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)
Col2a1(up in LSJL)
Fxyd2(up in LSJL)

Upregulated genes:

"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:

Genes downregulated:

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"

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