In the article about cholesterol, it was mentioned that the actin cytoskeleton(hence why Lateral Synovial Joint Loading can increase height after puberty) and Rho GTPases regulate chondrogenic differentiation. Ror-Alpha(a member of the Rho GTPase family) is regulated by Hypoxia Inducible Factor-1 alpha. Ror-alpha also modulates cholesterol signaling. Could Rho GTPases like Ror-alpha be another way to induce chondrogenic differentiation of stem cells like by actin cytoskeleton stimulation(by LSJL or LIPUS) or electromagnetic stimulation(PEMF)?
Biology and pathology of Rho GTPase, PI-3 kinase-Akt, and MAP kinase signaling pathways in chondrocytes.
"The processes of growth, differentiation, cell death and matrix remodeling are regulated by a network of cell signaling pathways in response to a variety of extracellular stimuli. These stimuli consist of soluble ligands, including growth factors and cytokines, extracellular matrix proteins, and mechanical factors that act in concert to regulate chondrocyte function through a variety of canonical and non-canonical signaling pathways. Key chondrocyte signaling pathways include, but are not limited to, the p38, JNK and ERK MAP kinases, the PI-3 kinase-Akt pathway[Good for height growth by promoting chondrocyte survival], the Jak-STAT pathway, Rho GTPases[RhoA seems to be for height growth] and Wnt-beta-catenin and Smad pathways. Modulation of the activity of any of these pathways has been associated with various pathological states in cartilage. This review focuses on the Rho GTPases, the PI-3 kinase-Akt pathway, and some selected aspects of MAP kinase signaling."
"Cycling between the two forms is regulated by dozens of other proteins, in particular guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs activate Rho GTPases by replacing GDP with GTP while GAPs increase the intrinsic GTPase activity of Rho proteins and thereby accelerate their inactivation. Activity of GEFs and GAPs in turn is controlled by many cell surface receptors, including tyrosine and serine/threonine kinase receptors, G protein-coupled receptors and integrins. Rho GTPases are of particular interest in chondrocytes because of their role in connecting signals from the extracellular matrix to the actin cytoskeleton and cellular morphology, which in turn appear to control cellular activities such as cell cycle progression, gene expression[thus Rho GTP-ases have height increase applications] and apoptosis"
"RhoA promotes fibroblastoid cell shape and the formation of actin stress fibers transversing the cell. Normally chondrocytes are rounded or polygonal and display characteristic cortical actin organization while the formation of actin stress fibers, at least in vitro, has been associated with de-differentiation of chondrocytes to a fibroblast-like phenotype[It would seem that RhoA reduces height by encouraging a non-chondrogenic shape and forming actin stress fibers that reduce mechanical load]]. Therefore, it was not unexpected to find that overexpression of RhoA inhibits both early chondrogenesis and hypertrophic chondrocyte differentiation, while inhibition of RhoA, or the immediate downstream kinases ROCK1/2, promotes chondrocyte maturation[Or does RhoA allow for more stem cell proliferation and chondrocyte proliferation before terminal differentiation]"
"insulin-like growth factor 1 was reported to repress RhoA activity"<-possible way that IGF-1 can enhance height growth
Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N-cadherin.
"hondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor beta (TGFbeta). Interestingly, TGFbeta can also induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGF beta 3 upregulated SMC genes, whereas cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA, but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGF beta 3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis."
So Rac1 inactivation helps encourage chondrogenesis but you don't want to inhibit Rac1 in the entire body as that would inhibit the body from producing smooth muscle cells.
The transcriptional activity of Sox9 in chondrocytes is regulated by RhoA signaling and actin polymerization.
"In this study, we demonstrate that dedifferentiation of round primary chondrocytes into a fibroblast morphology correlates with a profound induction of RhoA protein and stress fibers. Culture of dedifferentiated chondrocytes in alginate gel induces a precipitous loss of RhoA protein and a loss of stress fibers concomitant with the reexpression of the chondrocyte differentiation program. We have found that chondrogenesis in limb bud micromass cultures similarly entails a loss of RhoA protein and that expression of dominant negative RhoA in such cultures can markedly enhance chondrogenesis. Consistent with these results, expression of the Rho antagonist C3 transferase can restore chondrocyte gene expression in dedifferentiated chondrocytes grown on plastic. Transfection of cells with agents that block actin polymerization enhance the ability of either exogenous Sox9 or a Gal4 DBD-Sox9 fusion protein to activate gene expression. Interestingly, the enhancement of Sox9 function by actin depolymerization requires both protein kinase A (PKA) activity and a PKA phosphorylation site in Sox9 (S181) that is known to enhance Sox9 transcriptional activity. Lastly, we demonstrate that RhoA-mediated modulation of actin polymerization regulates the ability of Sox9 to both activate chondrocyte-specific markers and maintain its own expression in chondrocytes via a positive feedback loop."
"It has been appreciated that environmental factors that alter either the cell shape or the actin cytoskeleton can modulate the ability of prechondrogenic cells to undergo chondrogenesis"
"Polymerization of actin to generate stress fibers is regulated by the GTPase RhoA. When bound to GTP, RhoA interacts with and induces the activity of mDia, a member of the formin family, which directly binds to actin and stimulates its polymerization. In addition, GTP-bound RhoA activates RhoA kinase (ROCK), which consequently activates LIM kinase, which phosphorylates the actin depolymerizing protein cofilin and thereby stabilizes actin filaments within the cell[RhoA's function is to stabilize the actin filaments and LSJL destabilizes the actin filaments and since RhoA is bad for chondrogenesis...]. Studies from the Beier lab have established that overexpression of RhoA can block both Sox9 expression and chondrogenic differentiation of the ATDC5 chondrocyte cell line. In addition, this group has demonstrated that inhibition of ROCK signaling or pharmacological disruption of the actin cytoskeleton can promote both Sox9 expression and chondrogenic differentiation in some but not all cellular contexts"
Ror-Alpha works by stabilizing the actin cytoskeleton. LSJL(and LIPUS) work by destablizing the actin cytoskeleton by inducing fluid flow. Ror-alpha inhibits chondrogenesis by phosphorylating the actin cytoskeleton. Therefore, since LSJL can disrupt the actin cytoskeleton it can promote Sox9 expression and chondrogenic differentiation of stem cells. Chondrocytes can cause height growth by chondrocyte hypertrophy.
Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes.
"During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation[Rac1 activation you remember inhibits chondrogenesis], whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development."
Maybe there could be a way to partially inhibit Rac-1 which would increase height by inhibiting alkaline phosphatase activity. If Rac-1 was only partially inhibited then it would still be possible for stem cells to differentiate into smooth muscle cells as needed. But here's a study that says that Rac1 activation helps chondrogenesis...
Rac1 signaling stimulates N-cadherin expression, mesenchymal condensation, and chondrogenesis.
"We have recently shown that the small GTPase RhoA inhibits [the] process[of chondrogenesis]. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms."
So Rac1 is good at the beginning of development as it encourages chondrogenic differentiation of stem cells but bad at the end of development as it then encourages osteogenic differentiation of those hypertrophic chondrocytes. Therefore it's not worth it to mess around with Rac1. The smooth muscle study even contradicted this study by saying that Rac1 activation inhibits chondrogenesis.
"The formation of cartilage templates in endochondral ossification begins with condensation of mesenchymal cells, increased expression of the cell adhesion molecules N-cadherin and N-CAM, and therefore increased cell-cell interactions"<-hydrostatic pressure may also help condense mesenchymal cells
"Forcing spherical cell shape by pharmacological inhibition of actin polymerization is sufficient to stimulate dedifferentiated cells to re-express chondrogenic matrix molecules and promotes mesenchymal cells to commit to the chondrogenic lineage"<-inhibiting actin polymerization may help to grow taller
"We demonstrate in the present study that Rac1 and Cdc42 signaling pathways are positive regulators of chondrogenesis."<-both of those pathways could possibly be used to help you grow taller
Ror-alpha seems to be worth investigating. A Ror-alpha inhibitor could prevent actin stabilization which prevents stem cells from differentiating into chondrocytes. This same effect of course could be induced by performing LSJL.
Here's a study involving Ror-Alpha featuring two LSJL scientists: K Hamumura and H Yokota.
RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts.
"Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor[this would seem to relate to the osteogenic components of LSJL]. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 h flow treatment at 10 dyn/cm(2), we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress[since LSJL induces flow-induced shear stress RhoA is involved in LSJL]. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer (FRET) between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated PI3K and MAPK signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling as well as phosphorylation of p38 and ERK1/2 in MAPK signaling was induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2 and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase 2. Collectively, the result demonstrates that in response to 1 h flow treatment to MC3T3-E1 cells at 10 dyn/cm(2), RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway."
MAPK is mostly involved in osteogenic function so we don't care about that in terms of height growth. But the RhoA activation of PI3K might have height growth implications. Unfortunately, this study isn't out yet so I can't find any more LSJL tidbits.
Here's a study about how inhibition of RhoA can induce chondrogenesis.
Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells.
"Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. To further investigate the role of RhoA and ROCK in chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis."
And perhaps LSJL via fluid flow or hydrostatic pressure can help dissolute those actin stress fibers.
"While inhibition of RhoA by CT04, inhibition of MLCK by ML-7, and inhibition of myosin II by blebbistatin induced disappearance of actin stress fibers and chondrogenesis of mesenchymal cells, cell treated with these inhibitors exhibited different cell shapes from each other indicating that regardless of underlying mechanism, reorganization of actin stress fibers is important for chondrogenesis induction."
RhoA/Rho kinase signaling regulates transforming growth factor-β1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway.
"The aim of this study was to evaluate the activation of the RhoA/Rho kinase (ROCK) pathway, as well as the manner by which it may contribute to chondrogenesis and the actin cytoskeletal organization of rat temporomandibular SMSCs in response to transforming growth factor-β1 (TGF-β1). Primary isolated SMSCs[synovium derived MSCS] were treated with TGF-β1, and their actin organization was examined by fluorescein isothiocyanate-phalloidin staining. The specific biochemical inhibitors, C3 transferase, Y27632 and SB431542, were employed to evaluate the function of RhoA/ROCK and Smads. The effect of C3 transferase and Y27632 on the gene expression of chondrocyte-specific markers was evaluated by quantitative real-time polymerase chain reaction. The stimulation of TGF-β1 in SMSCs resulted in the activation of the RhoA/ROCK pathway and concomitantly induced cytoskeletal reorganization, which was specifically blocked by C3 transferase and Y27632. The TGF-β-induced gene expression of Sox9, type I collagen, type II collagen and aggrecan was also inhibited by both C3 transferase and Y27632, at different levels. Y27632 treatment reduced the phosphorylation of Smad2/3 in a concentration-dependent manner."
LSJL didn't effect Smad2/3 gene expression but it could have affected phosphorylation.
"RhoA activity negatively affects chondrogenesis in the ATDC5 chondrogenic cell line and mesenchymal cells"
"TGF-β1 addition caused a rapid increase in RhoA activation after 1 day of stimulation, reaching its peak on day 4, but gradually decreased as the time proceeded"<-No change in RhoA was observed in LSJL despite being 49 hours after initial stimulus.
"The treatment of SMSCs with TGF-β1 resulted in an upregulation of the ROCK1 and ROCK2 genes. The expression of these genes was low on day 1, but increased as chondrogenesis proceeded, peaked after 5 days, and was sustained at relatively high levels until day 7."
According to this study TGF-Beta1 had no effect on Smad2/3 levels only Smad2/3 phosphorylation.
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