Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signal-regulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2-induced JNK activation.

Bone morphogenetic proteins (BMPs) are known to induce ectopic bone. However, it is largely unknown how BMP signaling in osteoblasts directly regulates endogenous bone. This study investigated the mechanism by which BMP signaling through the type IA receptor (BMPR1A) regulates endogenous bone mass using an inducible Cre-loxP system. When BMPR1A in osteoblasts was conditionally disrupted during embryonic bone development, bone mass surprisingly was increased with upregulation of canonical Wnt signaling. Although levels of bone formation markers were modestly reduced, levels of resorption markers representing osteoclastogenesis were severely reduced, resulting in a net increase in bone mass. The reduction of osteoclastogenesis was primarily caused by Bmpr1a-deficiency in osteoblasts, at least through the RANKL-OPG pathway. Sclerostin (Sost) expression was downregulated by about 90% and SOST protein was undetectable in osteoblasts and osteocytes, whereas the Wnt signaling was upregulated. Treatment of Bmpr1a-deficient calvariae with sclerostin repressed the Wnt signaling and restored normal bone morphology. By gain of Smad-dependent BMPR1A signaling in mice, Sost expression was upregulated and osteoclastogenesis was increased. Finally, the Bmpr1a-deficient bone phenotype was rescued by enhancing BMPR1A signaling, with restoration of osteoclastogenesis. These findings demonstrate that BMPR1A signaling in osteoblasts restrain endogenous bone mass directly by upregulating osteoclastogenesis through the RANKL-OPG pathway, or indirectly by downregulating canonical Wnt signaling through sclerostin, a Wnt inhibitor and a bone mass mediator.

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine-and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1⌬N, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 ؊/؊ ) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 ؊/؊ ) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both CT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL-OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL-OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.

The bone morphogenetic protein (BMP) and Wnt signaling pathways both contribute essential roles in regulating bone mass. However, the molecular interactions between these pathways in osteoblasts are poorly understood. We recently reported that osteoblast-targeted conditional knockout (cKO) of BMP receptor type IA (BMPRIA) resulted in increased bone mass during embryonic development, where diminished expression of Sost as a downstream effector of BMPRIA resulted in increased Wnt/β-catenin signaling. Here, we report that Bmpr1a cKO mice exhibit increased bone mass during weanling stages, again with evidence of enhanced Wnt/β-catenin signaling as assessed by Wnt reporter TOPGAL mice and TOPFLASH luciferase. Consistent with negative regulation of the Wnt pathway by BMPRIA signaling, treatment of osteoblasts with dorsomorphin, an inhibitor of Smad-dependent BMP signaling, enhanced Wnt signaling. In addition to Sost, Wnt inhibitor Dkk1 also was downregulated in cKO bone. Expression levels of Dkk1and Sost were upregulated by BMP2 treatment and downregulated by Noggin. Moreover, expression of a constitutively active Bmpr1a transgene in mice resulted in the upregulation of both Dkk1 and Sost and partially rescued the Bmpr1a cKO bone phenotype. These effectors are differentially regulated by mitogen-activated protein kinase (MAPK) p38 because pretreatment of osteoblasts with SB202190 blocked BMP2-induced Dkk1 expression but not Sost. These results demonstrate that BMPRIA in osteoblasts negatively regulates endogenous bone mass and Wnt/β-catenin signaling and that this regulation may be mediated by the activities of Sost and Dkk1. This study highlights several interactions between BMP and Wnt signaling cascades in osteoblasts that may be amenable to therapeutic intervention for the modification of bone mass density. © 2010 American Society for Bone and Mineral Research

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-␤1 (TGF-␤1), a potent growth factor abundant in bone, the effect of LOX on TGF-␤1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-␤1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-␤1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-␤1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling. Lysyl oxidase (LOX)2 is a copper-dependent amine oxidase that initiates the process of covalent intra-and intermolecular cross-linking in collagen and elastin (1). The critical role of LOX in tissue stability is well exemplified by "lathyrism," the condition where deleterious effects in connective tissues are caused by lathyrogens such as ␤-aminopropionitrile (BAPN) (2). In lathyritic animals, bone is one of the most severely affected tissues revealing kyphoscoliosis, bone deformities, weakening of tendons and ligament attachments, dislocation of joints, impaired bone fracture healing, and ectopic bone exostoses (3, 4). BAPN is a potent and irreversible inhibitor of LOX catalytic activity and thus prevents cross-linking of immature collagen and elastin into mature, stable, and insoluble fibers. Therefore, it has been thought that the phenotypes seen in lathyritic animals are due primarily to the lack of collagen/elastin cross-linking.Recent reports, however, have revealed novel functions for LOX, including the regulation of gene transcription and cellular functions. Although the mechanisms are still not clear, those functions could be associated with its ability to oxidize substrates, other than collagen and elastin, such as basic fibroblast growth factor (5) as well as histone H1 and H2 (6, 7). Thus, lathyritic phenotypes could be due in part to the loss of LOX control of cellular functions. Indeed, several studies have reported that collagen synthesis/expression significantly increased when osteoblasts or chondrocytes were cultured in the presence of BAPN (8 -11), which is suggestive of such functions.In bone, there are several major growth factors, including transforming growth factor-␤ (TGF-␤), bone morphogenetic proteins (BMPs), insulin-like growth factor and platelet-derived growth factor, tumor necrosis factor-␣, and basic fibroblast growth factor. Among those factors, TGF-␤s and BMPs are a group of growth factors that are basic in nature (theoretical pI Ͼ 8.5) and up-regula...

Decorin (DCN) is one of the major matrix proteoglycans in bone. To investigate the role of DCN in matrix mineralization, the expression of DCN in MC3T3-E1 (MC) cell cultures and the phenotypes of MC-derived clones expressing higher (sense; S-DCN) or lower (antisense; AS-DCN) levels of DCN were characterized. DCN expression was significantly decreased as the mineralized nodules were formed and expanded in vitro. In S-DCN clones, in vitro matrix mineralization was inhibited, whereas in AS-DCN clones, mineralization was accelerated. At the microscopic level, collagen fibers in S-DCN clones were thinner while those of AS-DCN clones were thicker and lacked directionality compared to the controls. At the ultrastructural level, the collagen fibrils in S-DCN clones were markedly thinner, whereas those of AS-DCN clones were larger and irregular in shape. The results from Fourier transform infrared spectroscopy analysis demonstrated that in AS-DCN cultures the mineral content was greater but the crystallinity of mineral was poorer than that of the controls at early stage of mineralization. The in vivo transplantation assay demonstrated that no mineralized matrices were formed in S-DCN transplants, whereas they were readily detected in AS-DCN transplants at 3 weeks of transplantation. The areas of bone-like matrices in AS-DCN transplants were significantly greater than the controls at 3 weeks but became comparable at 5 weeks. The bonelike matrices in AS-DCN transplants exhibited woven bone-like non-lamellar structure while the lamellar bone-like structure was evident in the control transplants. These results suggest that DCN regulates matrix mineralization by modulating collagen assembly.

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2 ؉/-) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC-derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n ‫؍‬ 52), OPLs (n ‫؍‬ 32) and cell lines (n ‫؍‬ 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real-time quantitative RT-PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2-downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5-aza-2 -dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.