A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.
Glucocorticoids are well known to influence the secretion of TSH from the anterior pituitary gland, although it is uncertain whether its site of action is on the hypothalamus, pituitary, or both. To determine whether glucocorticoids can modulate the concentration of pro-TRH gene expression in hypothalamic hypophysiotropic neurons, we measured the content of pro-TRH messenger RNA (mRNA) in the paraventricular nucleus (PVN) of adrenalectomized and corticosterone- and dexamethasone-treated rats compared to that in control populations using in situ hybridization histochemistry. Adrenalectomy resulted in the expected increase in corticotropin-releasing hormone mRNA in the PVN and was accompanied by a parallel rise in pro-TRH mRNA (68.3%; P < 0.05). Conversely, corticosterone and dexamethasone both resulted in profound reduction in corticotropin-releasing hormone mRNA in the PVN and a parallel reduction in pro-TRH mRNA (43.2% and 73.2% respectively; P < 0.05). No significant differences were observed in pro-TRH mRNA in the lateral hypothalamus in any of the groups. These data suggest that glucocorticoids can influence the concentration of pro-TRH mRNA in a cell-specific manner and thereby could result in changes in the biosynthesis and release of TRH in hypophysiotropic neurons of the PVN.
There are three known isoforms of the retinoid-X receptor (RXR): RXR alpha, RXR beta, and RXR gamma. RXR alpha and RXR beta messenger RNAs are widely expressed, whereas RXR gamma messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is known about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we raised isoform-specific polyclonal antibodies against each of the known mouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each antibody was confirmed by immunoprecipitation, Western immunoblot analysis, and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed that anti-RXR alpha and anti-RXR beta antisera stained the nuclei of most pituitary cells. In contrast, anti-RXR gamma antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroid state, however, a marked increase in both the number and density of RXR gamma-immunostained nuclei were observed compared to those in the euthyroid state. Double immunostaining studies of hypothyroid rat pituitary with antibodies against pituitary hormones indicated that RXR gamma protein was predominantly expressed in thyrotropes. Antibody supershift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXR gamma antibody could alter the mobility of protein-DNA complexes formed only from nuclear extracts of rat whole pituitary and thyrotropic TtT-97 cells. In contrast, anti-RXR alpha and anti-RXR beta antibodies could supershift protein-DNA complexes formed from nuclear extracts of all cell lines tested. RXR gamma protein expression in TtT-97 cells also was observed by Western immunoblot analyses. Therefore, there is thyrotrope-predominant expression of RXR gamma protein. We speculate that RXR gamma may play a role in the regulation of thyroid hormone target genes in thyrotropes and possibly cell type differentiation in the pituitary.
In the acute phase of bacterial infection, a variety of cytokines, including interleukin-1 (IL-1), are elicited by bacterial endotoxin in both the periphery and the central nervous system. Bacterial endotoxin has been previously reported to profoundly activate the hypothalamic-pituitary-adrenal axis, resulting in elevated glucocorticoid secretion that may serve an important role as part of the inhibitory feedback mechanisms on the activated immune system. To determine whether IL-1 acts within the brain to mediate endotoxin-induced CRH gene expression in the hypothalamic paraventricular nucleus (PVN), we studied the effect of administering the human IL-1 receptor antagonist (IL-1ra) into the brain, a competitive inhibitor of IL-1, on CRH gene expression in the PVN after systemic lipopolysaccharide (LPS) treatment. Eight hours after the ip administration of LPS, the paraventricular CRH mRNA content was elevated 3-to 4-fold (P < 0.01) compared to the control value, and this elevation could be completely abolished by central IL-1ra pretreatment (P < 0.05 compared to LPS-treated group; P > 0.05 compared to controls). In contrast, systemic IL-1ra administration did not inhibit endotoxin-induced CRH gene expression in the PVN. These studies demonstrate that LPS stimulates hypothalamic CRH by a mechanism that involves the action of IL-1 within the central nervous system and may proceed independently of peripheral actions of IL-1 circulating in the bloodstream.
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