Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation.(Report)

Abstract

In the present study, we investigated the in vitro effect of resveratrol (RSVL), a polyphenolic phytoestrogen, on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL ([10.sup.-8]-[10.sup.-5]M) increased cell growth dose-dependently, as measured by [[.sup.3.H]]-thymidine incorporation, and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity, calcium deposition into the extracellular matrix, and the expression of osteoblastic markers such as RUNX2/CBFAI, Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL ([10.sup.-6] M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17[belta]-estrodial ([10.sup.-18] M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor, PD98059, significantly attenuated RSVL-induced ERK1/2 phosphorylation, consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast, SB203580, a p38 MAPK pathway blocker, blocked RSVL-induced p38 phosphorylation, but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Resveratrol; Mesenchymal stem cell; Estrogen receptor; ERK1/2; P38 MAPK

Introduction

Osteoporosis associated with estrogen deficiency after menopause is the most common cause of age-related bone loss (Kanis et al., 1994). Hormone replacement therapy (HRT) could effectively prevent postmenopausal osteoporosis (Lindsay et al., 1984) and reduce the incidence of fractures (Blank and Bockman, 1999). However, HRT also increases the risk of breast and endometrial cancer, in addition to other undesirable side effects (Vihtamaki et al., 1999). An alternative therapy of the classical HRT is needed. The phytoestrogens, therefore, are potentially important in the prevention of postmenopausal osteoporosis.

Resveratrol (3,4',5-trihydroxystilbene, RSVL), a known phytoestrogen, is a phenolic compound that occurs naturally in mulberries, peanuts and the berry skins of most grape cultivars (Jang et al., 1997) and possesses estrogenic activity (Gehm et al., 1997). Several biological effects of RSVL have been reported, including platelet aggregation attenuation (Wang et al., 2002), cardiovascular protection (Cao and Li, 2004), and anticancer activity (Savouret and Quesne, 2002). Recent studies show that RSVL stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells in vitro (Mizutani et al., 1998), and inhibited bone loss in ovariectomized rats in vivo (Mizutani et al., 2000). Most recently, RSVL was proved to act against Cyclosporin A (CsA) inhibition of proliferation and osteoblastic differentiation in mouse bone mesenchymal stem cells (Song et al., 2006). However, how RSVL positively regulates bone metabolism and cell functions in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures remains unclear.

There is evidence that the biological effects of RSVL were associated with MAPK signaling pathways. RSVL was reported to induce apoptosis in thyroid cancer cell lines via a MAPK- and p53-dependent mechanism (Shih et al., 2002), and reduce cell oxidation and proliferation in human retinal pigment epithelial cells via extracellular signal-regulated kinases (ERK) inhibition (King et al., 2005). Although some recent studies showed that MAPK pathways were involved in human bone metabolism, including the commitment of human bone marrow-derived mesenchymal stem cells (HBMSCs) to the osteogenic and adipogenic lineages (Jaiswal et al., 2000) and mechanical stimulus-induced cell proliferation and matrix mineralization of HBMSCs (Simmons et al., 2003; Riddle et al., 2006), whether the effect of RSVL on bone metabolism occurs via MAPK signaling is still unknown. In the current study, we investigated the in vitro effect of RSVL on proliferation and osteoblastic maturation of HBMSCs cultures, and explored the role of the MAPK signaling pathway in RSVL-mediated anabolic cell responses.

Materials and methods

Reagents

Alpha minimum essential medium ([alpha]-MEM), phenol red-free [alpha]-MEM, fetal bovine serum (FBS), and sodium dodecyl sulfate (SDS) were obtained from GIBCO BRL (Grand Island, NY, USA). Resveratrol (RSVL), 17[beta]-estrodial ([E.sub.2]), trypsin, ascorbic acid, [beta]-glycerophosphate, p-nitrophenyl phosphate (p-NPP), p-nitrophenol (p-NP), diethanolamine, dextran-coated charcoal, PD98059 and SB203580 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ICI182,780 was purchased from Tocris Cookson Inc. Ltd. (Avonmouth Bristol BS11 8TA, UK). [[.sup.3.H]]-thymidine ([[.sup.3.H]]-TDR) was purchased from Shanghai Institute of Nuclear Research (Shanghai, China). Bio-Rad …