Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N6-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors.

JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts.

INTRODUCTION: RANKL is known to be important not only for differentiation and activation of osteoclasts but also for their survival. Experimentally, apoptosis of osteoclasts is rapidly induced by the deprivation of RANKL. RANKL activates Elk-related tyrosine kinase (ERK), p38, c-Jun N-terminal kinase (JNK), and NF-kappaB pathways through TRAF6 in osteoclasts and the precursor cells. It has been shown that ERK is critical for regulation of osteoclast survival. However, an involvement of other RANKL signaling pathways such as JNK signaling in survival of osteoclasts has not been fully understood yet. MATERIALS AND METHODS: Osteoclasts derived from primary mouse bone marrow cells by soluble RANKL (sRANKL) were treated with a JNK inhibitor, SP600125, or infected with adenovirus carrying dominant-negative (DN)-c-jun, DN-c-fos, mitogen-activated protein kinase kinase 1 (MEKK1), I-kappaBalpha mutant, or NF-kappaB components, p50 and p65. Osteoclasts were cultured with or without sRANKL, and apoptotic phenotype was determined by TUNEL assay, DAPI staining, and expression of cleaved caspase 3 followed by TRACP staining. RESULTS: Overexpression of TRAF6 activated JNK and NF-kappaB signaling pathways and clearly prevented osteoclasts from apoptosis caused by abrogation of sRANKL. An anti-apoptotic effect of RANKL/RANK/TRAF6 signaling on osteoclast was inhibited by JNK-specific inhibitor SP600125 and by overexpression of dominant-negative JNK1, c-jun, and c-fos. Also, overexpression of MEKK1 inhibited apoptosis of osteoclasts even in the absence of sRANKL along with activation of JNK/c-jun signaling. On the other hand, blockade of NF-kappaB signaling by I-kappaBalpha mutant or overexpression of NF-kappaB components showed a marginal effect on apoptosis of osteoclasts. CONCLUSIONS: An important role of RANKL-induced activation of MEKK1/JNK/c-jun signaling in the regulation of apoptosis in osteoclasts was shown. Our study suggests that c-fos plays a role as a partner of activator protein-1 factor, c-jun, during the regulation of apoptosis in osteoclasts.