suppressed myotube fusion and differentiation marker gene expression were attenuated by blockade of Erk1/2 mitogen-activated protein kinase (MAPK) pathway through pretreatment with MAPK/Erk kinase 1 (MEK1) inhibitor PD98059, indicating that myostatin-stimulated activation of Erk1/2 negatively regulates myogenic differentiation. Activin receptor type IIb (ActRIIb) was previously suggested as the only type II membrane receptor triggering myostatin signaling. In this study, by using synthesized small interfering RNAs and dominant-negative ActRIIb, we show that myostatin failed to stimulate Erk1/2 phosphorylation and could not inhibit myoblast differentiation in ActRIIb-knockdown C2C12 cells, indicating that ActRIIb was required for myostatin-stimulated differentiation suppression. Altogether, our findings in this report provide the first evidence to reveal functional role of the Erk1/2 MAPK pathway in myostatin action as a negative regulator of muscle cell growth." 

So inhibiting ActRIIb can also enhance cellular proliferation as can inhibiting Ras. 

Enhanced muscle growth by plasmid-mediated delivery of myostatin propeptide. 

"Myostatin is a member of the transforming growth factor beta (TGF-beta) superfamily that functions as a negative regulator of skeletal muscle development and growth. Myostatin blockade therefore offers a strategy for promoting muscle growth in livestock production without resorting to genetic manipulation. In this report, we examined the effect of myostatin inhibition by plasmid-mediated delivery of a mutant myostatin propeptide (MProD76A), a natural inhibitor of myostatin, on the growth performance of mice. A significant increase in skeletal muscle mass was observed after a single intramuscular injection of naked plasmid DNA encoding MProD76A into mice. Enhanced muscle growth occurred because of myofiber hypertrophy, but no cardiac muscle hypertrophy and organomegaly was observed in the mice after myostatin inhibition by plasmid-mediated MProD76A delivery. These results demonstrate a promising approach to enhancing muscle growth that warrants further investigation in domestic animals." 

Now this peptide may only inhibit myostatin in myofiber muscle and not bone.  

Systemic myostatin inhibition via liver-targeted gene transfer in normal and dystrophic mice. 

"Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose-dependent manner. CONCLUSIONS/SIGNIFICANCE: Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle." 

Now this adeno-associated virus seemed to inhibit myostatin everywhere(everywhere includes bone) so this has more promise. 

Trichostatin A induces follistatin a myostatin inhibitor.  

Inhibition of myostatin promotes myogenic differentiation of rat bone marrow-derived mesenchymal stromal cells. 

"Mesenchymal stromal cells (MSC) have been thought to be attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of MSC myogenesis is very low. Thus MSC treatment for DMD is restricted. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. We hypothesized that inhibition of Mstn by using anti-Mstn antibody (Ab) would ameliorate the myogenic differentiation of MSC in vitro and in vivo. Methods MSC were isolated from rat bone marrow. Induced rat MSC (rMSC) were treated with various concentrations of anti-Mstn Ab. The expression of myogenic differentiation antigen (MyoD), myogenin and myosin heavy chain-type alpha (MHC-alpha) were estimated by immunofluorescence analysis and reverse transcription-polymerase chain reaction (RT-PCR). Adipogenic differentiation of rMSC inhibited by anti-Mstn Ab was evaluated by Oil Red O staining. The expression of dystrophin was detected 16 weeks after anti-Mstn Ab injection and rMSC transplantation by immunofluorescence staining, RT-PCR and Western blot. Motor function, serum creatine kinase (CK) and histologic changes were also evaluated. Results Five-azacytidine-mediated myogenic differentiation induced significant endogenous Mstn expression. Anti-Mstn Ab improved the expression of MyoD, myogenin and MHC-alpha and inhibited adipocyte formation. Sixteen weeks after transplantation, the inhibition of Mstn had improved motor function and muscle mass. In accordance with the increased motor function and muscle mass, dystrophin expression had increased. Furthermore, serum CK and centrally nucleated fiber (CNF) levels decreased slightly, suggesting specific pathologic features of the dystrophic muscle were partially restored. Conclusions Using anti-Mstn Ab, we found that inhibition of Mstn improved myogenic differentiation of rMSC in vitro and in vivo. A combination of Mstn blockade and MSC transplantation may provide a pharmacologic and cell-based strategy for the treatment of DMD." 

So there are anti-myostatin antibodies. There is some indication that aerobic exercise may inhibit myostatin... 

Myostatin Decreases with Aerobic Exercise and Associates with Insulin Resistance. 

"There is mounting evidence that skeletal muscle produces and secretes biologically active proteins or "myokines" that facilitate metabolic cross talk between organ systems. The increased expression of myostatin, a secreted anabolic inhibitor of muscle growth and development, has been associated with obesity and insulin resistance. Despite these intriguing findings, there have been few studies linking myostatin and insulin resistance. METHODS.: To explore this relationship in more detail, we quantified myostatin protein in muscle and plasma from 10 insulin-resistant, middle aged (53.1 +/- 5.5 years) men before and after 6 months of moderate aerobic exercise training (1200 kcal/wk at 40-55% peak VO2). To establish a case-effect relationship we also injected C57/Bl6 male mice with high-physiologic levels of recombinant myostatin protein. RESULTS.: Myostatin protein levels were shown to decrease in muscle (37%, P=0.042, n=10) and matching plasma samples (28.7 pre-training to 22.8 ng/ml post-training, P=0.003, n=9) with aerobic exercise. Furthermore, the strong correlation between plasma myostatin levels and insulin sensitivity (R2 = 0.82, P<0.001, n=9) suggested a cause-effect relationship that was subsequently confirmed by inducing insulin resistance in myostatin-injected mice. A modest increase (44%) in plasma myostatin levels was also associated with significant reductions in the insulin-stimulated phosphorylation of AKT (Thr308) in both muscle and liver of myostatin treated animals. CONCLUSIONS.: These findings indicate that both muscle and plasma myostatin protein levels are regulated by aerobic exercise and furthermore, that myostatin is in the causal pathway of acquired insulin resistance with physical inactivity." 

Exercise increases insulin sensitivity.  Is it the increase in insulin sensitivity that inhibits myostatin or something with the exercise itself?  Also, only myostatin in the muscle decreased; we don't know about the bone.  There is also evidence that resistance exercise inhibits myostatin too but does that include the bone or just the muscle..