Get a Taller Stature with TGF-Beta1

Getting a taller stature involves stem cells differentiating into chondrocytes.  Two ways to achieve that are:  Hydrostatic Pressure(LSJL) and LIPUS(Ultrasound)+TGF-Beta1.  LIPUS however does increase TGF-Beta synthesis.  Although one study of LIPUS, studied the effects of LIPUS on the Mandibular Condyle.  It reported that LIPUS on it's own(without TGF-Beta1) did not significantly affect cartilaginous layer thickness.  However, specific parts of that layer did change in thickness relative to before but the overall layer did not increase.  So it's unclear whether the increase in TGF-Beta1 induced by LIPUS is sufficient to induce mesenchymal stem cell differentiation.  You also need available mesenchymal stem cells that have sufficiently long telomeres also.

How does TGF-Beta work in endochondral ossification?  What are some ways we can manipulate TGF-Beta in order to get a taller stature? 

Smad signaling determines chondrogenic differentiation of bone-marrow derived mesenchymal stem cells: Inhibition of Smad 1/5/8P prevents terminal differentiation and calcification. 

"The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in TGF-β induced chondrogenic differentiation of bone-marrow derived mesenchymal stem cells (BMSCs) in order to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs[i.e. can you grow taller(preventing terminal differentiation) by manipulating Smad signaling pathways(which affect TGF-Beta)]. Terminally differentiated chondrocytes produced in-vitro by chondrogenic differentiation of BMSCs or studied ex-vivo during murine embryonic limb formation, stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex-vivo articular cartilage samples that lacked expression for MMP13 and collagen X only expressed Smad2/3P[So if there's only Smad2/3P there's no expression of MMP13 and Collagen X which are involved in terminal differentiation, this perhaps inhibiting Smad1/5/8Phosphorylation may help you grow taller]. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis[however both pathways are needed in early chondrogenesis, so for LSJL for example you would need both pathways but for a case like an active growth plate you would only want Smad2/3P]. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of MMP13, collagen X and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization." 

Many of the Smad pathways are involved in the phosphorylation of TGF-Beta.  The Smad2/3P is critical for proper bone deposition whereas Smad 1/5/8P is not essential and result in terminal differentiation.   Therefore, ways of inhibiting Smad 1/5/8 phosphorylation may be a mechanism of increasing height.   So you want to allow for Smad2/3P but not Smad 1/5/8P.  You want Smad 1/5/8 just not for it to be phosphorylated(deactivated).   However, with something like LSJL which involves early chondrogenesis you would want Smad 1/5/8P.

"This terminal differentiation of BMSCs may be advantageous for tissue engineering of bone through the endochondral route"<-And terminal differentiation may be needed for endochondral ossification.  But prolonging Smad 1/5/8P may help you grow taller longer.

"TGF-β signaling requires binding to complexes of type II and type I serine/threonine kinase receptors, followed by receptor-Smad phosphorylation at their C-terminus. The canonical TGF-β pathway is the Smad2/3 pathway. However, by use of an alternate type I receptor (ALK1 instead of ALK5) Smad1/5/8 is phosphorylated instead of Smad2/3. The Smad1/5/8 route is commonly known as the route activated by bone morphogenetic proteins (BMPs), also a member of the TGF-β superfamily and a very potent inducer of bone formation"  TGF-Beta normally is involved with Smad 2/3P whereas BMP-2 is involved with Smad1/5/8P.

"Although chondrogenesis and cartilage formation are achieved, it eventually leads to terminal differentiation of chondrocytes instead of the production of stable hyaline cartilage."<-So scientists have a way for endochondral ossification(which causes height growth) just not stable hyaline cartilage for the joint.

"both TGF-β and BMP are important for early chondrogenesis"<-TGF-Beta is upregulated by LSJL whereas there aren't definitive signs that BMP-2 is.  This could be a problem with LSJL effectivity.  Ways of upregulating BMP-2 may be needed to enhance LSJL effectiveness.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.

"TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors.
Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples.
Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation[Remember Smad1/5 phosphorylation is bad for height growth, and Smad2 phosphorylation is necessary for height growth therefore Endoglin is bad for height growth], Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage.
Endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes." 

Endoglin is a receptor for TGF-Beta. Finding a way to inhibit Endoglin may be a way to make people taller.

"Accompanying [the] morphological changes [to a more fibroblastic morphology] is the loss of the chondrogenic (differentiated) phenotype or ‘chondrocyte dedifferentiation’ which is characterized by a decrease in type II collagen expression"<-So type II collagen expression is key to maintaining chondrogenesis.  Col2A1 can induce CNP as well.

E-selectin ligand-1 regulates growth plate homeostasis in mice by inhibiting the intracellular processing and secretion of mature TGF-beta. 

"TGF-beta signaling is a critical determinant of growth plate homeostasis.  TGF-beta is synthesized as an inactive precursor that is cleaved to become mature in the Golgi apparatus.  Here, we report that a cysteine-rich protein, E-selectin ligand-1 (ESL-1), acts as a negative regulator of TGF-beta production by binding TGF-beta precursors in the Golgi apparatus in a cell-autonomous fashion, inhibiting their maturation. Furthermore, ESL-1 inhibited the processing of proTGF-beta by a furin-like protease, leading to reduced secretion of mature TGF-beta by primary mouse chondrocytes and HEK293 cells. In vivo loss of Esl1 in mice led to increased TGF-beta/SMAD signaling in the growth plate that was associated with reduced chondrocyte proliferation and delayed terminal differentiation."

Surprising, ESL knockout mice had shorter growth plates and height growth.  So ESL-1 is not something that can be specifically targeted for inhibition as TGF-Beta serves different functions at different stages of differentiation and ESL-1 function is needed to inhibit TGF-Beta signaling at certain growth plate stages.

"ADAMTSL2 mutations in geleophysic dysplasia patients have recently been reported to cause elevated TGF-β secretion and activity, leading to disproportionate short stature and brachydactyly in humans. In contrast, fibrillin1 mutations in Marfan syndrome exhibit increased TGF-β activity but result in tall stature. In earlier skeletal developmental stages, Esl1 is highly expressed in the perichondrium but at low levels in the cartilage. Similarly, fibrillin-1 and ADMTSL2 also exhibit strong expression in the perichondrium, where TGF-βs is abundantly synthesized, suggesting that the perichondrium is particularly important for production and regulation of TGF-β activity and regulation of the growth plate"-The perichondrium is a developing periosteum meaning that shear strain on the periosteum whether through LIPUS or LSJL may increase TGF-Beta activity. 

A role for age-related changes in TGFbeta signaling in aberrant chondrocyte differentiation and osteoarthritis. 

"We postulate that the dual effects of TGFbeta on chondrocytes can be explained by the fact that TGFbeta can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFbeta not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFbeta on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFbeta signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFbeta signaling in OA development." 

Smad 2/3 phosphorylation is good whereas Smad 1/5/8P is bad for height growth.  ALK1 stimulates MMP-13 chondrogenic expression.  Remember, F-spondin reduced height by as much as 30% and F-spondin increased levels of MMP-13. 

Inhibiting MMP-13, Smad 1/5/8P is a way to get a taller stature. Stimulating Smad's 2/3P is also a way to get a taller stature.

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

"Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation[prechondrogenic condensation may be dependent on ATP oscillations]. TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels."

"BMP signaling is more effective in condensed cells and has little effect on mesenchymal cells at low density"<-So BMP would likely have no effect on adult epiphyseal MSC's.

"TGF-β signaling drives Ca2+ oscillations by stimulating extracellular ATP signaling and modulating Ca2+ influx via VDCC and then TGF-β-driven Ca2+ oscillations subsequently generate ATP oscillations."<-This we can stimulate extracellular ATP signaling by other means to drive Ca2+ oscillations.

TGF-Beta may be able to induce significant height on it's own as shown by this mutation the resulted in a seven foot tall person.

Camurati-Engelmann disease: unique variant featuring a novel mutation in TGFβ1 encoding transforming growth factor beta 1 and a missense change in TNFSF11 encoding RANK ligand.

"We report a 32-year-old man and his 59-year-old mother with a unique and extensive variant of Camurati-Engelmann disease (CED) featuring histopathological changes of osteomalacia and alterations within TGFβ1 and TNFSF11 encoding TGFβ1 and RANKL, respectively. He suffered leg pain and weakness since childhood and reportedly grew until his late 20s, reaching 7 feet in height. He had deafness, perforated nasal septum, torus palatinus, disproportionately long limbs with knock-knees, low muscle mass, and pseudoclubbing. Radiographs revealed generalized skeletal abnormalities, including wide bones and cortical and trabecular bone thickening in keeping with CED, except that long bone ends were also affected. Lumbar spine and hip BMD Z-scores were + 7.7 and + 4.4, respectively. Biochemical markers of bone turnover were elevated. Hypocalciuria accompanied low serum 25-hydroxyvitamin D (25[OH]D) levels. Pituitary hypogonadism and low serum insulin-like growth factor (IGF)-1 were present. Karyotype was normal. Despite vitamin D repletion, iliac crest histology revealed severe osteomalacia. Exon 1 of TNFRSF11A (RANK), exons 2, 3, and 4 of LRP5, and all coding exons and adjacent mRNA splice junctions of TNFRSF11B (OPG), SQSTM1 (sequestosome 1), and TNSALP (tissue nonspecific alkaline phosphatase) were intact. His asymptomatic and less dysmorphic 5'11″ mother, also with low serum 25(OH)D, had milder clinical, radiological, biochemical, and histopathological findings. Both individuals were heterozygous for a novel 12-bp duplication (c.27_38dup, p.L10_L13dup) in exon 1 of TGFβ1, predicting four additional leucine residues in the latency-associated-peptide segment of TGFβ1, consistent with CED. The son was also homozygous for a single base transversion in TNFSF11, predicting a nonconservative amino acid change (c.107C > G, p.Pro36Arg) in the intracellular domain of RANKL that was heterozygous in his nonconsanguineous parents. This TNFSF11 variant was not found in the SNP Database, nor in published TNFSF11 association studies, but it occurred in four of the 134 TNFSF11 alleles (3.0%) we tested randomly among individuals without CED. Perhaps the unique phenotype of this CED family is conditioned by altered RANKL activity."

"[The] mutations in TGFβ1 seem to enhance TGFβ1 anabolic effects within bone by freeing TGFβ1 from the latency-associated-peptide"<-maybe we can do the same thing?

"His feet were remarkably small for his extreme height (shoe size 11)"

"His subnormal serum creatinine of 0.5 mg/dL (0.7–1.5 Nl) perhaps reflected low muscle mass. Plasma protein was slightly elevated at 8.7 g/dL (6.5–8.5 Nl). Serum total bilirubin was low at 0.2 mg/dL (0.3–1.1 Nl). Serum testosterone was deficient at 152 ng/dL (241–827 Nl) while follicle stimulating hormone was 1.6 IU/L (1.4–18 Nl for men), prolactin 5.2 ng/mL (2.1–18.0 Nl), and 17β-estradiol < 40 pg/mL (< 52 Nl for men). Serum random growth hormone was elevated at 2.17 ng/mL (0.01–0.97 Nl), but IGF-1 was low at 69 ng/mL (115–307 Nl), suggesting undernutrition. However, serum ferritin was 66 ng/mL (22–322 Nl)."

"Markers of bone turnover, including serum ALP, indicated rapid skeletal remodeling"

"A novel heterozygous TGFβ1 duplication in keeping with CED was identified in both individuals, but the son is also homozygous and his mother heterozygous for a missense change in TNFSF11 encoding RANKL."<-So maybe you need both mutations in RANKL and TGF-Beta to get the height gain.

"The pro-TGFβ1 precursor molecule dimerizes and undergoes proteolytic cleavage to separate the LAP and TGFβ1 moieties. However, the LAP(Latency-associated peptide) remains associated in the bone matrix with mature TGFβ1 until subjected to specific activation conditions. Release of LAP activates circulating TGFβ1 and presumably also TGFβ1 within bone."

"All 10 TGFβ1 mutations causing CED involve LAP; none are within TGFβ1 itself"<-So maybe you need to cause the release of LAP to get TGF-Beta1 to increase height.

The influence of delayed compressive stress on TGF-β1-induced chondrogenic differentiation of rat BMSCs through Smad-dependent and Smad-independent pathways.

"Col2α1 mRNA was increased by delayed dynamic compressive stress initiated at the 8th day of chondrogenic culture. The current work is to further study the possibility of using delayed mechanical stress to relay chondrogenesis initiated by exogenous TGF-β1. Mechanical stimulation was delivered from day 8 to day 14 of chondrogenic culture. It showed that delayed compressive stress not only stimulated gene expression and protein synthesis of chondrocyte-specific markers, but also stimulated the endogenous TGF-β1 gene transcription, protein expression and the subsequent activation even when exogenous TGF-β1 was discontinued. Furthermore, mechanical stress also promoted protein phosphorylation and nuclear translocation of Smad2/3, the TGF-β1 downstream effectors. Inhibition TGF-β with SB431542 significantly affected the stress-induced chondrogenic gene expression. In addition, phosphorylated-p38 {phosphorylated p38 is pro-chondrogenic} and RhoB{Rhobtb1 was downregulated by LSJL} were upregulated by delayed loading in a TGF-β-related manner. Phosphorylated-ERK1/2{p-ERK1 is anti chondrogenic} and Wnt7a were also increased, but in a TGF-β-independent way."

"TGF-β activates ERK, p38, RhoB, Wnt and PKC"

"p38 MAPK was activated by cyclic compressive force in a rapid and transient manner to mediate subsequent transcriptional regulation in chondrogenic differentiation of rat BMSCs"

"ompressive stress promoted gene expression of chondrogenic markers even in the absence of exogenous TGF-β1, and the stress-induced gene expression was affected by the TβRI{downregulated by LSJL} inhibitor, SB431542, in different degrees. The stress-induced mRNA level of Col2α1, Sox9 and Runx2 was respectively repressed to 72.4%, 61.6%, and 74.1% after being treated with SB431542 for 3 days. In contrast, the stress-induced gene expression of Aggrecan and Ihh was unaffected by SB431542"

" Inactivation of TGF-β1 by the specific inhibitor SB431542 abrogated the up-regulation of p-p38 and RhoB by dynamic stress at varying degrees. Mechanical stress also increased the levels of p-ERK1/2 and Wnt7a to certain extents, whereas p-ERK1/2 and Wnt7a were unaffected by SB431542 treatment"<-although downregulation of TGFBetaR1 could be part of the negative feedback mechanism to load and expression could be restored over time.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

"Supplementation with TGF-β1 could initiate and promote chondrogenesis of synovium-derived stem cell (SDSCs), but TGF-β1 alone was insufficient to fully differentiate SDSCs into chondrocytes. However, HDAC4 overexpression can promote TGF-β1-induced SDSC chondrogenesis but inhibit chondrogenically differentiated stem cell hypertrophy"

"C-type natriuretic peptide/NPR-B signaling pathway was activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may be involved in glycosaminoglycan synthesis during this process in a dose-dependent manner"

" The chondrogenesis of trabecular bone-derived MPCs was initiated and maintained by TGF-β1 through the differential chondro-stimulatory activities of p38, ERK-1, and JNK. TGF-β1-mediated MAPK activation also controlled wnt-7a gene expression and WNT-mediated signaling through the intracellular β-catenin-TCF pathway, which probably regulated the expression of cell adhesion protein, N-cadherin"

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells.

"This study investigated the role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10(-8) and 10(-7) M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10(-6) M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect."

So this study confirms that not only is TGF-Beta capable of inducing chondrogenic differentiation of epiphyseal bone marrow MSCs but that TGF-Beta stimulates CNP.

"CNP increases the number of chondrogenic condensations, induces the expression of N-cadherin, stimulates GAG synthesis, but does not alter the expression of the chondrogenic transcription factors Sox9, -5, and -6, or of the main ECM genes encoding collagen type II and aggrecan of mouse embryonic limb bud cells in micromass culture. "

Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway.

"This study investigated the effect of mechanical load on human mesenchymal stem cell (hMSC) differentiation under different exogenous transforming growth factor-beta1 (TGF-beta(1)) concentrations (0, 1 or 10 ng/ml).  Human MSCs were seeded into fibrin-biodegradable polyurethane scaffolds at a cell density of 5 x 10(6) cells per scaffold and stimulated using our bioreactor. One hour of surface motion superimposed on cyclic compression was applied once a day over seven consecutive days. Scaffolds were analysed for gene expression, DNA content and glycosaminoglycan amount. Addition of TGF-beta(1) in the culture medium was sufficient to induce chondrogenesis of hMSCs. Depending on the TGF-beta(1) concentration of the culture medium, mechanical load stimulated chondrogenesis of hMSCs compared to the unloaded scaffolds, with a much stronger effect on gene expression at lower TGF-beta(1) concentrations. With TGF-beta(1) absent in the culture medium, mechanical load stimulated gene transcripts and protein synthesis of TGF-beta(1) and TGF-beta(3). TGF-beta type I receptor inhibitor LY364947 blocked the up-regulation on TGF-beta(1) and TGF-beta(3) production stimulated by mechanical load, and also blocked the chondrogenesis of hMSCs. Mechanical load promotes chondrogenesis of hMSCs through TGF-beta pathway by up-regulating TGF-beta gene expression and protein synthesis."

LSJL stimulates the TGF-Beta pathway.

"Dimeric ligands of the TGF-β superfamily signal across cell membranes by assembling heterotetrameric complexes of structurally related serine/threonine–kinase receptor pairs, designated types I and II. TGF-β complexes assemble cooperatively through recruitment of the low-affinity (type I) receptor by the ligand-bound high-affinity (type II) pair. The type II receptor phosphorylates the type I receptor, which in turn activates type I receptor kinase activity"

"The loaded group was exposed to ball oscillation of ±25° at 1 Hz. Simultaneously, dynamic compression was applied at 1 Hz with 10% sinusoidal strain, superimposed on a 10% static offset strain, resulting in an actual strain amplitude of 10–20%. Mechanical loading was performed 1 hr a day over 7 consecutive days. The group of unloaded constructs served as controls. After 7 days in pre-culture and 7 days of loading the top 10% of the construct was used for gene expression analysis."

TGFB1 increased TGFB3, Sp7, Col1, Aggrecan, and Col2.  It decreased PRG4 expression.  Mechanical loading also increased TGFB3.

"The standard chondrogenic TGF-β1 level (10 ng/ml) [used to induce chondrogenesis] is non-physiological and artificially high. In this situation the effects of mechanical load on the chondrogenesis of hMSCs are masked, as seen by very small increase in the type II collagen (1.2-fold) and aggrecan (1.5-fold) gene level between control and loaded samples in our study"

"Dexamethasone is necessary in the process of chondrogenic differentiation stimulated by mechanical load. We performed an experiment with neither dexamethasone nor TGF-β1 in the culture medium. Mechanical load did not affect the gene expression profile except for 7.45-fold increase of PRG4 mRNA expression"

Shearing of synovial fluid activates latent TGF-β.

"TGF-β is synthesized in an inactive latent complex that is unable to bind to membrane receptors, thus unable to induce a cellular biological response until it has been activated. In addition to activation by chemical mediators, mechanical forces may activate latent TGF-β via integrin-mediated cellular contractions, or mechanical shearing of blood serum. Since TGF-β is present in synovial fluid in latent form, and since normal diarthrodial joint function produces fluid shear, this study tested the hypothesis that the native latent TGF-β1 of synovial fluid can be activated by shearing.

Synovial fluid from 26 bovine joints and three adult human joints was sheared at mean shear rates up to 4000 s(-1) for up to 15 h.

Unsheared synovial fluid was found to contain high levels of latent TGF-β1 (4.35 ± 2.02 ng/mL bovine, 1.84 ± 0.89 ng/mL human; mean ± radius of 95% confidence interval) and low amounts (<0.05 ng/mL) of the active peptide. Synovial fluid concentrations of active TGF-β1 increased monotonically with shear rate and shearing duration, reaching levels of 2.64 ± 1.22 ng/mL for bovine and 0.60 ± 0.39 ng/mL for human synovial fluid. Following termination of shearing, there was no statistical change in these active levels over the next 8 h for either species, demonstrating long-term stability of the activated peptide. The unsheared control group continued to exhibit negligible levels of active TGF-β1 at all times."

Will shear have the same impact in the bone marrow?

"In [the] latent complex, mature TGF-β 25 kDa peptide is linked non-covalently to a 70 kDa latency associated peptide (LAP), together forming the small latent complex (SLC). This complex may be disulfide-bonded to a latent TGF-β binding protein (LTBP, ∼180 kDa), constituting a configuration termed the large latent complex (LLC). In growth plate cartilage, both SLC and LLC are secreted by chondrocytes; the LLC can bind to the ECM via the LTBP "<-So shear strain can definitely activate the latent TGF-Beta in growth plate chondrocytes.

"matrix-bound latent TGF-β can be activated by stromelysin-1 (MMP-3){upregulated in LSJL} and collagenase-3 (MMP-13) released from cellular matrix vesicles during endochondral ossification"

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture.

"we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture"

"In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed."  TGFBeta3 was upregulated during dedifferentitation

"The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation [and were inactivated during chondrogenic differentiation], whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation."

"TGFb3-production [during dedifferentiation] might be a signal to stimulate other cells or themselves in an autocrine way to redifferentiate."

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation.

"we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation."

"when plated in micromass cultures under either TGF-β1 or BMP-2 stimulation, C3H10T1/2 cells condense and differentiate along the chondrocytic lineage"

"The addition of TGF-β1 and Col2α1 increased Sox9 expression in mesenchymal progenitor cells, while treatment with TGF-β1 and type I collagen (Col1α2) reduced Sox9 and Agc expression "

"CTGF stimulation alone or in conjunction with TGF-β1 cause a significant increase in Col1α2 expression both in vivo "

From tall to short: the role of TGFβ signaling in growth and its disorders.

"The acromelic dysplasia group is characterized by short stature, short hands and feet, stiff joint, and "muscular" build. Four disorders can now be ascribed to this group, namely Weill-Marchesani syndrome (WMS), geleophysic dysplasia (GD), acromicric dysplasia (AD), and Myhre syndrome (MS). Although closely similar, they can be distinguished by subtle clinical features and their pattern inheritance. WMS is characterized by the presence of dislocation of microspherophakia and has autosomal dominant or recessive mode of inheritance. GD is the more severe one, with a progressive cardiac valvular thickening, tracheal stenosis, bronchopulmonary insufficiency, often leading to an early death. AD has an autosomal dominant mode of inheritance, distinct facial and skeleton features (a hoarse voice and internal notch of the femoral head). Finally, MS is sporadic, characterized by prognathism, deafness, developmental delay, thickened calvarium, and large vertebrae with short and large pedicles. We first identified mutations in Fibrillin-1 (FBN1) in the dominant form of WMS and then mutations in A Disintegrin-like And Metalloproteinase domain with ThromboSpondin type 1 repeats 10 (ADAMTS10) in the recessive form of WMS.  ADAMTS10 [interacts with] FBN1. We then identified mutations in ADAMTSL2 in the recessive form of GD and a hotspot of mutations in FBN1 in the dominant form of GD and in AD (exon 41-42, encoding TGFβ binding protein-like domain 5 (TB5) of FBN1). Using a yeast double hybrid screen, we identified latent transforming growth factor-β (TGFβ) binding protein 1 as a partner of ADAMTSL2. We found an increased level of active TGFβ in the fibroblast medium from patients with FBN1 or ADAMTSL2 mutations and an enhanced phosphorylated SMAD2 level, allowing us to conclude at an enhanced TGFβ signaling in GD and AD. Finally, a direct interaction between ADAMTSL2 and FBN1 was demonstrated suggesting a dysregulation of FBN1/ADAMTSL2 interrelationship as the underlying mechanism of the short stature phenotypes. Using exome sequencing in MS probands, we identified de novo SMAD4 missense mutations, all involving isoleucine residue at position 500, in the MH2 domain. In MS fibroblasts, we found decreased ubiquitination level of SMAD4 and increased level of SMAD4 supporting a stabilization of SMAD4 protein. Functional SMAD4 is required for canonical signal transduction through the oligomerization with phosphorylated SMAD2/3 and SMAD1/5/8. We therefore studied the nuclear localization of mutant SMAD complexes and found that the complexes translocate to the nucleus. We finally observed a decreased expression of downstream TGFβ target genes supporting impaired TGFβ driven transcriptional control in MS. Short stature phenotypes [involve] TGFβ signaling. TGFβ signaling [is enhanced] in Marfan phenotypes[specifically via FBN1]."