Journal of Cellular Biochemistry 85:825–836 (2002) Leptin Stimulates Human Osteoblastic Cell Proliferation, De Novo Collagen Synthesis, and Mineralization: Impact on Differentiation Markers, Apoptosis, and Osteoclastic Signaling Jan O. Gordeladze, 1 * Christian A. Drevon, 2 Unni Syversen, 3 and Janne E. Reseland 2 1 Department of Medical Biochemistry, University of Oslo, Oslo, Norway 2 Institute for Nutrition Research, University of Oslo, Oslo, Norway 3 Department of Intra-Abdominal Diseases, University of Science & Technology, Trondheim, Norway Abstract Anabolic hormones, mechanical loading, and the obese protein leptin play separate roles in maintaining bone mass. We have previously shown that leptin, as well as its receptor, are expressed by normal human osteoblasts. Consequently, we have investigated how leptin affects proliferation, differentiation, and apoptosis of human osteoblasts. Iliac crest osteoblasts, incubated with either leptin (100 ng/ml), calcitriol (1,25(OH) 2 D 3 ; 10 9 M) or 1–84 human parathyroid hormone (PTH; 10 8 M), were cultured for 35 consecutive days and assayed for expression of various differentiation-related marker genes (as estimated by RT-PCR), de novo collagen synthesis, proliferation, in vitro mineralization, and osteoclast signaling. The effects of leptin on protection against retinoic acid (RA; 10 7 M) induced apoptosis, as well as transition into preosteocytes, were also tested. Leptin exposure enhanced cell proliferation and collagen synthesis over both control condition and PTH exposure. Leptin inhibited in vitro calcified nodule production after 1–2 weeks in culture, however, subsequent to 4–5 weeks, leptin significantly stimulated mineralization. The mineralization profile throughout the entire incubation period was almost undistinguishable from the one induced by PTH. In comparison, 1,25(OH) 2 D 3 generally reduced proliferation and collagen production rates, whereas minera- lization was markedly enhanced. Leptin exposure (at 2 and 5 weeks) significantly enhanced the expression of TGFb, IGF-I, collagen-Ia, ALP, and osteocalcin mRNA. Leptin also protected against RA-induced apoptosis, as estimated by soluble DNA fractions and DNA laddering patterns subsequent to 10 days of culture. The expression profiles of Bax-a and Bcl-2 mRNAs indicated that leptin per se significantly protected against apoptosis throughout the entire incubation period. Furthermore, the osteoblast marker OSF-2 was diminished, whereas the CD44 osteocyte marker gene expression was stimulated, indicating a transition into preosteocytes. In terms of osteoclastic signaling, leptin significantly augmented the mRNA levels of both interleukin-6 (IL-6) and osteoprotegerin (OPG). In summary, continuous leptin exposure of iliac crest osteoblasts, promotes collagen synthesis, cell differentiation and in vitro mineralization, as well as cell survival and transition into preosteocytes. Leptin may also facilitate osteoblastic signaling to the osteoclast. J. Cell. Biochem. 85: 825–836, 2002. ß 2002 Wiley-Liss, Inc. Key words: proliferation; collagen synthesis; mineralization; differentiation; apoptosis; osteoclast stimulation Obesity yields an unequivocally protective effect on bone mass, and has been ascribed to high body fat content in relation to enhanced leptin plasma levels [Klein et al., 1998]. Thus, leptin, which is a gene product synthesized and released from adipocytes [Zhang et al., 1994; Stephens et al., 1995], may serve as an important signal to modulate osteoblastic func- tion. In the natural leptin knock-out model, ob/ ob mice, leptin administration increases bone density and mineral content, length of limbs and brain mass [Steppan and Swick, 1999], as well as total body bone area, bone mineral contents, and density [Steppan et al., 1999]. ß 2002 Wiley-Liss, Inc. Grant sponsor: Anders Jahre Medical Research Founda- tion, Norway; Grant sponsor: Freia-Marabou Nutritional Research Foundation, Norway; Grant sponsor: Norwegian Society for Fighting Cancer, Norway. *Correspondence to: Jan O. Gordeladze, Department of Medical Biochemistry, University of Oslo, P.O. Box 1112, Blindern, N-0316 Oslo, Norway. E-mail: j.o.gordeladze@basalmed.uio.no Received 11 December 2001; Accepted 30 January 2002 DOI 10.1002/jcb.10156