Role of PACAP in the physiology and pathology of the sympathoadrenal system.

Sympathetic neurons and chromaffin cells derive from common sympathoadrenal precursors which arise from the neural crest. Cells from this lineage migrate to their final destination and differentiate by acquiring a catecholaminergic phenotype in response to different environmental factors. It has been shown that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its PAC1 receptor are expressed at early stages of sympathetic development, and participate to the control of neuroblast proliferation and differentiation. PACAP also acts as a neurotransmitter to stimulate catecholamine and neuropeptide biosynthesis and release from sympathetic neurons and chromaffin cells, during development and in adulthood. In addition, PACAP and its receptors have been described in neuroblastoma and pheochromocytoma, and the neuropeptide regulates the differentiation and activity of sympathoadrenal-derived tumoral cell lines, suggestive of an important role in the pathophysiology of the sympathoadrenal lineage. Transcriptome studies uncovered genes and pathways of known and unknown roles that underlie the effects of PACAP in the sympathoadrenal system.

Selenoprotein T is a PACAP-regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion.

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.

Tumor necrosis factor (TNF)-alpha persistently activates nuclear factor-kappaB signaling through the type 2 TNF receptor in chromaffin cells: implications for long-term regulation of neuropeptide gene expression in inflammation.

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.

Identification of potential gene markers and insights into the pathophysiology of pheochromocytoma malignancy.

CONTEXT: Pheochromocytomas are catecholamine-producing tumors that are generally benign but that can also present as or develop into malignancy. Occurrence of malignant pheochromocytomas can only be asserted by imaging of metastatic lesions. OBJECTIVES: We conducted a gene expression profiling of benign and malignant tumors to identify a gene signature that would allow us to discriminate benign from malignant pheochromocytomas and to gain a better understanding of tumorigenic pathways associated with malignancy. DESIGN: A total of 36 patients with pheochromocytoma was studied retrospectively. There were 18 (nine benign and nine malignant) tumors used for gene expression profiling on pangenomic oligonucleotide microarrays. RESULTS: We identified and validated a set of predictor genes that could accurately distinguish the two tumor subtypes through unsupervised clustering. Most of the differentially expressed genes were down-regulated in malignant tumors, and several of these genes encoded neuroendocrine factors involved in prominent characteristics of chromaffin cell biology. In particular, the expression of two key processing enzymes of trophic peptides, peptidylglycine alpha-amidating monooxygenase and glutaminyl-peptide cyclotransferase, was reduced in malignant pheochromocytomas. CONCLUSION: The gene expression profiling of benign and malignant pheochromocytomas clearly identified a set of genes that could be used as a prognostic multi-marker and revealed that the expression of several genes encoding neuroendocrine proteins was reduced in malignant compared with benign tumors.

Maternal perinatal undernutrition alters neuronal and neuroendocrine differentiation in the rat adrenal medulla at weaning.

Epidemiological studies suggest that chronic adult diseases, such as type 2 diabetes and hypertension, can be programmed during fetal and early postnatal life. The nervous system regions governing vegetative functions and the hypothalamic-pituitary-adrenal axis are particularly sensitive to the perinatal nutritional status. Despite recent reports demonstrating that the activity of the sympathoadrenal system can be altered by early life events, the effects of maternal nutrient restriction on the adrenal medulla remain unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, immunohistochemical experiments revealed alterations in chromaffin cell aggregation and in nerve fiber fasciculation in the adrenal medulla of FR50 pups. These morphological changes were associated with enhanced circulating levels of catecholamines after decapitation (epinephrine by 55% and norepinephrine by 41%). Using macroarrays, we identified several genes whose expression was affected by maternal nutrient restriction. Semiquantitative RT-PCR confirmed the overexpression of four genes involved in neuroendocrine differentiation and neuronal plasticity (chromogranin B, growth-associated protein 43, neurofilament 3, and Slit2) in the adrenal glands of FR50 rats. Using in situ hybridization, we showed that these genes are solely expressed in the adrenal medulla. Together, our results suggest that perinatal maternal undernutrition markedly alters the differentiation of the adrenal medulla during postnatal life, resulting in enhanced activity of chromaffin cells at weaning. These alterations may persist in adulthood and participate to the programming of chronic adult diseases.

Possible implication of the transcriptional regulator Id3 in PACAP-induced pro-survival signaling during PC12 cell differentiation.

PACAP inhibits cell proliferation and promotes cell survival and neurite outgrowth of pheochromocytoma PC12 cells. Transcriptome analysis of PACAP-treated PC12 cells allowed to identify potential genes implicated in this differentiation process. Among the genes whose expression is up-regulated by PACAP, we identified the Inhibitor of DNA binding 3 (Id3). Id3 is a member of the helix-loop-helix (HLH) family of transcription factors which acts as a negative dominant inhibitor of basic HLH factors. Time-course studies revealed that Id3 is an early PACAP response gene (8-fold after 1 h of stimulation), and that the up-regulation of its expression persists over 12 h after the onset of PACAP treatment. The stimulatory effect of PACAP on Id3 mRNA levels was mimicked by adenylate cyclase/PKA activators like forskolin and dibutyryl cyclic AMP. Moreover, PACAP-induced Id3 gene expression was inhibited by phosphatidylinositol 3'-OH-kinase and p38 MAP kinase blockers. Northern blot analysis of Id3 distribution in rat tissues showed a strong expression of this gene in the adrenal medulla. Overexpression of Id3 increased the number of living PC12 cells, in basal condition and after exposure to oxidative stress. These results indicate that Id3 is a cAMP-responsive gene whose up-regulation could be involved in PACAP-induced pro-survival signaling during sympathoadrenal cell differentiation.

Microarray and suppression subtractive hybridization analyses of gene expression in pheochromocytoma cells reveal pleiotropic effects of pituitary adenylate cyclase-activating polypeptide on cell proliferation, survival, and adhesion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects on several neuronal, neuroendocrine, and endocrine cells. To gain insight into the pattern of the transcriptional modifications induced by PACAP during cell differentiation, we studied the effects of this neuropeptide on rat pheochromocytoma PC12 cells. We first analyzed the transcriptome of PC12 cells in comparison to that of terminally differentiated rat adrenomedullary chromaffin cells, using a high-density microarray, to identify genes associated with the proliferative phenotype that are possible targets of PACAP during differentiation of sympathoadrenal normal and tumoral cells. We then studied global gene expression in PC12 cells after 48 h of exposure to PACAP, using both cDNA microarray and suppression subtractive hybridization technologies. These complementary approaches resulted in the identification of 75 up-regulated and 70 down-regulated genes in PACAP-treated PC12 cells. Among the genes whose expression is modified in differentiated cells, a vast majority are involved in cell proliferation, survival, and adhesion/motility. Expression changes of most of these genes have been associated with progression of several neoplasms. A kinetic study of the effects of PACAP on some of the identified genes showed that the neuropeptide likely exerts early as well as late actions to achieve the gene expression program necessary for cell differentiation. In conclusion, the results of the present study underscore the pleiotropic role of PACAP in cell differentiation and provide important information on novel targets that could mediate the effects of this neuropeptide in normal and tumoral neuroendocrine cells.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.