Citations

ABSTRACT Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclast-specific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. © 2015 American Society for Bone and Mineral Research.

In young adults, bone lost through osteoclast-mediated resorption is precisely replaced in both location and amount. Understanding how these two processes are coupled is crucial to advancing treatments for osteoporosis, a disease that progresses when the processes become uncoupled. We documented that osteoclasts secrete the mammalian integration 1 gene that is the homolog of Drosophila Wngless (Wnt) 10b, bone morphogenetic protein 6 (BMP6), and the chemokine sphingosin 1 phosphate (S1P) to promote mesenchymal cell mineralization in vitro. During bone resorption, TGF-β1 is released from the bone extracellular matrix and activated by osteoclasts. Thus, TGF-β1 levels are elevated during the resorption phase of bone turnover. We therefore investigated the influences of TGF-β1 on osteoclast-mediated support of mineralization. TGF-β1 increased osteoclast production of Wnt10b, but not BMP6 or S1P. Blocking Wnt10b activity with the Wnt signaling inhibitor Dickkoph-related protein 1 suppressed the ability of TGF-β–treated osteoclast-conditioned media to promote osteoblast mineralization. Examination of TGF-β signaling in osteoclasts revealed that induction of Wnt10b expression was dependent on Smad2/3 activation and independent from TGF-β1 stimulation of protein kinase B (AKT) or MAPK kinase. TGF-β1–treated osteoclast-conditioned media from cells with blocked Smad signaling exhibited a reduced ability to support mineralization, demonstrating the importance of Smad signaling in this response. Parallel cultures with suppressed TGF-β activation of AKT or MAPK kinase signaling retained their ability to elevate mineralization. These results demonstrate that TGF-β1 stimulates Wnt10b production in osteoclasts, which may enhance restoration of the bone lost during the resorptive phase of bone turnover.

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Local release of TGF-β during times of high bone turnover leads to elevated levels within the bone microenvironment, and we have shown that TGF-β suppresses osteoclast apoptosis. Therefore, understanding the influences of TGF-β on bone resorbing osteoclasts is critical to the design of therapies to reduce excess bone loss. Here we investigated the mechanisms by which TGF-β sustains suppression of osteoclast apoptosis. We found TGF-β rapidly increased leukemia inhibitory factor (LIF) expression and secretion by phosphorylated mothers against decapentaplegic-dependent and -independent signaling pathways. TGF-β also induced suppressor of cytokine signaling 3 (SOCS3) expression, which was required for TGF-β or LIF to promote osteoclast survival by. Blocking LIF or SOCS3 blocked TGF-β promotion of osteoclast survival, confirming that LIF and SOCS3 expression are necessary for TGF-β-mediated suppression of osteoclast apoptosis. Investigation of the mechanisms by which LIF promotes osteoclast survival revealed that LIF-induced expression of Bcl-XL and repressed Bcl-2 interacting domain expression by activating MAPK kinase, AKT, and nuclear factor-κB pathways. Suppression of Janus kinase/signal transducer and activator of transcription signaling further increased Bcl-XL expression and enhanced osteoclast survival, supporting that this pathway is not involved in prosurvival effects of TGF-β and LIF. These data show that TGF-β coordinately induces LIF and SOCS3 to promote prosurvival signaling. This alters the ratio of prosurvival Bcl2 family member Bcl-XL to proapoptotic family member Bcl-2 interacting domain, leading to prolonged osteoclast survival.

Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.

Abstract 2-Methoxyestradiol (2ME2), a physiologic metabolite of 17β-estradiol (estrogen), has emerged as a promising cancer therapy because of its potent growth-inhibitory and proapoptotic effects on both endothelial and tumor cells. 2ME2 also suppresses osteoclast differentiation and induces apoptosis of mature osteoclasts, and has been shown to effectively repress bone loss in an animal model of postmenopausal osteoporosis. Given these observations, we have examined whether 2ME2 could effectively target metastasis to bone, osteolytic tumors, and soft tissue tumors. A 4T1 murine metastatic breast cancer cell line was generated that stably expressed Far Red fluorescence protein (4T1/Red) to visualize tumor development and metastasis to bone. In an intervention study, 4T1/Red cells were injected into bone marrow of the left femur and the mammary pad. In the latter study, 2ME2 (10, 25, and 50 mg/kg/d) treatment began on the same day as surgery and was continued for the 16-day duration of study. Tumor cell growth and metastasis to bone were monitored and bone volume was determined by micro-computed tomography. 2ME2 inhibited tumor growth in soft tissue, metastasis to bone, osteolysis, and tumor growth in bone, with maximum effects at 50 mg/kg/d. Furthermore, tumor-induced osteolysis was significantly reduced in mice receiving 2ME2. In vitro, 2ME2 repressed osteoclast number by inducing apoptosis of osteoclast precursors as well as mature osteoclasts. Our data support the conclusion that 2ME2 could be an important new therapy in the arsenal to fight metastatic breast cancer. [Cancer Res 2007;67(21):10106–11]

Abstract Purpose: Although metastasis is the primary determinant of poor survival of patients with osteogenic sarcoma, some patients live much longer than others, indicating metastatic heterogeneity underlying survival outcome. The purpose of the investigation was to identify genes underlying survival outcome of patients with osteogenic sarcoma metastasis. Experimental Design: We have used microarray to first compare mRNA expression between normal bone and osteogenic sarcoma specimens, identified genes overexpressed in osteogenic sarcoma, and compared expression of the selected gene between a poorly metastatic (SAOS) and two highly metastatic cell lines (LM8 and 143B). Finally, expression of the selected gene was assessed by immunostaining of osteogenic sarcoma samples with known survival outcome. Results: Microarray analysis revealed 5.3-fold more expression of WT1 mRNA in osteogenic sarcoma compared with normal bone and >2-fold overexpression in 143B and LM8 cells compared with SAOS. Furthermore, WT1 mRNA was absent in normal bone (10 of 10) by reverse transcription-PCR but present in osteogenic sarcoma–derived cell lines (5 of 8). One hundred percent (42 of 42) of low-grade osteogenic sarcoma specimens expressed no WT1 as determined by immunostaining; however, 24% (12 of 49) of the high-grade specimens showed intense staining. Mean survival of patients with high-grade metastatic osteogenic sarcoma but low WT1 staining (27 of 37) was 96.5 ± 129.3 months, whereas mean survival of patients with high-grade metastatic osteogenic sarcoma having intense staining (10 of 37) was 18.3 ± 12.3 months (P > 0.0143). All splice variants of WT1 mRNA, including a hitherto unknown variant (lacking exons 4 and 5), were found to be expressed in osteogenic sarcoma. Conclusion: WT1 seems to be associated with very poor survival of patients with osteogenic sarcoma metastasis.

Abstract Background: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) α and β are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERα or ERβ in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERα and ERβ expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. Methods: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERα and ERβ, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERα and ERβ with smooth muscle actin, a marker of VSMCs. Results: ERβ and ERα were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERβ expression exceeded ERα expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERβ expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERα expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERα but not ERβ declined with age (P < 0.01) in HT women only. Age had no effect on ERα or ERβ expression in non-HT or premenopausal women. Conclusions: ERβ is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERβ expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.