Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs.
Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant. (+info)
Strain-specific steroidal control of pituitary function.
We have previously shown that 7B2 null mice on the 129/SvEvTac (129) genetic background die at 5 weeks of age with hypercorticosteronemia due to a Cushing's-like disease unless they are rescued by adrenalectomy; however, 7B2 nulls on the C57BL/6NTac (B6) background remain healthy, with normal steroid levels. Since background exerts such a profound influence on the phenotype of this mutation, we have evaluated whether these two different mouse strains respond differently to high circulating steroids by chronically treating wild-type 129 and B6 mice with the synthetic steroid dexamethasone (Dex). Dex treatment decreased the dopamine content of the neurointermediate lobes (NIL) of 129 mice, leading to NIL enlargement and increased total D(2)R mRNA in the 129, but not the B6, NIL. Despite the decrease in this inhibitory transmitter, Dex-treated 129 mice exhibited reduced circulating alpha-melanocyte-stimulating hormone (alpha-MSH) along with reduced POMC-derived peptides compared with controls, possibly due to reduced POMC content in the NIL. In contrast, Dex-treated B6 mice showed lowered cellular ACTH, unchanged alpha-MSH and beta-endorphin, and increased circulating alpha-MSH, most likely due to increased cleavage of NIL ACTH by increased PC2. Dex-treated 129 mice exhibited hyperinsulinemia and lowered blood glucose, whereas Dex-treated B6 mice showed slightly increased glucose levels despite their considerably increased insulin levels. Taken together, our results suggest that the endocrinological response of 129 mice to chronic Dex treatment is very different from that of B6 mice. These strain-dependent differences in steroid sensitivity must be taken into account when comparing different lines of transgenic or knockout mice. (+info)
Hes1 is required for pituitary growth and melanotrope specification.
Rathke's pouch contains progenitor cells that differentiate into the endocrine cells of the pituitary gland. It gives rise to gonadotrope, thyrotrope, somatotrope, corticotrope and lactotrope cells in the anterior lobe and the intermediate lobe melanotropes. Pituitary precursor cells express many members of the Notch signaling pathway including the downstream effector gene Hes1. We hypothesized that Hes1 regulates the timing of precursor differentiation and cell fate determination. To test this idea, we expressed Hes1 in differentiating pituitary cells and found that it can inhibit gonadotrope and thyrotrope differentiation. Pituitaries of Hes1 deficient mice have anterior lobe hypoplasia. All cells in the anterior lobe are specified and differentiate, but an early period of increased cell death and reduced proliferation causes reduced growth, evident as early as e14.5. In addition, cells within the intermediate lobe differentiate into somatotropes instead of melanotropes. Thus, the Hes1 repressor is essential for melanotrope specification. These results demonstrate that Notch signaling plays multiple roles in pituitary development, influencing precursor number, organ size, cell differentiation and ultimately cell fate. (+info)
Identification and characterization of two novel (neuro)endocrine long coiled-coil proteins.
We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway. (+info)
Disparate effects of p24alpha and p24delta on secretory protein transport and processing.
BACKGROUND: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). METHODOLOGY/PRINCIPAL FINDINGS: Here we find that transgene expression of p24alpha(3 )or p24delta(2) specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3) greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2)-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation. CONCLUSIONS/SIGNIFICANCE: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing. (+info)
Pituitary adenylate cyclase-activating polypeptide regulates brain-derived neurotrophic factor exon IV expression through the VPAC1 receptor in the amphibian melanotrope cell.