Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts G P Thomas, S U K Baker, J A Eisman and E M Gardiner Bone and Mineral Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia (Requests for offprints should be addressed to G P Thomas, Bone and Mineral Research Program, Garvan Institute of Medical Research, 384 Victoria St, Sydney, New South Wales 2010, Australia; Email: g.thomas@garvan.org.au) Abstract Osteoblast–osteoclast coordination is critical in the main- tenance of skeletal integrity. The modulation of osteoclas- togenesis by immature cells of the osteoblastic lineage is mediated through receptor activator of NFB (RANK), its ligand RANKL, and osteoprotegerin (OPG), a natural decoy receptor for RANKL. Here, the expression of OPG and RANKL in primary mouse osteoblastic cultures was investigated to determine whether the osteoclastogenic stimulus depended on the stage of osteoblastic differentia- tion and the presence of the calciotrophic hormone 1,25- dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). OPG mRNA expression was increased in osteoblastic cultures after the onset of mineralisation relative to less mature cultures, but did not alter in response to 1,25- (OH) 2 D 3 treatment. In contrast, basal RANKL mRNA expression did not change during differentiation but was significantly enhanced by 1,25-(OH) 2 D 3 treatment at all times. The stimulatory effects of 1,25-(OH) 2 D 3 on RANKL were lessened in more mature cultures, however. The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by 1,25-(OH) 2 D 3 treat- ment at all stages of osteoblastic differentiation, but to a lesser degree in cultures after the onset of mineralisation. Thus the 1,25-(OH) 2 D 3 -driven increase in osteoclas- togenic potential of immature osteoblasts appears to be mediated by increased RANKL mRNA expression, with mature osteoblasts having relatively decreased osteoclas- togenic activity due to increased OPG mRNA expression. These findings suggest a possible mechanism for the recently proposed negative regulatory role of mature osteoblasts on osteoclastogenesis and indicate that the relative proportions of immature and mature osteoblasts in the local microenvironment may control the degree of resorption at each specific bone site. Journal of Endocrinology (2001) 170, 451–460 Introduction Skeletal structure is continually adapting to metabolic and mechanical demands. Osteoblastic (bone forming) and osteoclastic (bone resorbing) cells maintain the integrity of bone during this remodelling process, with tight regulation and coordination of their activities. In vivo histological and in vitro culture studies suggested a role for osteoblastic cells in the direct regulation of osteoclast activity (Rodan & Martin 1981, Martin & Ng 1994). Resorption is stimulated by a number of factors, includ- ing the hormones 1,25-dihydroxyvitamin D 3 (1,25- (OH) 2 D 3 ) and parathyroid hormone (Teti et al. 1988, Flanagan et al. 1995, Suda et al. 1997). Enhancement of osteoclast differentiation and activity by 1,25-(OH) 2 D is largely mediated via the immature cells of the osteoblastic lineage (Martin & Ng 1994). Regulatory factors involved in this interaction have been identified. Stromal and osteoblast precursor cells express a member of the tumour necrosis factor ligand family, receptor activator of NFB ligand (RANKL), also identified as osteoclast differentia- tion factor (ODF), osteoprotegerin ligand (OPG-L) and tumour necrosis factor-related, activation-induced cy- tokine (TRANCE) (Anderson et al. 1997, Wong et al. 1997, Lacey et al. 1998, Yasuda et al. 1998b). This cell-surface ligand stimulates osteoclastogenesis and osteo- clast activity by binding to its cognate receptor, RANK, also termed ODF receptor (ODFR), on the surface of osteoclast precursors (Hsu et al. 1999). Addition of the cleaved and thus soluble form of RANKL can increase osteoclast formation and activity in vitro (Lacey et al. 1998, Matsuzaki et al. 1998, Yasuda et al. 1998b, Burgess et al. 1999). A further level of regulation is provided by the production of a RANKL ‘decoy receptor’, osteoprotegerin (OPG) or osteoclastogenesis inhibitory factor (OCIF), by osteoblasts and other cell types (Simonet et al. 1998, Yasuda et al. 1998a). OPG binds to RANKL, blocking its interaction with RANK on osteoclast precursors and thus inhibiting osteoclast activity (Lacey et al. 1998, Yasuda et al. 1998b, Hsu et al. 1999). The importance of this pathway has been demonstrated in OPG knockout mice, which exhibit severe osteoporosis (Bucay et al. 1998, 451 Journal of Endocrinology (2001) 170, 451–460 0022–0795/01/0170–451  2001 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org