Rabbit Anti-Human ATG4B monoclonal antibody, clone 2ID7MD20

Effects of 17 beta-estradiol (E2), testosterone, and 5 alpha-dihydrotestosterone (DHT) on protein turnover and proteolytic gene expression were determined in rainbow trout (Oncorhynchus mykiss) primary myocytes and white muscle tissue. E2 reduced rates of protein synthesis and increased rates of protein degradation in primary myocytes by 45% and 27%, respectively. DHT reduced rates of protein synthesis by 27%. Testosterone did not affect protein synthesis and neither testosterone nor DHT affected rates of protein degradation. Single injections of E2 increased expression of ubiquitin ligase genes fbxo32, fbxo25, and murf1, and the proteasome subunit psmd6 by 24 h after injection. Within the cathepsin-lysosome pathway, E2 increased expression of cathepsins ctsd and ctsl, as well as autophagy-related genes atg4b and lc3b. Additionally, E2 injection up-regulated the expression of casp3 and casp9 caspase genes. Incubation of primary myocytes with E2 also increased expression of ubiquitin ligase genes. Therefore, catabolic effects of E2 on protein turnover result in part from E2-induced increases in proteolytic gene expression directly in muscle. Injection of testosterone increased milli-calpain (capn2) and casp3 expression, and DHT increased ctsd expression in vivo, whereas both androgens up-regulated fbxo32 expression in primary myocytes. These results suggest that effects of androgens on protein turnover in muscle are not driven primarily by direct effects of these hormones in this tissue. Published by Elsevier Inc.

Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O-2 in vitro, and autophagy activation was also observed in EVTs at the early feto-maternal interface in vivo. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl2), which induces hypoxia inducible factor 1 alpha (HIF1 alpha) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B(C74A) cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl2 treatment through the suppression of MMP9 level, although CoCl2 did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B(C74A) cells treated with CoCl2 showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8- mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B(C74A) cells treated with CoCl2 was neutralized by ATP supplementation to the level of HTR8-ATG4B(C74A) cells treated without CoCl2. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1 alpha-mediated cellular energy consumption in EVTs.