Journal of Cellular Biochemistry Supplement 36:144±155 (2001) Estrogen Modulates Estrogen Receptor a and b Expression, Osteogenic Activity, and Apoptosis in Mesenchymal Stem Cells (MSCs) of Osteoporotic Mice Shuanhu Zhou, 1 Yoram Zilberman, 1 Karsten Wassermann, 2 Steven D. Bain, 3 Yoel Sadovsky, 4 and Dan Gazit 1 * 1 Molecular Pathology Laboratory, Hebrew University-Hadassah Medical and Gene Therapy Center, Jerusalem, Israel 2 Novo Nordisk, Malov, Denmark 3 SkeleTech Inc., Bothell, Washington 4 Washington University, St. Louis Abstract In the mouse, ovariectomy (OVX) leads to signi®cant reductions in cancellous bone volume while estrogen (17b-estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2- dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) a and b expression of primary culture MSCs isolated from OVX and sham-operated mice. MSCs, treated in vitro with 10 7 M E2, displayed a signi®cant increase in ERa mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERb mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up-regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF-b1, BMP-2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERa was the predominant form expressed in MSCs obtained from both OVX and sham-operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERa expression, osteogenic gene expression and, inhibits apoptosis and ERb expression in MSCs obtained from OVX and sham-operated mice. Co- expression of ERa, but not ERb, and osteogenic differentiation markers might indicate that ERa function as an activator and ERb function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERa activation. J. Cell. Biochem. Suppl. 36:144±155, 2001. ß 2001 Wiley-Liss, Inc. Key words: estrogen; estrogen receptor; mesenchymal stem cells (MSCs); osteogenesis; apoptosis; osteoporosis Estrogen deprivation results in postmeno- pausal osteoporosis; long-term treatment with estrogen in appropriate doses reduces the risk of hip fractures by 50 to 60% and the risk of vertebral deformation by 90% [Notelovitz, 1997]. Bone loss in ovariectomized (OVX) animals is similar to that in postmenopausal women [Frost and Jee, 1992]. Estrogen main- tains bone mass by preserving the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [Turner et al., 1994]. The protective effect of estrogen on the skeleton of postmenopausal women appears to be mediated by suppression of bone resorption, with little evidence to suggest that conventional doses of estrogen also stimulate osteoblast activity. However, recent studies demonstrate that prolonged exposure of postmenopausal women to relatively high doses of estrogen results in sustained stimulation of osteoblast function [Tobias and Compston, 1999]. Systemically administered 17b-estradiol (E2) enhanced bone formation in animals [Takano-Yamamoto and Rodan, 1990; Chow ß 2001 Wiley-Liss, Inc. This article published online in Wiley InterScience, March 20, 2001. Abbreviations used: MSCs, mesenchymal stem cells; ER, estrogen receptor; E2, 17b-estradiol; ALP, alkaline phos- phatase; BrdU, bromodeoxyuridine; OVX, ovariectomy. Grant sponsor: Novo Nordisk, Malov, Denmark. *Correspondence to: Dan Gazit, D.M.D., Ph.D., Molecular Pathology Lab, Hebrew University-Hadassah Medical and Gene Therapy Center, P.O.B. 12272 Jerusalem 91120, Israel. E-mail: dgaz@cc.huji.ac.il Received 31 October 2000; Accepted 21 December 2000