Monday, July 11, 2011

Fluid flow: LSJL v osteocytes & osteoblasts v chondrocytes

Since LSJL samples likely included osteocytes in the gene expression analysis. It should be useful to know which genetic upregulation is osteocyte specific.

Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes.

"MC3T3-E1 osteoblasts were studied as a positive control for the MLO-Y4 osteocyte response to mechanical loading. MLO-Y4 osteocytes and MC3T3-E1 osteoblasts were submitted to 1-h PFF (0.7 +/- 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5-3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real-time polymerase chain reaction (PCR). In MLO-Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c-jun, connexin 43, and CD44 at 1-3-h PI. In MC3T3-E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c-jun at 0.5-3-h PI. In MLO-Y4 osteocytes, gene expression of PFF-induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L-NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF-induced Wnt production. The response to PFF differed in MC3T3-E1 osteoblasts."

"The Wnt canonical pathway is activated when a Wnt molecule binds to the cell surface receptor complexes consisting of human lowdensity lipoprotein (LDL) receptor-related protein 5/6 (LRP5/6) and frizzled transmembrane proteins (Fzd)"<-This leads to increased Beta-catenin levels.

"Messenger RNA expression of Wnt3a and the Wnt antagonist SFRP4 was observed in both osteocytes and osteoblasts. Wnt5a gene expression was quantifiable in MC3T3-E1 osteoblasts, but not in MLO-Y4 osteocytes."

"PFF significantly increased NO production at 5 min in MLO-Y4 osteocytes by 4.6-fold, and in MC3T3-E1 osteoblasts by 3.4-fold. The stimulatory effect continued up to 60 min in both cell types"

Voltage profile generation for simultaneous multi-protein detection in western blot analysis

"Protein samples were harvested from MC3T3 osteoblast like cells. Cells were cultured on collagen coated glass slides in MEM medium containing 10% fetal bovine serum and antibiotics. At 80% confluence, cells were serum starved for 12 h, and they were subjected to uni- form flow shear stress at 10 dyn/cm2 for 1 h"

Akt signaling increased in osteoblasts between 0-40 minutes of fluid shear stress.  Flow dramatically increased p-38, p-ERK, and slightly increased p-Akt.  Thus some of the increase in ERK-p and p-38 detection in LSJL may be due to it's stimulation in osteoblasts but not so much for Akt.

Differential Activation and Inhibition of RhoA by Fluid Flow Induced Shear Stress in Chondrocytes.

"Activities of GTPase RhoA in chondrocytes are dependent on intensities of flow induced shear stress. RhoA activities can be either elevated or reduced by selecting different levels of shear stress intensities. C28/I2 chondrocytes have increased RhoA activities in response to high shear stress (10 or 20 dyn/cm(2) ), whereas a decrease in activity was seen with an intermediate shear stress of 5 dyn/cm(2). No changes were seen under low shear stress (2 dyn/cm(2) ). The observed 2-level switch of RhoA activities is closely linked to the shear stress-induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA-V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. In chondrocytes, expression of various metalloproteinases is, in part, regulated by shear and normal stresses through a network of GTPases."

"Moderate mechanical loading, for instance, is reported to decrease proteolytic activities of degenerative enzymes in the articular cartilage, while excessive loading may lead to an increase in expression of matrix metalloproteinases"<-This isn't necessarily a bad thing as some MMP's like MMP13 are associated with endochondral ossification.

"Rho-associated kinase (ROCK), promotes the assembly of actin cytoskeleton and phosphorylation of myosin light chains. By regulating intracellular tension through the cytoskeleton, this RhoA-ROCK signaling alters cell shape, and migration patterns as well as cellular differentiation"

"To test the role of intracellular tension in shear stress-induced RhoA activity, we used ML-7 to inhibit myosin light chain kinase or blebbistatin (Bleb) to inhibit non-muscle myosin II. Pretreating with ML-7 (25 μM) or Bleb (50 μM) also prevented shear stress-induced RhoA activation and inhibition at corresponding shear stress levels"

"myosin II-dependent, tensed actin cytoskeleton is necessary for selective RhoA regulation by shear stress regardless of the shear stress magnitude."

"Chondrocytes are in general rich in cortical actin but poor in cytosolic stress fibers. When they dedifferentiate to fibroblast-like cells, they are reported to develop stress fibers. Shapes and differentiation states of chondrocytes are regulated differentially by intermediate and high shear stresses"

Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts.

"Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified."

" β-catenin in the cytoplasm is stabilized by the inactivation of a destruction complex such as axin and GSK3β (glycogen synthase kinase 3β) and translocated to the nucleus. The β-catenin in the nucleus associates with TCF/LEF (T-cell factor/lymphocyte enhancing factor) transcription factors, leading to the activation of TCF/LEF and induction of expression of Wnt target genes"

That was a Yokota study.  So Beta-Catenin is likely stabilized in the osteoblasts by LSJL.  How that can apply to height increase is unclear.

Another Yokota study:

RhoA GTPase interacts with beta-catenin signaling in clinorotated osteoblasts.

"Does unloading suppress an activation level of RhoA GTPase and β-catenin signaling in osteoblasts? If yes, what is the role of RhoA GTPase and actin filaments in osteoblasts in regulating β-catenin signaling? Using a fluorescence resonance energy transfer (FRET) technique with a biosensor for RhoA together with a fluorescent T cell factor/lymphoid enhancer factor (TCF/LEF) reporter, we examined the effects of clinostat-driven[microgravity simulator] simulated unloading. Both RhoA activity and TCF/LEF activity were downregulated by unloading. Reduction in RhoA activity was correlated to a decrease in cytoskeletal organization of actin filaments. Inhibition of β-catenin signaling blocked unloading-induced RhoA suppression, and dominant negative RhoA inhibited TCF/LEF suppression. On the other hand, a constitutively active RhoA enhanced unloading-induced reduction of TCF/LEF activity. The TCF/LEF suppression by unloading was enhanced by co-culture with osteocytes, but it was independent on the organization of actin filaments, myosin II activity, or a myosin light chain kinase. Collectively, the results suggest that β-catenin signaling is required for unloading-driven regulation of RhoA, and RhoA, but not actin cytoskeleton or intracellular tension, mediates the responsiveness of β-catenin signaling to unloading."

"simulated unloading by clinorotation reduces β-catenin signaling activity of MC3T3-E1 cells"

"RhoA is activated by fluid flow and its activation mediates fluid flow-induced PI3K and MAPK signaling"

Fluid flow in the osteocyte mechanical environment: a fluid-structure interaction approach.

"Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. The objective of this study is to employ fluid-structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. By simulating loading levels representative of vigorous physiological activity (3000mupu compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities(60.5mupu/s and average maximum shear stresses  surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. The greatest mechanical stimulation of the osteocyte occurs in the cell processes, the most mechanosensitive area of the cell."

"Loading the bone matrix surrounding osteocytes generates a pressure differential that drives flow of interstitial fluid within the lacunar–canalicular network. The fluid flow generates a shear stress on the osteocyte cell membrane"

"pressure gradients within individual canaliculi [can be] as high as 1 Pa/nm, the equivalent of an approximately 800 Pa pressure gradient along the length of a single canaliculus"

"within the range of 0.1–2.2 Pa, which has been shown in cell culture studies of osteoblastic cells to result in increased nitric oxide (NO), prostaglandin ( PGE 2) and osteopontin production"<-These chemicals can affect MSCs and chondrocytes.  Calcium signaling also increases which too can impact MSCs.

Tuesday, July 5, 2011

Grow taller with Wif-1?

LSJL alters Wif-1 expression.  On the pathway diagram WIF-1 is highlighted and since WIF-1 is the only protein associated with that node.  LSJL likely alters WIF-1 in some fashion.  LSJL also alters either SFRP1, 2, 4, or 5.

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis.

"Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone.  Wif-1 [has specific binding] to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Wif-1 [blocks] Wnt3a-mediated activation of the canonical Wnt signalling pathway. Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells."

So we may want to counteract Wif-1 if it is upregulated by LSJL.  LSJL upregulates Wnt2 expression.

"Wnt3a and Wnt7a are expressed in nonchondrogenic limb tissue and, similarly to Wnt1, prevent chondrogenesis in micromass culture "

"Wnt5a and Wnt5b support formation of cartilage nodules in micromass cultures"<-LSJL upregulates Fzd2 and Fzd3 which can serve as frizzled receptors.

"Secreted antagonists of Wnt signalling, such as Dkk1 and sFRP1, have been identified as important factors that control the activity of Wnt proteins in skeletal development"<-LSJL alters Dkk expression.

"Dkk1-deficient mice exhibit abnormalities in skeletal development such as duplication and fusion of limb digits"

"Targeted mutation of the gene encoding secreted frizzled-related protein 1 (Sfrp1) in the mouse leads to a rather mild skeletal phenotype characterised by enhanced endochondral ossification. Chondrogenesis of murine embryonic fibroblasts isolated from Sfrp1-deficient mice is also accelerated"<-so Sfrp1 may be a way to grow taller.

"Wif-1 expression in the mesenchyme surrounding cartilage elements forming in the limb during early embryogenesis"

"WIF1 [is] a gene that was strongly downregulated in chondrocytes after their retinoic acid-induced dedifferentiation."

"Wif1 mRNA was absent from the growth plate"<-maybe Wif1 is a senescence related mechanism and restoring Wif1 can inhibit growth plate senescence.

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity.

"Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. [Wif-1] is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes.  CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling."

" Wif-1 is predominantly expressed in the peripheral chondrocyte layers of epiphyseal and articular cartilage. It is capable to interfere with Wnt3a-mediated signalling in chondrogenic cells; for example, it releases Wnt3a-dependent blockade of chondrogenesis"<-But target deletion of Wif-1 does not cause modultion of skeletal phenotypes.

"CTGF [enhances] expression of aggrecan (Acan) mRNA in chondrocytes"

"CTGF expression in peripheral layers of epiphyseal and articular cartilage may play a role in stabilising the cartilage surface."

"CTGF does not affect canonical Wnt signalling in HEK293T cells or 4C3 chondrocytes."

Can height increase due to diosgenin?

11-Keto-Diosgenin is a part of Alkoclar's new CNP inducing formula.  CNP does have height increasing potential as it inhibits FGFR3(which can cause dwarfism) and stimulates ECM extracellular matrix synthesis which helps you grow taller[stem cells have the ability to respond to the extracellular matrix environment].  Forms of Diosgenin are available for sale like Dioscorea Villosa (Dioscorea Villosa (Potency: 15C)).  The effectiveness of such compounds can not be verified.

Does Diosgenin increase CNP expression?  Does Diosgenin have other height augmenting effects other than increasing CNP expression?  It seems that the growth plate beneficial effects are mostly from reducing things like inflammation.  I don't know if it would have any impact on adults.

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells.

"Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa).  murine MC3T3-E1 osteoblastic cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay[Diosgenin stimulates osteoblast proliferation]. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment. Calcium deposition within the ECM also showed the same pattern. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation."

The fact that Diosgenin inhibits Runx2 at higher concentrations could be a good thing for height growth as Zfp521 which inhibits Runx2 and augments height growth.  Diosgenin does not inhibit Beta-Catenin and to grow taller you always want Beta-Catenin levels to be higher than your Sox 9 levels.

Diosgenin may inhibit parts of the Nitric Oxide pathway.  There are good and bad parts of the NO pathway(bad being inflammatory cytokines).  The Nitric Oxide pathway includes cGMP which is needed for CNP to function properly.

Nitric oxide inhibitory substances from the rhizomes of Dioscorea membranacea.

"Thai medicinal plants locally known as Hua-Khao-Yen were examined for their inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cell lines. Among the plant species studied, an ethanolic extract of Dioscorea membranacea exhibited the most potent inhibitory activity, with an IC(50) value of 23.6 microg/ml. From this extract, eight compounds [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4-6) and two steroidal saponins (7, 8)] were isolated and further investigated for their inhibitory properties of NO production. It was found that diosgenin-3-O-alpha-L-rhamnosyl (1-->2)-beta-D-glucopyranoside (7) possessed the highest activity (IC(50)=3.5 microM), followed by dioscoreanone (3, IC(50)=9.8 microM) and dioscorealide B (2, IC(50)=24.9 microM). Regarding structural requirements of diosgenin derivatives for NO production inhibitory activity, compound 7 which has a rhamnoglucosyl moiety at C-3 exhibited much higher activity than compounds that have either a diglucosyl substitution (8) or its aglycone (9); whereas, hydroxyl substitution at position 8 of naphthofuranoxepin derivatives conferred higher activity than the methoxyl group. I diosgenin-3-O-alpha-L-rhamnosyl (1-->2)-beta-D-glucopyranoside (7), dioscoreanone (3) and dioscorealide B (2) are active principles for NO inhibitory activity of Dioscorea membranacea. Compounds 1-3 were also tested for the inhibitory effect on LPS-induced TNF-alpha release in RAW 264.7 cells. The result revealed that 3 possessed potent activity against TNF-alpha release with an IC(50) value of 17.6 microM, whereas, 1 and 2 exhibited mild activity."

"Nitric oxide (NO) is one of the inflammatory mediators causing inflammation in many organs. This inorganic free radical has been implicated in physiologic and pathologic processes, such as vasodilation, non-specific host defense and acute or chronic inflammation. NO is produced by the oxidation of l-arginine by NO synthase (NOS). In the family of NOS, inducible NOS (iNOS) is involved in pathological aspects, and can be expressed in response to pro-inflammatory agents such as tumor necrosis factor-α (TNF-α), interleukin 1-β (IL-1β) and lipopolysaccahride (LPS) in various cell types including macrophages. NO acts as a host defense by damaging pathogenic DNA and as a regulatory molecule with homeostatic activities[one of the good attributes of NO]. However, excessive production of this free radical is pathogenic to the host tissue itself, since NO can bind with other superoxide radicals and acts as a reactive radical which directly damages the function of normal cells"<-So you want an equilibrium amount of NO or you want to augment the good features of NO while inhibiting the bad.

Diosgenin seems to uniformly inhibit all parts of the Nitric Oxide Pathway.  However, it is unclear whether it lowers the NO pathway below equilibrium(meaning it decreases levels too much so the positive benefits of NO start to be reduced).  If Diosgenin has another benefit on CNP expression outside of the NO pathway then it would be very good as Diosgenin would boost CNP(the good part of NO) while avoiding inflammatory cytokines(bad part of NO except for IL-6).

According to Effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyrocytes., diosgenin decreases cyclin D1 and causes cell cycle arrest.

Diosgenin may also inhibit Stat3, which is a good thing for height growth, according to Diosgenin, a steroidal saponin, inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells.

Methyl Protodioscin is a part of Dioscorea.  Protodioscin does stimulate the NO pathway and increases testosterone levels.  Dioscin's direct effect on CNP has not been studied.  Diosgenin may help keep the inflammatory inducing parts of Protodioscin in check  The effect of diosgenin has not been studied at all on chondrocytes so there may again be stuff there.

Advances in the pharmacological activities and mechanisms of diosgenin

"Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor (ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases."<-decreasing oxidative stress and preventing inflammatory events may help with height growth too.

Couldn't get this full study.

Diosgenin prevents bone loss on retinoic acid-induced osteoporosis in rats<-I need this full study because it measures diosgenin effects on femur length.  But in the study it said that diosgenin partially reversed some of the retinoic acid reductions in femur length.

According to Effects of diosgenin on the skeletal system in rats with experimental type 1 diabetes, diosgenin reduced some of the negative effects on the growth plate from diabetes.

How LSJL deforms the bone

Measurement of Strain Distributions in Mouse Femora with 3D-Digital Speckle Pattern Interferometry

"During measurements, we applied 5 steps of loading in 10 seconds interval with an increment of 11.8 V to the piezoelectric actuator. This voltage step generated a step force of 0.2 N, and the total force applied to the epiphysis was 1 N"

"strain in the femoral diaphysis is significantly smaller (nearly zero) than that in the distal epiphysis at the loading site"

Monday, July 4, 2011

height by inhibiting asporin?

LSJL upregulates asporin by 3 fold.

Mechanisms for asporin function and regulation in articular cartilage.

"Asporin binds to transforming growth factor-beta (TGF-beta) and inhibits TGF-beta-induced chondrogenesis.  Asporin blocks chondrogenesis and inhibits TGF-beta1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-beta1; in turn, TGF-beta1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-beta1 form a regulatory feedback loop{So asporin upregulation in LSJL is also a sign of TGF-Beta signaling}. Asporin inhibits TGF-beta/Smad signaling upstream of TGF-beta type I receptor activation in vivo by co-localizing with TGF-beta1 on the cell surface and blocking its interaction with the TGF-beta type II receptor."

"Knockdown of asporin significantly increased type II collagen and aggrecan mRNA expression"

"Decorin inhibits TGF-β function"

"Decorin blocks interaction of TGF-β with its cell-surface receptor(s)"

"Decorin inhibits TGF-β-induced expression of plasminogen activator inhibitor-1 via activation of the Ca2+/calmodulin-dependent protein kinase II, which results in phosphorylation of Smad2 at Ser204, an important negative regulatory site."

"Asporin co-localizes with TGF-β in the human articular cartilage extracellular space, inhibiting TGF-β signaling upstream of TβRI"

"Asporin inhibits TGF-β1 binding to TβRII"

"A serum glycoprotein, fetuin, also inhibits TGF-β activity by competing with TGF-β for binding to TβRII"

"[Asporin is a] class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes.  Interleukin-1β and tumor necrosis factor-α downregulated ASPN, whereas transforming growth factor-β1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. The transcription factor Sp1 [regulates] ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity[thus we want to reduce Sp1]. Sp1 mediated [ASPN's] effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene."

"Asporin [is] also called periodontal ligament–associated protein-1 (PLAP-1)"

"GFβ exerts its positive effect on ASPN only when cells are cultured in the absence or with very weak levels of FCS"

"ASPN is the indirect target of Smad3, and hence, its expression could be influenced by GADD45, a factor that upregulates biglycan upon TGFβ treatment and is a target gene of Smad3"

"ASPN mRNA [is present] in the perichondrium/periosteum of long bones{thus periosteal stimulation may affect height growth and ability to form new growth plates with TGF-Beta}, but [is absent] in articular cartilage and growth plates. ASPN protein expression [is predominantly] in the perichondrium/periosteum. TGF-beta1 induced endogenous ASPN mRNA expression over time in vitro, and this induction was suppressed by the TGF-beta type I receptor kinase inhibitor SB431542. Inhibition of Smad3 significantly reduced TGF-beta1-induced ASPN expression, whereas overexpression of Smad3 augmented the induction. Characterization of the human ASPN promoter region revealed a region from -126 to -82 that is sufficient for full promoter activity; however, TGF-beta1 failed to increase activity through the ASPN promoter."

"TGF-β1 induces ASPN expression through ALK5 activity (TGF-β type I receptor)"

According to Systems analysis of matrix metalloproteinase mRNA expression in skeletal tissues., "[elements with a] stimulatory role [are] cis-acting elements such as AP1, NFY, PEA3, and Sp1 [whereas AP2 has an inhibitory role]".  But they are stimulatory on transcription not necessarily on chondrogenesis.

Sunday, July 3, 2011


Here's some details about the Entheses.
Annexin A5 involvement in bone overgrowth at the enthesis.

"Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibiae and femur were increased after 7 weeks of age. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. bone overgrowth at the enthesis requires mechanical forces. We further examined effects of AnxaA5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. AnxaA5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in AnxaA5 KO mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators."<-so maybe if we knockout anxa5 we can induce bone growth at the enthesis.

"Tendons and ligaments are connected to bones through a specialized transitional tissue called the enthesis"

"Anxa5 mediates the Ca2+ influx into matrix vesicles secreted from hypertrophic chondrocytes in the growth plate by interacting with extracellular type II and type X collagens, thereby initiating cartilage calcification"

"the Anxa5–/– mice were viable and fertile, and showed no significant differences in body weight and longitudinal tibial bone length until 40 weeks of age when compared to Anxa5+/+ and Anxa5+/– mice. However, under a binocular microscope, we found that several portions of tendon insertions to the bones were projected abnormally in the Anxa5-/- mice compared to the wild-type mice. For example, the bone ridge at the insertion of the tibialis anterior muscle, and that at the trochanter
tertius of the proximal femur was prominent in the Anxa5-/- mice "

"To assess whether the localized phenotype at the entheses is caused by changes in the
genetically regulated pattern formation of bone outlines or involves mechanical force-driven
local growth of fibrocartilaginous tissues, we used tenotomy to eliminate tibial muscle
contractile force transmission. Microscopic observation revealed that bone ridge overgrowth at the enthesis was suppressed at tenotomized sites to a similar extent in Anxa5–/– and Anxa5+/+ mice "

" AnxaA5KD in chondrocytes resulted in up-regulation of Sox9, whereas it increased Scx and Tnmd
expression in tenocytes , suggesting that AnxaA5KD promotes chondrocyte and tenocyte
differentiation. AnxaA5KD in osteoblasts resulted in decreased Runx2 and osteopontin expression, suggesting that AnxaA5KD inhibits osteoblastic differentiation "