Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.     

Immunoblotting technique to study release of melanophore-stimulating hormone from individual melanotrope cells of the intermediate lobe of Xenopus laevis.

The melanotrope cells in the pars intermedia in the pituitary of Xenopus laevis synthesize and release the melanophore-stimulating hormone (alpha MSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous alpha MSH-release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit alpha MSH-release, this messenger is used to study the regulation of the heterogeneity. To quantify alpha MSH-release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small alpha MSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released alpha MSH to a nitrocellulose filter. After immunostaining, the amount of alpha MSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in alpha MSH-release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of alpha MSH-release. Dopamine treatment strongly inhibits alpha MSH-release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose-dependent as a low dopamine concentration has only a moderate effect on the alpha MSH-release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in alpha MSH-release.     

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.     

Dynamics of cyclic-AMP efflux in relation to alpha-MSH secretion from melanotrope cells of Xenopus laevis.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.     

A proteome map of the pituitary melanotrope cell activated by black‐background adaptation of Xenopus laevis

Upon transfer of Xenopus laevis from a white to a black background, the melanotrope cells in the pituitary pars intermedia secrete α‐melanocyte‐stimulating hormone, which stimulates dispersion of melanin pigment in skin melanophores. This adaptive behavior is under the control of neurotransmitters and neuropeptides of hypothalamic origin. The α‐melanocyte‐stimulating hormone‐producing cells and their hypothalamic control system provide an interesting model to study proteins required for biosynthetic and secretory processes involved in peptide hormone production and for brain–pituitary signaling. We present a 2‐D PAGE‐based proteome map of melanotrope cells from black‐adapted animals, identifying 204 different proteins by MS analysis.     

Lung specific developmental expression of the Xenopus laevis surfactant protein C and B genes

Efforts to characterize the mechanisms underlying early lung development have been confounded by the absence of a model that permits study of lung development prior to the onset of endodermal differentiation. Since Xenopus laevis development occurs in an extrauterine environment, we sought to determine whether the classical molecular markers of lung development and function, surfactant protein genes, are expressed in X. laevis. Surfactant protein C (SP-C) is a specific marker for lung development, expressed early in development and exclusively in the lung. Surfactant protein B (SP-B) expression is essential for life, as its absence results in neonatal death in mice and gene mutations have been associated with neonatal respiratory failure in humans. Here, we report the cloning of the first non-mammalian SP-C and SP-B genes (termed xSP-C and xSP-B) using the Xenopus model. The processed mature translated regions of both xSP-C and xSP-B have high homology with both human and mouse genes. xSP-C and xSP-B are both expressed throughout the lung of the X. laevis swimming tadpoles soon after the initiation of lung development as assessed by RT-PCR and whole mount in situ hybridization. The temporal expression patterns of xSP-C and xSP-B are consistent with the expression patterns in mammalian models of lung development. In both the tadpole and the adult X. laevis, xSP-C and xSP-B are expressed only in lung. Knowledge of the sequence and expression pattern of these two surfactant proteins in Xenopus might allow for use of this organism to study early lung development.     

Gene expression profiling of pituitary melanotrope cells during their physiological activation

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.     

Transgene expression of prion protein induces crinophagy in intermediate pituitary cells

The cellular form of the prion protein (PrP(C)) is a plasma membrane-anchored glycoprotein whose physiological function is poorly understood. Here we report the effect of transgene expression of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP-PrP(C)) specifically in the neuroendocrine intermediate pituitary melanotrope cells of Xenopus laevis. In the transgenic melanotrope cells, the level of the prohormone proopiomelanocortin (POMC) in the secretory pathway was reduced when the cells were (i) exposed for a relatively long time to the transgene product (by physiologically inducing transgene expression), (ii) metabolically stressed, or (iii) forced to produce unfolded POMC. Intriguingly, although the overall ultrastructure was normal, electron microscopy revealed the induction of lysosomes taking up POMC secretory granules (crinophagy) in the transgenic melanotrope cells, likely causing the reduced POMC levels. Together, our results indicate that in neuroendocrine cells transgene expression of PrP(C) affects the functioning of the secretory pathway and induces crinophagy.     

Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

Both Xenopus laevis oocytes and mammalian cells are widely used for heterologous expression of several classes of proteins, and membrane proteins especially, such as ion channels or receptors, have been extensively investigated in both cell types. A full characterization of a specific protein will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels.     

Gut specific expression using mammalian promoters in transgenic Xenopus laevis.

The recent development of transgenic methods for the frog Xenopus laevis provides the opportunity to study later developmental events, such as organogenesis, at the molecular level. Our studies have focused on the development of the tadpole gut, where tissue specific promoters have yet to be identified. We have used mammalian promoters, for the genes elastase, pancreatic duodenal homeobox-1, transthyretin, and intestinal fatty acid binding protein to drive green fluorescent protein expression in live tadpoles. All of these were shown to drive appropriate tissue specific expression, suggesting that the molecular mechanisms organising the gut are similar in amphibians and mammals. Furthermore, expression from the elastase promoter is initiated in the pancreatic buds before morphological definition becomes possible, making it a powerful tool for the study of pancreatic determination.     

Particular processing of pro-opiomelanocortin in Xenopus laevis intermediate pituitary. Sequencing of alpha- and beta-melanocyte-stimulating hormones

alpha- and beta-melanocyte-stimulating hormones (alpha-MSH and beta-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular alpha-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian alpha-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus beta-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian beta-MSHs.     

Endogenous production of nitric oxide and effects of nitric oxide and superoxide on melanotrope functioning in the pituitary pars intermedia of Xenopus laevis.

Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of beta-Ala-Lys-N(epsilon)-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO(-)(2)) or peroxynitrite (ONOO(-)) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [(3)H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

The CRF-related peptide sauvagine stimulates and the GABAB receptor agonist baclofen inhibits cyclic-AMP production in melanotrope cells of Xenopus laevis.

Release of alpha-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates alpha-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABAB receptor agonist, gave a pertussis toxin-sensitive decrease in the cyclic-AMP level and an inhibition of alpha-MSH release. We conclude that sauvagine stimulates alpha-MSH secretion through activation of adenylate cyclase and that GABAB receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of alpha-MSH. This observation supports the conclusion that GABAB receptor activation inhibits cyclic-AMP production.     

Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesis

The cellular prion protein (PrP(C)) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrP(C) functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrP(C), the newly synthesized GFP-PrP(C)K81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrP(C), the mature GFP-PrP(C)K81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrP(C) at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrP(C)DeltaGPI and GFP-PrP(C)octa mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrP(C) for its correct biosynthesis.     

Developmentally regulated expression of a mitogen-activated protein kinase in Xenopus laevis.

Mitogen-activated protein (MAP) kinases are activated in somatic cells in response to many extracellular stimuli and in oocytes during meiotic maturation. We have examined the tissue specificity of expression of a MAP kinase (Xp42) in adult and larval Xenopus laevis. MAP kinase RNA and protein were abundant in the nervous system and lymphoid tissues and were readily detected in most other organs. A remarkably high level of RNA was detected in ovary. Fractionation of oocytes showed that MAP kinase RNA is expressed at the highest level in small oocytes, suggesting that it is a maternal RNA that is stored for early embryogenesis. The levels of MAP kinase RNA and protein did not change from the time of fertilization through to late blastula. The results are consistent with functions for MAP kinases in signal transduction in embryonic as well as adult cells.     

Kinetic behavior of 30S rRNA intermediate labeled in developing embryonic cells of Xenopus laevis

In Xenopus neurula cells, "30S" RNA was found to be labeled with 3H-uridine after a relatively short labeling period. Results obtained from cumulative labeling and pulse-labeling and chase experiments with cells from late gastrulae, yolk plug-stage embryos, and neurulae showed that the 30S RNA is an intermediate in rRNA processing and is derived from 40S pre-rRNA and processed to 28S rRNA. The half-life of the 30S rRNA intermediate was about 7.5 min or less at the three stages examined.