Loss of Myeloid-Specific TGF-? Signaling Decreases CTHRC1 to Downregulate bFGF and the Development of H1993-Induced Osteolytic Bone Lesions.
ABSTRACT: The role of myeloid cell-specific TGF-? signaling in non-small-cell lung cancer (NSCLC)-induced osteolytic bone lesion development is unknown. We used a genetically engineered mouse model, Tgfbr2LysMCre knockout (KO), which has a loss of TGF-? signaling specifically in myeloid lineage cells, and we found that the area of H1993 cell-induced osteolytic bone lesions was decreased in Tgfbr2LysMCre KO mice, relative to the area in control littermates. The bone lesion areas were correlated with tumor cell proliferation, angiogenesis, and osteoclastogenesis in the microenvironment. The smaller bone lesion area was partially rescued by bFGF, which was expressed by osteoblasts. Interestingly, bFGF was able to rescue the osteoclastogenesis, but not the tumor cell proliferation or angiogenesis. We then focused on identifying osteoclast factors that regulate bFGF expression in osteoblasts. We found that the expression and secretion of CTHRC1 was downregulated in osteoclasts from Tgfbr2LysMCre KO mice; CTHRC1 was able to promote bFGF expression in osteoblasts, possibly through the Wnt/?-catenin pathway. Functionally, bFGF stimulated osteoclastogenesis and inhibited osteoblastogenesis, but had no effect on H1993 cell proliferation. On the other hand, CTHRC1 promoted osteoblastogenesis and H1993 cell proliferation. Together, our data show that myeloid-specific TGF-? signaling promoted osteolytic bone lesion development and bFGF expression in osteoblasts; that osteoclast-secreted CTHRC1 stimulated bFGF expression in osteoblasts in a paracrine manner; and that CTHRC1 and bFGF had different cell-specific functions that contributed to bone lesion development.
Project description:Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared with patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.
Project description:The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes, and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin (SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influenceaA osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines" from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin (OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2 (LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain. We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.
Project description:Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into severe combined immunodeficient (SCID) or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism-activation of p38 signaling in myeloma cells-by which myeloma cells induce osteolytic bone lesions, and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.
Project description:Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable and are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared with normal and benign breast tumors and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclasts and Tie2 is present in a certain population of these cells, we isolated Tie2(+) and Tie2(-) myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2(-) compared with Tie2(+) CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclasts and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 as a therapeutic target for controlling not only tumor angiogenesis but also osteolytic bone metastasis in breast cancer.
Project description:The clinical evidence indicated that cyclophosphamide (CPD), one of the chemotherapy drugs, caused severe deteriorations in bones of cancer patients. However, the exact mechanisms by which CPD exerts effects on bone remodeling is not yet fully elucidated. Therefore, this study was performed to investigate the role and potential mechanism of CPD in osteoblastogenesis and osteoclastogenesis. Here it was found that CPD treatment (100mg/kg/day) for 7 days led to osteoporosis phenotype in male mice. CPD inhibited osteoblastogenesis as shown by decreasing the number and differentiation of bone mesenchymal stem cells (MSCs) and reducing the formation and activity of osteoblasts. Moreover, CPD suppressed the osteoclastogenesis mediated by receptor activator for nuclear factor-? B ligand (RANKL) as shown by reducing the maturation and activity of osteoclasts. At the molecular level, CPD exerted inhibitory effect on the expression of components (Cyclin D1, ?-catenin, Wnt 1, Wnt10b) of Wnt/?-catenin signaling pathway in MSCs and osteoblasts-specific factors (alkaline phosphatase, Runx2, and osteocalcin). CPD also down-regulated the expression of the components (tumor necrosis factor receptor-associated factor 6, nuclear factor of activated T-cells cytoplasm 1, c-Fos and NF-?B) of RANKL signaling pathway and the factors (matrix metalloproteinase 9, cathepsin K, tartrate-resistant acid phosphates and carbonic anhydrase II) for osteoclastic activity. Taken together, this study demonstrated that the short-term treatment of CPD induced osteoporosis in mice and the underlying mechanism might be attributed to its marked suppression on osteoblastogenesis and osteoclastogenesis, especially the effect of CPD on bone formation might play a dominant role in its detrimental effects on bone remodeling.
Project description:Activation of calcineurin-dependent nuclear factor of activated T cells c1 (NFATc1) is convergent for normal bone homeostasis. NFATc1 regulates both osteoclastogenesis and osteoblastogenesis. Here we investigated the roles of regulator of calcineurin (RCAN) genes in bone homeostasis. RCANs function as potent physiological inhibitors of calcineurin. Overexpression of RCANs in osteoclast precursor cells attenuated osteoclast differentiation, while their overexpression in osteoblasts enhanced osteoblast differentiation and function. Intriguingly, opposing effects of RCANs in both cell types were shown by blocking activation of the calcineurin-NFATc1 pathway. Moreover, the disruption of RCAN1 or RCAN2 in mice resulted in reduced bone mass, which is associated with strongly increased osteoclast function and mildly reduced osteoblast function. Taken together, RCANs play critical roles in bone homeostasis by regulating both osteoclastogenesis and osteoblastogenesis, and they serve as inhibitors for calcineurin-NFATc1 signaling both in vivo and in vitro.
Project description:p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in human myeloma, has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we show that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1, or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1, it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition may offer an effective therapeutic approach to treat osteolytic bone lesions in patients with myeloma.
Project description:Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.
Project description:Osteoblasts express two key molecules for osteoclast differentiation, receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG), a soluble decoy receptor for RANKL. RANKL induces osteoclastogenesis, while OPG inhibits it by blocking the binding of RANKL to RANK, a cellular receptor of RANKL. OPG-deficient (OPG-/-) mice exhibit severe alveolar bone loss with enhanced bone resorption. WP9QY (W9) peptide binds to RANKL and blocks RANKL-induced osteoclastogenesis. W9 is also reported to stimulate bone formation in vivo. Here, we show that treatment with W9 restores alveolar bone loss in OPG-/-mice by suppressing osteoclastogenesis and enhancing osteoblastogenesis. Administration of W9 or risedronate, a bisphosphonate, to OPG-/-mice significantly decreased the osteoclast number in the alveolar bone. Interestingly, treatment with W9, but not risedronate, enhanced Wnt/?-catenin signaling and induced alveolar bone formation in OPG-/-mice. Expression of sclerostin, an inhibitor of Wnt/?-catenin signaling, was significantly lower in tibiae of OPG-/-mice than in wild-type mice. Treatment with risedronate recovered sclerostin expression in OPG-/-mice, while W9 treatment further suppressed sclerostin expression. Histomorphometric analysis confirmed that bone formation-related parameters in OPG-/-mice, such as osteoblast number, osteoblast surface and osteoid surface, were increased by W9 administration but not by risedronate administration. These results suggest that treatment of OPG-/-mice with W9 suppressed osteoclastogenesis by inhibiting RANKL signaling and enhanced osteoblastogenesis by attenuating sclerostin expression in the alveolar bone. Taken together, W9 may be a useful drug to prevent alveolar bone loss in periodontitis.