Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling:AT1R SIGNALING IN OSTEOBLAST DIFFERENTIATION

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also reduce loss of bone mineral density. Here, we identified in a genetic screening the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also improve loss of bone mineral density. Here we identified in a genetic screen the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors

Identification of genes that regulate bone marrow stromal cell differentiation by RNA interference

Mesenchymal stem cells (MSCs) can be isolated from bone marrow and can differentiate in a variety of cell types, including osteoblasts, adipocytes, chondrocytes, myoblasts, hepatocytes, and neural cells. For this reason, MSCs are very promising for the development of cell-based therapy for degenerative diseases. Understanding the mechanisms behind MSC cell fate determination is not easy, since the molecular processes that drive differentiation are complex and poorly understood. Even though in the recent years many improvements have been done, many mechanism need to be elucidated. We planned to investigate on differentiation mechanisms of MSC and, in particular, we focused our activities towards osteoblast differentiation; at this aim, we have silenced specific mRNAs using a mouse shRNA library in a 96 well plate-based screening strategy. With this methodology we identified sevral genes that are possible candidates for a role in osteoblast differentiation. Among the candidate genes, a consistent fraction was represented by genes whose function is still unknown: we focused our attention on a short number of these genes that we named ObI (Osteoblast inducer). In this thesis project we focused our experiments on a particular gene, that we named ObI-1. We also considered genes identified during the screening with a known function but an unknown role during the osteogenic differentiation process. Among these genes we focused our attention on Angiotensin receptor 1 (AT1R), whose role in osteogenic differentiation had been previously suggested. For both candidates that we analyzed for this project we confirmed the effects of silencing on osteogenic differentiation and we analyzed expression in tissues and in our cell culture system. Furthermore, we characterized function and mechanism of action in osteoblastogenesis for both genes