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.