Thursday, August 9, 2012

Why doesn't LSJL upregulate Smad3?

TGF-Beta induces chondrogenesis via Smad3 but Smad2 or 3 were not altered above threshold by LSJL. Note that just because LSJL does not upregulate Smad3 does not mean it does not phosphorylate Smad3. And Smad 3 was shown as not having a fold change so lack of observation of Smad3 was not likely to be possible.  It should be noted though that bone samples were harvested and not cells from the bone marrow specifically although there were likely some bone marrow cells contained within.

Upregualtion of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signaling through activating of Smurf1/Smad6 complex.

"TGF-β1-induced ROCK elevation suppressed the antagonizing effects evoked by BMP-2[LSJL BMP-2 upregulation is predicted] and strengthened fibrotic response. Exogenous BMP-2 supply could attenuate TGF-β1 signaling through Smad6-Smurf-1 complex activation[LSJL alteration of Smurf1 is predicted]. In vitro, mechanical stretch upregulated cardiac TGF-β1 and TGF-β1-dependent ROCK. While BMP-2 level was upregulated after blocking TGF-β1 signaling by SB-431542 or inhibition of ROCK by Y-27632. Dose-dependent TGF-β1 induction could activate ROCK and suppress endogenous BMP-2 level in cardiomyocytes. Knock-down BMP-2 in cardiomyocytes enhanced TGF-β1-mediated PKC-δand Smad3 signaling cascades. In contrast, application of Y-27632 or SB-431542 suppressed TGF-β1 pathway, but BMP-2 was only upregulated by Y-27632. BMP-2 silencing abolished Y-27632 but not SB-431542 mediated suppression of TGF-β1 pathway. Further experiments showed that the inhibitory Smad6 and Smurf1 were required for BMP-2-evoked antagonizing effects. Smad6 overexpression attenuated TGF-β1-induced activation of PKC-δand Smad3, promoted TGF-β RI degradation in BMP-2 knock-down cardiomyocytes Smad6 or Smurf1 siRNA could abolish the antagonizing effect of BMP-2 on TGF-β1 pathway, in which Smad6/Smurf1 complex formation was critically involved. In vivo data showed that pressure overload-induced collagen deposition were attenuated and TGF-β1-dependent activation of PKC-δand Smad3 were reduced after 2 weeks treatment with rhBMP-2(0.5mg/kg) or Y-27632 (10mg/kg) in mice underwent surgical transverse aortic constriction."

"Overexpression BMP-2 in renal interstitial fibroblast cells effectively antagonized TGF-β1 mediated fibrosis by enhancing the catabolism of type I TGF-β receptors (TGF-β RI)."

"pressure overload activated Rho related kinase (ROCK) and suppressed the endogenous BMP-2 expression"<-So maybe more pressure is needed to suppress BMP-2 signaling and induce Smad3.

According to Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation., Smurf1 reduces Smad5 levels but has no effect on Smad3 levels.

Inihibiting Estrogen may inhibit Smurf1.

Estrogen inhibits transforming growth factor beta signaling by promoting Smad2/3 degradation.

"Estrogen is a growth factor that stimulates cell proliferation. The effects of estrogen are mediated through the estrogen receptors, ERalpha and ERbeta, which function as ligand-induced transcription factors and belong to the nuclear receptor superfamily. TGF-beta acts as a cell growth inhibitor, and its signaling is transduced by Smads. ERalpha inhibits TGF-beta signaling by decreasing Smad protein levels. ERalpha-mediated reductions in Smad levels did not require the DNA binding ability of ERalpha, implying that ERalpha opposes the effects of TGF-beta via a novel non-genomic mechanism. Our analysis revealed that ERalpha formed a protein complex with Smad and the ubiquitin ligase Smurf, and enhanced Smad ubiquitination and subsequent degradation in an estrogen-dependent manner."

So you may want to inhibit estrogen to allow for TGF-Beta to induce Smad3 signaling which can induce chondrogenesis while performing LSJL.  Note that despite no Smad3 signaling, there were still signs of chondrogenesis in LSJL like chondrogenic ECM protein production.

Ski may be involved in Smad3 signaling:

Ski inhibits TGF-β/phospho-Smad3 signaling and accelerates hypertrophic differentiation in chondrocytes

"Since transforming growing factor-β (TGF-β)/Smad signaling inhibits chondrocyte maturation, endogenous negative regulators of TGF-β signaling are likely also important regulators of the chondrocyte differentiation process. One such negative regulator, Ski, is an oncoprotein that is known to inhibit TGF-β/Smad3 signaling via its interaction with phospho-Smad3 and recruitment of histone deacetylases (HDACs) to the DNA binding complex. Based on this, we hypothesized that Ski inhibits TGF-β signaling and accelerates maturation in chondrocytes via recruitment of HDACs to transcriptional complexes containing Smads. We tested this hypothesis in chick upper sternal chondrocytes (USCs), where gain and loss of Ski expression experiments were performed. Over-expression of Ski not only reversed the inhibitory effect of TGF-β on the expression of hypertrophic marker genes such as type X collagen (colX) and osteocalcin, it induced these genes basally as well. Conversely, knockdown of Ski by RNA interference led to a reduction of colX and osteocalcin expression under basal conditions. Furthermore, Ski blocked TGF-β induction of cyclinD1 and caused a basal up-regulation of Runx2, consistent with the observed acceleration of hypertrophy. Regarding mechanism, not only does Ski associate with phospho-Smad2 and 3, but its association with phospho-Smad3 is required for recruitment of HDAC4 and 5. Implicating this recruitment of HDACs in the phenotypic effects of Ski in chondrocytes, the HDAC inhibitor SAHA reversed the up-regulation of colX and osteocalcin in Ski over-expressing cells. These results suggest that inhibition of TGF-β signaling by Ski, which involves its association with phospho-Smad3 and recruitment of HDAC4 and 5, leads to accelerated chondrocyte differentiation."

So Ski is likely bad for height growth but we don't know for sure.

"Ski is required for the association between pSmad3 and HDAC4 and HDAC5"

"inhibition of the TGF-β pathway by Smad7 leads to reduced chondrocyte proliferation and proteoglycan synthesis in chondrocyte cultures. Inhibition of BMP signaling with either Smad6 or noggin leads to deceleration of the chondrocyte hypertrophic program in vitro"

Anisomycin can downregulate Ski according to Downregulation of Ski and SnoN co-repressors by anisomycin.

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