Yankel Gabet

Regulation of Adult Bone Turnover by Sex Steroids

Recent reports reveal increasing complexity of mechanisms underlying the bone sparing effects of sex steroids. This review focuses on mechanisms by which sex steroids attenuate endocortical and trabecular adult bone turnover, perhaps their most important property as bone mass regulators. Clearly, estrogen withdrawal increases osteoclast number and bone resorption; however, important open questions are the extent to which osteoblasts and their precursors are involved, and the relative contributions of the RANK/RANKL/OPG system, Fas ligand and Runx2. In addition to reviewing these aspects of estrogen action, we also discuss proskeletal effects of androgens on the adult male skeleton, including aromatization to estrogens and male‐specific mechanisms. Detailed understanding of skeletal site‐ and gender‐dependent mechanisms by which sex steroids protect the adult skeleton will provide the foundation for improved risk assessment, prevention and management of osteoporosis. J. Cell. Physiol. 224: 305–310, 2010.

Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling.

The CB1 cannabinoid receptor has been implicated in the regulation of bone remodeling and bone mass. A high bone mass (HBM) phenotype was reported in CB1-null mice generated on a CD1 background (CD1CB1-/- mice). By contrast, our preliminary studies in cb1-/- mice, backcrossed to C57BL/6J mice (C57CB1-/- mice), revealed low bone mass (LBM). We therefore analyzed CB1 expression in bone and compared the skeletons of sexually mature C57CB1-/- and CD1CB1-/- mice in the same experimental setting. CB1 mRNA is weakly expressed in osteoclasts and immunoreactive CB1 is present in sympathetic neurons, close to osteoblasts. In addition to their LBM, male and female C57CB1-/- mice exhibit decreased bone formation rate and increased osteoclast number. The skeletal phenotype of the CD1CB1-/- mice shows a gender disparity. Female mice have normal trabecular bone with a slight cortical expansion, whereas male CD1CB1-/- animals display an HBM phenotype. We were surprised to find that bone formation and resorption are within normal limits. These findings, at least the consistent set of data obtained in the C57CB1-/- line, suggest an important role for CB1 signaling in the regulation of bone remodeling and bone mass. Because sympathetic CB1 signaling inhibits norepinephrine (NE) release in peripheral tissues, part of the endocannabinoid activity in bone may be attributed to the regulation of NE release from sympathetic nerve fibers. Several phenotypic discrepancies have been reported between C57CB1-/- and CD1CB1-/- mice that could result from genetic differences between the background strains. Unraveling these differences can provide useful information on the physiologic functional milieu of CB1 in bone.

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

BackgroundProstate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide mRNA expression changes in PCa cells in response to Runx2.ResultsWe engineered a C4-2B PCa sub-line called C4-2B/Rx2dox, in which Doxycycline (Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal.ConclusionsThe effects of Runx2 in C4-2B/Rx2dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Modulation of Runx2 Activity by Estrogen Receptor-α: Implications for Osteoporosis and Breast Cancer

The transcription factors Runx2 and estrogen receptor-alpha (ERalpha) are involved in numerous normal and disease processes, including postmenopausal osteoporosis and breast cancer. Using indirect immunofluorescence microscopy and pull-down techniques, we found them to colocalize and form complexes in a ligand-dependent manner. Estradiol-bound ERalpha strongly interacted with Runx2 directly through its DNA-binding domain and only indirectly through its N-terminal and ligand-binding domains. Runx2s amino acids 417-514, encompassing activation domain 3 and the nuclear matrix targeting sequence, were sufficient for interaction with ERalphas DNA-binding domain. As a consequence of the interaction, Runx2s transcriptional activation activity was strongly repressed, as shown by reporter assays in COS7 cells, breast cancer cells, and late-stage MC3T3-E1 osteoblast cultures. Metaanalysis of gene expression in 779 breast cancer biopsies indicated negative correlation between the expression of ERalpha and Runx2 target genes. Selective ER modulators (SERM) induced ERalpha-Runx2 interactions but led to various functional outcomes. The regulation of Runx2 by ERalpha may play key roles in osteoblast and breast epithelial cell growth and differentiation; hence, modulation of Runx2 by native and synthetic ERalpha ligands offers new avenues in selective ER modulator evaluation and development.

Endosseous implant anchorage is critically dependent on mechanostructural determinants of peri-implant bone trabeculae

Low bone mass is highly prevalent among patients receiving endosseous implants. In turn, the implantation prognosis in low‐density skeletal sites is poor. However, little is known about the mechanostructural determinants of implant anchorage. Using metabolic manipulations that lead to low bone density and to its rescue, we show here that anchorage is critically dependent on the peri‐implant bone (PIB). Titanium implants were inserted horizontally into the proximal tibial metaphysis of adult rats 6 weeks after orchiectomy (ORX) or sham ORX. Systemic intermittent administration of human parathyroid hormone (1–34) [iahPTH(1–34)] or vehicle commenced immediately thereafter for 6 weeks. The bone‐implant apparatus was then subjected to image‐guided failure assessment, which assesses biomechanical properties and microstructural deformation concomitantly. Anchorage failure occurred mainly in PIB trabeculae, 0.5 to 1.0 mm away from the implant. Mechanically, the anchorage performed poorly in ORX‐induced low‐density bone, attributable mainly to decreased trabecular number. iahPTH(1–34) rescued the PIB density and implant mechanical function by augmenting trabecular thickness (Tb.Th). However, implant biomechanical properties in low‐density bone were relatively insensitive to implant surface treatment that affected only the osseointegration (%bone‐implant contact). These results support a model wherein anchorage failure involves buckling of the weakest trabecular struts followed by sequential failure of the stronger trabeculae. Treatment with iahPTH(1–34) induced thicker struts, which were able to delay and even prevent failure of individual elements, thus implicating trabecular thickness as a prime target for enhancing implant anchorage by systemic bone anabolic therapy.

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS‐degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh‐heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh‐heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild‐type (WT) and hpa‐transgenic (hpa‐tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa‐tg mice. Heparanase expression was absent in osteoclasts. Micro‐computed tomographic and histomorphometric skeletal analyses in male and female hpa‐tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase. J. Cell. Physiol.

Lef1 Haploinsufficient Mice Display a Low Turnover and Low Bone Mass Phenotype in a Gender- and Age-Specific Manner

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4- and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20(+/-) mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20(+/-) splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis.

Erythropoietin directly stimulates osteoclast precursors and induces bone loss

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57B16 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%‐61% trabecular bone loss caused by increased bone resorption (+60%‐88% osteoclast number) and reduced bone formation rate (‐19 to ‐74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bone monocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels.—Hiram‐Bab, S., Liron, T., Deshet‐Unger, N., Mittelman, M., Gassmann, M., Rauner, M., Franke, K., Wielockx, B., Neumann, D., Gabet, Y. Erythropoietin directly stimulates osteoclast precursors and induces bone loss. FASEB J. 29, 1890‐1900 (2015). www.fasebj.org