Original Full Length ArticleEstrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association
Introduction
Runx2 is as an osteoblast master regulator that is required for bone formation. Initially identified based on its interaction with the bone-specific Osteocalcin promoter in vitro [1], [2], [3], the pivotal role of Runx2 in osteogenesis in vivo was demonstrated by the absence of differentiated osteoblasts and failure of skeletal mineralization in Runx2-deficient mice [4], [5]. Furthermore, inhibition of Runx2 in vitro abrogates expression of osteoblast markers, and its forced expression in non-osteoblasts induces bone-like cellular phenotypes [2], [3]. Contrasting the role of Runx2 as a master regulator of osteoblast differentiation and embryonic bone development, its function in bone resorption is less appreciated. Over-expression of Runx2 in osteoblasts resulted in increased osteoclast differentiation from co-cultured pre-osteoclasts in vitro [6], [7], [8] and exaggerated bone resorption in vivo [6], [9]. Conversely, expression of a dominant negative RUNX2 decreased osteoclastogenesis in co-culture assays and decreased bone resorption in vivo [7]. Accordingly, osteoclast number decreased in mice with either global or osteoblast-specific Runx2 ablation [4], [10]. Thus, RUNX2 promotes both osteoblastogenesis and osteoblast-driven osteoclastogenesis.
Regulation of osteoclastogenesis by osteoblasts constitutes a fundamental principle in the coupling of bone resorption to bone formation [11]. Among osteoblast-borne signals mediating this coupling is the quintessential osteoclastogenic factor RANKL [12], [13], [14]. Although RUNX2 can increase RANKL mRNA levels in smooth muscle cells [15], stimulation of osteoclastogenesis by RUNX2 does not necessarily involve the regulation of Rankl mRNA levels in osteoblasts [8], [16], [17]. Indeed, using primary osteoblast cultures, the present work demonstrates that RUNX2 influences RANKL through regulating its association with the cell membrane without significantly affecting its mRNA expression.
Postmenopausal osteoporosis inflicts a pathological fracture on two in every five women over the age of fifty [18]. It is mostly attributable to reduced stimulation of estrogen receptor α (ERα) in osteoblasts and osteoclasts [19]. Accordingly, estrogens and selective estrogen receptor modulators (SERMs) constitute therapeutic options for the preservation of bone mass in postmenopausal women, and some SERMs have beneficial effects on the skeleton when used for the management of breast cancer [20]. Based on previous reports on inhibition of RUNX2 activity by ERα [21], as well as resistance to ovariectomy-induced bone loss in mice expressing of a dominant negative RUNX2 isoform [7], we hypothesized that activation of ERα in osteoblasts attenuates RUNX2-driven osteoclastogenic signal(s). Indeed, we show that estrogen signaling in osteoblasts abrogates RUNX2-mediated RANKL membrane association and differentiation of co-cultured splenocytes into mature osteoclasts.
Section snippets
Animals
C57BL/6 JAX® mice from Jackson Laboratory (Sacramento, CA) were used for the extraction of both osteoblasts and splenocytes without regard to mouse gender. Splenocytes were isolated from either wild type or ERα knockout (ERKO) animals. Mice were housed in microisolator-type cages at the vivaria of University of Southern California (USC) or University of California Los Angeles (UCLA). The respective Institutional Animal Care and Use Committees approved all experimental procedures with animals.
Reagents
Dox-inducible Runx2 expression in newborn mouse calvarial osteoblasts (NeMCOs)
RUNX2 promotes not only osteoblast differentiation and bone formation, but also osteoblast-driven osteoclastogenesis [6], [7], [8], [9], [10], [28]. Because estrogens inhibit RUNX2 activity [21], we asked whether they would inhibit osteoclast differentiation driven by expression of RUNX2 in co-cultured osteoblasts. First, we transduced newborn mouse calvarial osteoblasts (NeMCOs) with lentiviruses encoding doxycycline (dox)-inducible FLAG-RUNX2 [24] and treated the so-called NeMCO/Rx2dox cells
Discussion
It is well established that accelerated bone turnover increases fracture risk, with postmenopausal osteoporosis serving a prime example. Bone loss that occurs at physiological turnover rates is slow and usually transpires without pathological consequences because coupling mechanisms secure the replacement of most of the resorbed bone with newly deposited material. Adding to classical coupling mechanisms of signaling from osteoblasts to osteoclasts and back, the regulation of both osteoblast
Acknowledgments
This work was supported by grants RO1 DK071122 and RO1 DK071122S1 from NIDDK to BF, holder of the J. Harold and Edna L. LaBriola Chair in Genetic Orthopaedic Research. AM was supported by a training grant T32 DE021982 from NIDCR, and YG was supported by a Meyer Young Investigator Fellowship from The Arthritis Foundation. We thank Drs. Ebrahim Zandi (USC), Dr. Masashi Honma and Dr. Hiroshi Suzuki (University of Tokyo) for reagents, the UCLA Vector Core (supported by CURE/P30DK041301) for
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Present Address: Department of Orthopaedic Surgery, University of Tennessee Health Science Center, 19 S. Manassas St, Memphis, TN 38163, USA.