Osteogenic Differentiation of Normotensive Rats Depends on the β‐adrenergic Receptor

Abstract

Bone remodeling is a complex process that depends on the balance between formation and resorption bone, a process which is regulated by bone cells and systemic factors, like the Sympathetic Nervous system (SNS). The mediators of these system are able to regulate bone metabolism through adrenergic receptors on the surface of the osteoblasts. However, the role of β‐adrenergic receptors is not clear in the osteogenic differentiation process. Thus, in this study we aimed to evaluate the role of B‐adrenergic receptor on osteoblastic differentiation of bone marrow mesenchymal stem cells from normotensive and Spontaneously Hypertensive Rats (SHR).Methods70‐days‐old male Wistar and SHR rats were used for bone marrow collection from femurs, which was placed in cell culture flasks and after in to 24‐well plates, where they received osteogenic medium (OM: MEM, plus 50 μg/mL ascorbic acid, 10 mM β glycerophosphate, and 10−8 M dexamethasone) and the treatment with Carvedilol (1μM), non‐selective adrenergic receptor antagonist, or Nebivolol (0,1 μM), β1‐adrenergic receptor antagonist. Cell proliferation (MTT assay) and alkaline phosphatase specific activity (Alp) were analyzed at day 7, 10 and 14. Mineralization was evaluated at day 14, by Alizarin Red S. Gene expression of osteogenic markers and B1 and B2‐adrenergic receptor were evaluated at day 7, by real time‐RT‐PCR. The proteolytic activity of matrix metalloproteinase 2 and 9 (MMP‐2 and MMP‐9) were evaluated at day 7 using gelatin zymography. The protocol was approved by Institutional Animal Care and Use Committees (School of Dentistry of Araçatuba; Process 00686‐2016).ResultsAlp activity increased in OM group from Wistar rats at day 7 and the Nebivolol group reduced those activity at day 7 and at day 10. SHR OM group did not present Alp increase, and the treatment with both drugs did not change it. Nebivolol treatment decreased Runx2, Osterix and β‐catenin in Wistar compared to OM. In SHR, only Osterix was reduced. Alp, Osteocalcin and Bone sialoprotein have significative decrease in cells from Wistar treated with Nebivolol compared to OM. Pro and active MMP‐2 was reduced by Nebivolol treatment just at Wistar cells. Besides that, Nebivolol reduced Adrb1 gene expression at day 7 in Wistar group compared to OM. Mineralization showed that Nebivolol reduced mineral deposition in Wistar compared to OM.Conclusionβ1 adrenergic receptor seems to be involved in the osteogenic differentiation of cells from Wistar rats.Support or Funding InformationFAPESP (Grant: #2015/03965‐2) and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.