Selective modulation of PPARgamma activities in marrow mesenchymal stem cells and their effects on bone.

摘要

Thiazolidinediones (TZDs), a class of oral anti-diabetic agents, sensitize cells to insulin through a specific activation of PPARgamma nuclear receptor, however their prolonged use causes bone loss and increases fracture risk. In this study, the effects of three different TZDs (rosiglitazone, pioglitazone, and troglitazone) on osteoblast and adipocyte differentiation, phenotype-specific gene expression, and TGFbeta/BMP signaling pathways were evaluated and showed that all tested TZDs simultaneously inhibited osteoblast differentiation and stimulated adipocyte development. They suppressed the expression of osteoblast-specific transcription factors and decreased expression of components of the TGFbeta/BMP pathways. In conclusion, TZDs affect an activity of TGFbeta/BMP signaling pathways which may account for their inhibitory effect on osteoblast differentiation and bone formation. Microarray analysis done to determine the pathways involved in PPARgamma2 mediated pro-adipocytic and anti-osteoblastic activities indicated that among the early responders BMP4 a ligand for the osteoinductive signaling pathway was downregulated and Tob1, a suppressor of activity of BMP signaling pathway was upregulated. Tob1 upregulation with Rosiglitazone (Rosi) activated PPARgamma2 was correlated with the downregulation of members of the TGFbeta/BMP pathway, along with downregulation of its target genes. Based on the profile of Tob1 expression and its function as a suppressor of TGFbeta/BMP signaling, we hypothesize that Tob1 mediates PPARgamma2 anti-osteoblastic activity. To test this hypothesis, we regulated the expression of Tob1 using overexpression vectors or gene silencing techniques. Results indicate that Tob1 doesn't mimic PPARgamma2 anti-osteoblastic activity completely and indeed it inhibits both osteoblast and adipocytic phenotypes. This might suggest that Tob1 may play an important role in maintaining the mesenchymal stem cells (MSCs) and inhibiting its differentiation towards any of the lineages i.e. it protects the "stemness" of MSCs. As there was no single candidate gene which completely mediates the anti-osteoblastic activity of activated PPARgamma2, there is a need to identify the PPARgamma2 ligands which can selectively activate the beneficial anti-diabetic/pro-adipocytic but not anti-osteoblastic activities. One such selective PPARgamma modulator is the Telmisartan (Tel) which is an anti-hypertensive drug inhibiting the activity of angiotensin II receptor. Tel acts as a selective PPARgamma agonist and improves insulin resistance and lipid profile in both humans and mice; however no clinical evidence or scientific research has been done to investigate the effects of Tel on bone mass. We hypothesize that Tel does not have any effect on osteoblast phenotype by its own and can protect bone from the negative effects of Rosi by protecting the integrity of TGFbeta/BMP pathway . We investigated this, and results indicate that Tel did not affect osteoblast phenotype, however it induced adipocyte phenotype and it protected against the anti-OB effect of Rosi. Tel did not affect the expression of genes or ALP activity in vitro and in vivo and even it protected from Rosi's anti-osteoblastic effect. Unlike Rosi activated PPARgamma2 which suppresses the activity of TGFbeta/BMP pathway, Tel did not affect pathway activity and protected from negative effects of Rosi. When administered to hyperglycemic and diabetic Avy/a mice, both drugs had a similar anti-diabetic effect. However, administration of Rosi resulted in 60% decrease in trabecular bone mass, whereas Tel did not have any effect on bone. Moreover, when mice received combination of both drugs, Tel prevented bone loss induced by Rosi. Infact, our lab is the first one to investigate the effects of Tel on bone mass and the mechanisms involved in the protection. Thus, Tel selectively induces PPARgamma2 anti-diabetic activity without affecting osteoblastic phenotype in bone MSCs and in mice. Thus, Tel represents a paradigm of multifunctional drug, which can be used alone or in combination with Rosi, for simultaneous treatment of hypertension and diabetes without adverse effects on bone.