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.

The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.

Expression of group IIA secretory phospholipase A 2 (sPLA 2 -IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA 2 -IIA is associated with neurodegeneration. However, how sPLA 2 -IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA 2 -IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA 2 activity was increased in the CTX. An sPLA 2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA 2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA 2 -IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA 2 -IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA 2 -IIA liberated arachidonic acid (AA) and generated prostaglandin D 2 (PGD 2 ), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD 2 generation. Indoxam prevented neurons from sPLA 2 -IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA 2 -IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA 2 -IIA-induced PGD 2 generation and neuronal cell death. In conclusion, sPLA 2 -IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD 2 . Furthermore, sPLA 2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA 2 -IIA inhibitors for stroke.

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G 1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21 WAF1/Cip1 . Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes.

These results demonstrate that PFD can attenuate both renal fibrosis and renal damage in this model, and suggest that PFD can be clinically useful for preventing progressive, irreversible renal failure.

Recent clinical studies have investigated postural sway characteristics in anterior cruciate ligament (ACL)-deficient knees, but the relative contributions of vision and ACL remain unclear. In the current study, we measured and compared postural sway during one-leg standing with eyes open and closed to assess the difference between legs with and without ACL injury, and we discuss the contribution of the ligament relative to vision and to postural sway in patients. We examined 32 patients (17 males, 15 females) with ACL injury before surgery from March 2001 through January 2004. None presented obvious dysfunction in the lower limbs or central nervous system. Using a gravicorder, we measured locus length per time (LG) and environmental area (AR) as the factors of postural sway during two-leg and one-leg standing with eyes open or closed. In the ACL-injured knee, the amount of postural sway increased significantly during injured leg standing with eyes closed (LG, P < 0.0001; AR, P < 0.0001), but it did not increase significantly with eyes open. There were no significant differences with respect to sex or general joint laxity. There was no correlation between postural sway and the anterior translation of the tibia measured by arthrometer KT2000 or between the muscle strength around the knee. We concluded that the amount of postural sway in the ACL-injured knee increased significantly on injured leg standing with eyes closed, and that vision appears to be dominant in compensating for the decreased contribution of the injured ACL.

No abstract

Epicardin/capsulin/Pod-1, expressed in skeletal myoblasts within brachial arches and in the condensing mesenchyme, is a member of the basic helix-loop-helix (bHLH) transcription factor family that is involved in various cell differentiation processes. In this study, we examined the functional properties of epicardin/capsulin/Pod-1 in differentiation. The yeast and mammalian two-hybrid systems showed physical associations between epicardin/capsulin/Pod-1 and E2A, both of which were present in the nuclei. The bHLH domains mediated this association. Ectopic expression of epicardin/capsulin/Pod-1 inhibited E2A-dependent activation of the exogenous and endogenous expression of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) gene, and the muscle creatine kinase gene that encodes the predominant creatine kinase isoform expressed in mammalian skeletal muscle. Transfection with epicardin/capsulin/ Pod-1 small interfering RNA abolished the epicardin/ capsulin/Pod-1-mediated suppression of E12-dependent activation of the p21 promoter. Chromatin immunoprecipitation assay showed that epicardin/capsulin/Pod-1 was physically associated with the muscle creatine kinase promoter in vivo. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of epicardin/capsulin/Pod-1. These inhibitory functions of epicardin/capsulin/Pod-1 closely resemble those of the bHLH inhibitor Twist protein. These results indicate that epicardin/capsulin/Pod-1 functions as a negative regulator of differentiation of myoblasts through transcription in at least two distinct steps, cell growth arrest and lineage-specific differentiation.