Selenoproteins contain the essential trace element selenium, the deficiency of which is associated with cancer or accelerated aging. Although selenoproteins are thought to be instrumental for the effects of selenium, the biological function of many of these proteins remains unknown. Here, we studied the role of selenoprotein T (SelT), a selenocysteine (Sec) -containing protein with no known function, which we have identified as a novel target gene of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) during PC12 cell differentiation. SelT was found to be ubiquitously expressed throughout embryonic development and in adulthood in rat. Immunocytochemical analysis revealed that SelT is mainly localized to the endoplasmic reticulum through a hydrophobic domain. PACAP and cAMP induced a rapid and long-lasting increase in SelT gene expression in PC12 cells, in a Ca(2+)-dependent manner. These results suggested a possible role of SelT in PACAP signaling during PC12 cell differentiation. Indeed, overexpression of SelT in PC12 cells provoked an increase in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) that was dependent on the Sec residue. Conversely, SelT gene knockdown inhibited the PACAP-induced increase in [Ca(2+)](i) and reduced hormone secretion. These findings demonstrate the implication of a selenoprotein in the regulation of Ca(2+) homeostasis and neuroendocrine secretion in response to a cAMP-stimulating trophic factor.

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.

Selenoproteins contain the essential trace element selenium whose deficiency leads to major disorders including cancer, male reproductive system failure, or autoimmune thyroid disease. Up to now, 25 selenoprotein-encoding genes were identified in mammals, but the spatiotemporal distribution, regulation, and function of some of these selenium-containing proteins remain poorly documented. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein, is regulated by the trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) in differentiating but not mature adrenomedullary cells. In fact, our analysis revealed that, in rat, SelT is highly expressed in most embryonic structures, and then its levels decreased progressively as these organs develop, to vanish in most adult tissues. In the brain, SelT was abundantly expressed in neural progenitors in various regions such as the cortex and cerebellum but was undetectable in adult nervous cells except rostral migratory-stream astrocytes and Bergmann cells. In contrast, SelT expression was maintained in several adult endocrine tissues such as pituitary, thyroid, or testis. In the pituitary gland, SelT was found in secretory cells of the anterior lobe, whereas in the testis, the selenoprotein was present only in spermatogenic and Leydig cells. Finally, we found that SelT expression is strongly stimulated in liver cells during the regenerative process that occurs after partial hepatectomy. Taken together, these data show that SelT induction is associated with ontogenesis, tissue maturation, and regenerative mechanisms, indicating that this PACAP-regulated selenoprotein may play a crucial role in cell growth and activity in nervous, endocrine, and metabolic tissues.

Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic β- and δ-cells and is involved in the control of glucose homeostasis.

Systemic delivery of self-complementary (sc) adeno-associated-virus vector of serotype 9 (AAV9) was recently shown to provide robust and widespread gene transfer to the central nervous system (CNS), opening new avenues for practical, and non-invasive gene therapy of neurological diseases. More recently, AAV of serotype rh10 (AAVrh10) was also found highly efficient to mediate CNS transduction after intravenous administration in mice. However, only a few studies compared AAV9 and AAVrh10 efficiencies, particularly in the spinal cord. In this study, we compared the transduction capabilities of AAV9 and AAVrh10 in the brain, the spinal cord, and the peripheral nervous system (PNS) after intravenous delivery in neonatal mice. As reported in previous studies, AAVrh10 achieved either similar or higher transduction than AAV9 in all the examined brain regions. The superiority of AAVrh10 over AAV9 appeared statistically significant only in the medulla and the cerebellum, but a clear trend was also observed in other structures like the hippocampus or the cortex. In contrast to previous studies, we found that AAVrh10 was more efficient than AAV9 for transduction of the dorsal spinal cord and the lower motor neurons (MNs). However, differences between the two serotypes appeared mainly significant at low dose, and surprisingly, increasing the dose did not improve AAVrh10 distribution in the spinal cord, in contrary to AAV9. Similar dose-related differences between transduction efficiency of the two serotypes were also observed in the sciatic nerve. These findings suggest differences in the transduction mechanisms of these two serotypes, which both hold great promise for gene therapy of neurological diseases.

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N-and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N-and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the constitutive secretory pathway. The fundamental feature of neuroendocrine and endocrine cells is the occurrence of dense-core secretory granules (DCGs), 3 which are key cytoplasmic organelles responsible for secretion of hormones, neuropeptides, and neurotransmitters through the regulated secretory pathway (RSP). Storage at high concentrations of these secretory products is required for their finely tuned release in response to extracellular stimulation (1, 2). DCG biogenesis starts with the budding of immature secretory granules (ISGs) from the trans-Golgi network (TGN) through interactions between lipid rafts and protein components, in a similar manner to constitutive vesicle budding (2, 3). The ISG budding is followed by a multistep maturation process to form the mature secretory granules, including removal of the constitutive secretory proteins and lysosomal enzymes inadvertently packaged into ISGs (4).Despite increasing knowledge of the various steps of DCG formation, the nature of the sorting signals for entry of proteins into the DCGs and the molecular machinery required to generate secretory granules are not fully elucidated (5, 6). Several recent studies highlighted the role of members of the granin family, which may represent the driving force for granulogenesis in the TGN (2), although this notion has been a matter of debate (7). Granins are soluble acidic proteins widely distributed in endocrine and neuroendocrine cells, which are characterized by the ability to aggregate at acidic pH and a high Ca 2ϩ environment (8, 9). These conditions are found in the lum...

Chromaffin cells of the adrenal medulla elaborate and secrete catecholamines and neuropeptides for hormonal and paracrine signaling in stress and during inflammation. We have recently documented the action of the cytokine TNF-alpha on neuropeptide secretion and biosynthesis in isolated bovine chromaffin cells. Here, we demonstrate that the type 2 TNF-alpha receptor (TNF-R2) mediates TNF-alpha signaling in chromaffin cells via activation of nuclear factor (NF)-kappaB. Microarray and suppression subtractive hybridization have been used to identify TNF-alpha target genes in addition to those encoding the neuropeptides galanin, vasoactive intestinal polypeptide, and secretogranin II in chromaffin cells. TNF-alpha, acting through the TNF-R2, causes an early up-regulation of NF-kappaB, long-lasting induction of the NF-kappaB target gene inhibitor kappaB (IkappaB), and persistent stimulation of other NF-kappaB-associated genes including mitogen-inducible gene-6 (MIG-6), which acts as an IkappaB signaling antagonist, and butyrate-induced transcript 1. Consistent with long-term activation of the NF-kappaB signaling pathway, delayed induction of neuropeptide gene transcription by TNF-alpha in chromaffin cells is blocked by an antagonist of NF-kappaB signaling. TNF-alpha-dependent signaling in neuroendocrine cells thus leads to a unique, persistent mode of NF-kappaB activation that features long-lasting transcription of both IkappaB and MIG-6, which may play a role in the long-lasting effects of TNF-alpha in regulating neuropeptide output from the adrenal, a potentially important feedback station for modulating long-term cytokine effects in inflammation.

Pheochromocytomas are catecholamine-producing tumors which are generally benign, but which can also present as or develop into malignancy. Molecular pathways of malignant transformation remain poorly understood. Pheochromocytomas express various trophic peptides which may influence tumoral cell behavior. Here, we investigated the expression of trophic amidated peptides, including pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), and adrenomedullin (AM), and their receptors in benign and malignant pheochromocytomas in order to assess their potential role in chromaffin cell tumorigenesis and malignant transformation. PACAP, NPY, and AM are expressed in the majority of pheochromocytomas studied; NPY exhibiting the highest mRNA levels relative to reference genes. Although median gene expression or peptide levels were systematically lower in malignant compared to benign tumors, no statistically significant difference was found. Among all the receptors of these peptides that were analyzed, only the AM receptor RDC1 displayed a differential expression between benign and malignant pheochromocytomas. This receptor exhibited a fourfold higher expression in malignant than in benign tumors. AM and stromal cell-derived factor 1, which has also been described as a ligand for RDC1, increased the number of human pheochromocytoma cells in primary culture and exerted anti-apoptotic activity on rat pheochromocytoma PC12 cells. In addition, RDC1 gene silencing decreased the number of viable PC12 cells. This study shows the expression of several trophic peptides and their receptors in benign and malignant pheochromocytomas, and suggests that AM and its RDC1 receptor could be involved in chromaffin cell tumorigenesis through pro-survival effects. Therefore, AM and RDC1 may represent valuable targets for the treatment of malignant pheochromocytomas.