Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia

Abstract

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.Multicellular organisms maintain tissue homeostasis through response of their cells to extracellular signals that either promote their proliferation and differentiation or induce their death. Evidence is accumulating that extracellular stimuli, such as growth factors and cytokines, operate via complex signal transduction networks that ultimately control cellular fates (1); however, the complete molecular mechanisms need to be elucidated.One convenient system for studying the molecular mechanisms governing cell survival and death is spermatogenesis. It is mediated not only by cell proliferation and differentiation but also by programmed cell death or apoptosis, culminating in the production of spermatozoa. Apoptosis is necessary for eliminating unwanted cells and adjusting cell numbers in multicellular organisms to ensure tissue homeostasis.In the testis of the Japanese red-bellied newt, Cynops pyrrhogaster, primary spermatogonia proliferate through seven mitotic divisions, and then in the 8th generation the spermatogonia differentiate into primary spermatocytes and initiate meiosis in the spring when the ambient temperature is high; in contrast, in the autumn when the ambient temperature is low, the spermatogonia often undergo apoptosis in the 7th generation, resulting in the cessation of spermatogenesis (2). These findings suggest the existence of molecular mechanisms regulating the fate of 7th generation spermatogonia.We have shown in vitro and in vivo that the cellular fate in the spermatogonia is mainly regulated by changes in the endogenous levels of two peptide hormones secreted from the pituitary gland as follows: follicle-stimulating hormone (FSH) 2 th...

Loss of programmed cell death 4 induces apoptosis by promoting the translation of procaspase-3 mRNA

“…Pdcd4 protein decreased in cells treated with any apoptosis inducers, where PARP cleavage indicative of apoptosis occurred ( Figure 1a). We also observed decreased Pdcd4 protein levels during the prolactin-dependent apoptosis in newt spermatogonia 17 and during the STS-stimulated apoptosis in various cell types, such as human neuronal cells (SH-SY5Y), murine Sertoli cells (Sertoli B), 18 murine spermatogonia (GC-1), and murine myoblasts (C2C12), where PARP was cleaved and thereby apoptosis progressed ( Figure 1b). Next, the time course of Pdcd4 decrease was compared by western blotting in HeLa cells with that of procaspase-3 expression, its activation to active caspase-3, and PARP cleavage following STS treatment.…”

“…The difference between changes in the Pdcd4 mRNA and protein expression during apoptosis is consistent with the previous reports suggesting that changes in the mRNA levels are not always paralleled by concomitant changes in the protein levels. 17,27 This observation may be explained by our data implicating miR-199a-5p-operated translational repression in the apoptotic downregulation of the Pdcd4 protein expression independent of the mRNA expression: even though Pdcd4 mRNA is continuously expressed, its translation is inhibited by action of miR-199a-5p, resulting in the decrease of the protein. In addition, this observation may be supported by the previous report describing the involvement of an RNA-binding protein in the Pdcd4 mRNA stability and translation.…”

“…In addition, this observation may be supported by the previous report describing the involvement of an RNA-binding protein in the Pdcd4 mRNA stability and translation. 17 Likewise, there are conflicting results concerning Pdcd4 function in tumor suppression and apoptosis. Although the loss of Pdcd4 is correlated with tumor progression in lung cancer, 28 high expression of Pdcd4 is found in bladder and breast carcinoma; 29 overexpression of Pdcd4 induces apoptosis in human breast cancer cells 30 and inhibits proliferation in human endocrine tumor cells, 31 but does not alter cell cycle or induce apoptosis in colon carcinoma cells.…”

“…These findings suggest that Pdcd4 affects multiple mechanisms including signal transduction pathways and mediates diverse responses to various factors including genetic backgrounds, pathophysiological conditions such as tumor grade, and apoptosis inducers in different cell types, 15,25 although they are not fully clarified yet. Overall, alterations in the Pdcd4 protein expression could be governed by interactions of the RNA-binding protein, 17 microRNA molecules such as miR-199a-5p and miR-21, 16,23 and Pdcd4 mRNA, leading to divergent functions of Pdcd4.…”

The RNA-binding protein xCIRP2 is involved in apoptotic tail regression during metamorphosis in Xenopus laevis tadpoles

“…It belongs to a family of glycine-rich RNA-binding proteins consisting of RNA-binding motifs at their amino-terminal region and a glycine-rich domain at their carboxyl-terminal region. CIRP homologs were identified from human (Homo sapiens), rat (Rattus norvegicus), Mexican axolotl (Ambystoma mexicanum), bullfrog (Rana catesbeiana), African clawed frog (Xenopus laevis), and Japanese red-berried newt (Cynops pyrrhogaster) [3,6,15,22,30,33]. To date three Xenopus CIRP have been isolated: XCIRP, XCIRP-1, and xCIRP2 [10,19,30].…”