Alpha, beta, and gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus.

Two different types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors. MR exhibit the highest affinity for the endogenous glucocorticoid in the rat, corticosterone. During development, low corticosterone levels influence neurogenesis, and these effects are probably MR mediated. Three MR complementary DNA clones, alpha, beta, and gamma, have been identified in the rodent. All of these MR complementary DNA clones have identical coding regions, but differ significantly at the 5'-untranslated end. Although the functional significance of these three messenger RNA (mRNA) species remains unknown, one hypothesis is that they reflect the ability of the brain to regulate the expression of MR, allowing multiple factors to differentially control transcription in a tissue- and time-specific manner. To investigate this possibility, we examined the presence of these distinct mRNA forms in the developing rat hippocampus (HC). In situ hybridization with specific alpha, beta, and gamma complementary RNA probes was performed in the HC of 3-, 5-, 7-, 12-, 14-, 28-, 35-, and 65-day-old animals. We found that there is differential expression of these forms in each of the HC subfields from infancy to adulthood. y expression appears to be associated with periods of cell birth and increased axonal sprouting. beta expression, on the other hand, may be best linked to periods of synaptogenesis, growth of commissural and associative terminal fields, and possibly active pruning. To explore the possibility that the differential gene expression may be related to corticosterone environment, adrenalectomy was performed. A rapid modulation of the MR mRNA variants (14 h) in an age- and site-specific fashion was seen. These findings suggest that the variation in expression and regulation during development of the multiple MR transcripts could reflect a complex pattern of developmental regulation that may involve a multitude of factors unique to each postnatal age and to the different neuronal populations within the hippocampal formation.

hVH-5: a protein tyrosine phosphatase abundant in brain that inactivates mitogen-activated protein kinase.

A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen-activated protein kinase. The catalytic region of hVH-5 was expressed as a fusion protein and was shown to hydrolyze p-nitrophenylphosphate and inactivate mitogen-activated protein kinase, thus proving that hVH-5 possessed phosphatase activity. A unique proline-rich region distinguished hVH-5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH-5 from related phosphatases was that hVH-5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH-5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH-5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate-early gene, suggesting a possible role in the signal transduction cascade.

Multiple dual specificity protein tyrosine phosphatases are expressed and regulated differentially in liver cell lines.

An emerging subclass of protein-tyrosine phosphatases (PTPases) exhibits sequence identity to the vaccinia H-1 (VH-1) gene product. These VH-1-like PTPases possess the canonical HCXAGXXR(S/T) sequence common to all PTPases, but unlike other PTPases they exhibit dual catalytic activity toward phosphotyrosine and nearby phosphothreonine residues in substrate proteins. We have isolated a novel VH-1-like PTPase, hVH-3, from the human placenta and compared various aspects of its expression with previously isolated members of this subfamily. The mammalian members of this subfamily including hVH-3 commonly localize to the nucleus and exhibit catalytic activity toward phosphorylated extracellular signal-regulated kinase. However, while the expression of some VH-1-like PTPases is extremely transient and independent of protein synthesis, hVH-3 expression is sustained over 3 h after being cell stimulated. Tissue-specific expression of hVH-3 is also distinct from other VH-1-like PTPases. Although VH-1-like PTPases have overlapping substrate specificity, there are differences in their mRNA regulation, response to extracellular stimuli, and tissue-specific expression, suggesting they serve specific roles in cellular function.

Negative feedback regulation following administration of chronic exogenous corticosterone.

Administration of exogenous glucocorticoids is known to suppress the HPA axis and has been reported to occupy brain glucocorticoid receptors, eventually leading to down-regulation. To determine the effects of chronic corticosterone administration on HPA axis function, corticosterone was administered as both 25% and 50% corticosterone/cholesterol pellets. Rats were sacrificed 6 days after corticosterone pellet implantation. The 25% corticosterone pellets produced a small increase in morning corticosterone concentrations but no change in evening ACTH or corticosterone secretion. The 50% corticosterone pellets produced constant corticosterone concentrations of 5-6 micrograms/dl, with no circadian variation in corticosterone, indicating inhibition of evening ACTH and corticosterone secretion. The 25% corticosterone pellets produced no significant decrease in thymus weight or in adrenal weight; 50% corticosterone pellets produced significant decreases in thymus weight and adrenal weight. Neither 25% nor 50% corticosterone pellets produced significant decreases in GR in hippocampus and cortex. The 50% corticosterone pellets treatment resulted in a decrease in anterior pituitary POMC mRNA levels, a decrease in baseline and oCRH stimulated ACTH release from the anterior pituitary, and a near complete inhibition of the AM and PM response to restraint stress. These results suggest that: 1) the HPA axis was able to adjust to the small increase in glucocorticoids produced by the 25% cort pellets with minimal disturbances in function and 2) 50% corticosterone pellets exert a significant inhibitory effect on stress and diurnal ACTH secretion which appears to be exerted at the pituitary as well as possible inhibitory effects on brain.

Isolation and characterization of a human dual specificity protein-tyrosine phosphatase gene.

Vaccinia phosphatase VH-1 and its mammalian counterparts, including protein-tyrosine phosphatases (PTPase) CL100 and VHR, constitute a novel subfamily of protein-tyrosine phosphatases that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates. The expression of human VH-1-like PTPase CL100 is rapidly inducible by mitogen stimulation and oxidative stress, suggesting that this gene is transcriptionally regulated. In order to study the mechanism underlying this transcriptional regulation, we isolated the first human gene of this subfamily, the CL100 gene, and characterized its promoter. The gene consists of four exons intervened by three short introns 400-500 base pairs in length. Analysis of the protein sequence encoded by each exon revealed that there is a second region of similarity between CL100 protein and cdc25 in addition to the PTPase catalytic domain. Promoter analysis of the CL100 gene indicates that an 800-base pair region flanking the transcriptional initiation site is sufficient to confer a transcriptional response to serum and 12-O-tetradecanoylphorbol-13-acetate stimulation. The CL100 gene is expressed in numerous tissues, including nonmitotic cells in the brain. Within the brain, CL100 mRNA is localized in discrete neuronal populations, suggesting that this PTPase is likely to play a key role in neurotransmission as well as in mitotic signaling. Finally, although extracellular signal-regulated kinase has recently been shown to act as substrate for CL100 in vitro, we find no clear correspondence between the distribution of extracellular signal-regulated kinase and CL100 mRNA in the brain. The potential significance of a second cdc25 homology domain of CL100 is discussed.

5'-Heterogeneity of the mineralocorticoid receptor messenger ribonucleic acid: differential expression and regulation of splice variants within the rat hippocampus.

The mineralocorticoid receptor (MR) cDNA we previously isolated from the rat hippocampus differs from the clone isolated from the kidney at the 5'-untranslated (5'UT) region. The kidney clone (alpha MR mRNA) and the hippocampal clone (beta MR mRNA) possess unique 5'UT sequences of 220 and 300 nucleotides, respectively, but share an invariant peptide-coding domain and appear to encode an identical MR protein. The two mRNA variants may represent tissue-specific forms of the MR or may be coexpressed in the rat hippocampus along with other 5'UT variants. Here, we report that three mRNA subtypes were found in the hippocampus; their relative abundance was as follows: alpha = beta >> gamma. The three mRNA variants were differentially distributed within the hippocampal subfields, with the alpha form being highly enriched in CA2, dentate, the fasciculum cinereum, and the indusium griseum, whereas beta and gamma forms were evenly distributed through CA1-4. Adrenalectomy selectively increased alpha MR mRNA content, but the changes were restricted to CA1, CA2, and CA3 regions. We conclude that multiple MR mRNAs are differentially expressed in the rat hippocampus. The expression of alpha MR mRNA is specifically increased during adrenalectomy, suggesting that the increase in total MR mRNA content documented previously arises from a substantial increase in a single MR variant that elevates the total MR mRNA content, with the apparent elevation reflecting the average of regulated and unregulated transcripts. It is suggested from our data that a complex mechanism involving transcription and translation regulates MR expression in the rat hippocampus.

A novel receptor-type protein tyrosine phosphatase is expressed during neurogenesis in the olfactory neuroepithelium.

Tyrosine phosphorylation plays a central role in the control of neuronal cell development and function. Yet, few neuronal protein tyrosine phosphatases (PTPs) have been identified. We examined rat olfactory neuroepithelium for expression of novel PTPs potentially important in neuronal development and regeneration. Using the polymerase chain reaction with degenerate DNA oligomers directed to the conserved tyrosine phosphatase domain, we identified 6 novel tyrosine phosphatases. One of these, PTP NE-3, is a receptor-type PTP expressed selectively in both rat brain and olfactory neuroepithelium. In the olfactory neuroepithelium, PTP NE-3 expression is restricted to neurons and describes a novel pattern of expression with a high level in the immature neurons and a lower level in mature olfactory sensory neurons.

Corticosteroids regulate brain hippocampal 5-HT1A receptor mRNA expression.

Using in situ hybridization techniques, the expression of 5-HT1A receptor mRNA was measured within the hippocampal formation after bilateral adrenalectomy (ADX). After 24 hr ADX, 5-HT1A receptor mRNA expression was significantly increased in all hippocampal subfields in ADX animals relative to sham-operated controls (SHAM). The magnitude of the increase was most pronounced within CA2 (127%) and CA3/4 (94%)-subfields of dorsal hippocampus, intermediate in the dentate gyrus (73%), and least within CA1 (60%). Administration of exogenous corticosterone (CORT) at the time of ADX maintained the level of 5-HT1A receptor mRNA expression within the range of SHAM animals. In vitro receptor autoradiographic analysis of 5-HT1A receptors in adjacent sections from the same animals indicated a simultaneous increase in 5-HT1A binding throughout the hippocampus in response to ADX. 5-HT1A binding increased to a similar extent (approximately 30%) in CA subfields and dentate gyrus but remained within SHAM levels in CORT-replaced animals. 5-HT1A receptor mRNA levels were also increased in hippocampal subregions of 1 week ADX animals relative to SHAM animals. Within both CA1 and CA2 subfields, the increments were approximately double those observed after 1 d ADX. 5-HT1A receptor binding was increased in every hippocampal subfield to a similar extent as that observed after 1 d ADX. Increases in both 5-HT1A receptor mRNA expression and 5-HT1A receptor binding were preventable by administration of exogenous CORT at the time of ADX. Hippocampal 5-HT1C receptor mRNA and D1 receptor mRNA expression were not significantly altered by either acute or chronic ADX treatment. These data indicate that adrenal steroids may selectively regulate hippocampal 5-HT1A receptors at the level of 5-HT1A receptor mRNA expression.

Diurnal CRH mRNA rhythm in the hypothalamus: decreased expression in the evening is not dependent on endogenous glucocorticoids.

Levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA and plasma glucocorticoids vary diurnally as a result of circadian influences on the hypothalamopituitary-adrenal axis. CRH mRNA expression increases from morning to afternoon in rats but decreases rapidly near the onset of dark as glucocorticoids reach peak concentrations in plasma. Since glucocorticoids are normally inhibitory on hypothalamic CRN mRNA expression, we determined whether the glucocorticoid secretion at the diurnal peak reduced CRH mRNA concentration in the evening. We found that adrenalectomy did not prevent the decrease in CRH mRNA levels near the onset of dark. It appears that the drop in CRH mRNA expression occurs via a steroid-independent mechanism. While the mean CRH mRNA level increased after adrenalectomy, the shape of the CRH mRNA rhythm remained unchanged except in the morning. Interestingly, adrenalectomy increased CRH mRNA levels disproportionately in the morning, producing a sharp rise followed by a plateau during the light phase instead of the gradual rise observed in intact animals. We subsequently treated adrenalectomized animals with corticosterone pellets to determine whether a constant steroid signal was sufficient in restoring the normal shape of the mRNA rhythm during the light phase. Results indicate that the endogenous steroid rhythm is not necessary for generating the normal CRH mRNA rhythm during the light phase. Instead, a constant exposure to corticosterone at approximately 50% of the daily mean (2.4-3 micrograms/dl) appears to be sufficient for regulation of the mRNA rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal corticotropin-releasing hormone mRNA variation in the hypothalamus exhibits a rhythm distinct from that of plasma corticosterone.

The hypothalamopituitary-adrenal axis exhibits a diurnal rhythm as witnessed by the daily excursion of corticosterone in plasma. The rhythm appears to be mediated largely by the stimulation of CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus. In the present study, we investigated the effects of circadian influence on CRH mRNA levels in the paraventricular hypothalamus. Animals were sacrificed through a 24-hour period to establish a detailed time course of CRH mRNA fluctuations. Levels of both type I and type II corticosterone receptor mRNAs were also measured in this area to see whether changes correlate with that of CRH mRNA. Plasma levels of ACTH were quantified as an index for CRH peptide secretion. The results indicate that changes in ACTH closely paralleled alterations in corticosterone levels with an increasing trend starting at 1 PM, suggesting that the diurnal secretory drive commences around this time. The CRH mRNA rhythm as determined by RNase protection assays appeared to change in an anticipatory fashion to these endocrine fluctuations, increasing during the light phase and reaching maximal levels just prior to dark (5-6 PM). An abrupt decrease of 30% in the CRH mRNA content was detected in the hypothalamus within 2 h after dark (8 PM) and coincided with the peak of plasma corticosterone levels. However, other periodic variations in the CRH mRNA content were not accompanied by changes in plasma corticosterone. Neither types of corticosterone receptor mRNAs showed any diurnal change suggesting that the expression of steroid receptors in the hypothalamus is not regulated by circadian influences. We conclude that CRH mRNA levels fluctuate diurnally but are inversely related to corticosterone levels only in the early evening.

Changes in proopiomelanocortin primary transcript levels in the anterior pituitary accompany increased adrenocorticotropin secretion during the diurnal surge.

Proopiomelanocortin (POMC) gene transcription in the anterior pituitary varies during stress and glucocorticoid feedback. These changes appear to parallel alterations in peptide release. The diurnal rhythm of the hypothalamo-pituitary-adrenal axis also involves the periodic excursion of adrenocorticotropin (ACTH) levels in plasma, but it is not clear whether the diurnal release is accompanied by changes at the transcriptional level. In the present study, we have initially characterized the heteronuclear species of POMC (hnPOMC) RNA found in the anterior pituitary by a Northern blot analysis and subsequently used this method to quantitate relative changes in the levels of heteronuclear transcript during diurnal stimulation. Two species of RNA migrating at 6.0 kb and 4.1 kb were found in the nuclear fraction of the anterior pituitary. Successive probing by various POMC cRNAs indicated that the 6.0 kb fragment was the primary transcript and the 4.1 kb fragment corresponded to the intron A-containing processing intermediate of POMC. The nuclear species were quantitated after acute swim stress and during the diurnal ACTH secretion. Acute swim increased plasma ACTH levels by 243% after 30 min. This was paralleled by a 214% increase in the primary transcript RNA levels. Endogenous circadian stimulation in the evening produced a smaller rise of plasma ACTH (79%), and was accompanied by a 34% increase in POMC hnRNA levels. Nuclear processing intermediate (4.1 kb) and the mRNA levels did not vary during the evening. These results suggest that the diurnal mechanism transiently increases ACTH release as well as POMC gene transcription in the anterior pituitary. Release and transcription appear to be tightly coupled during circadian activation as well as during stress.

Pro-opiomelanocortin mRNA and peptide co-expression in the developing rat pituitary.

Pro-opiomelanocortin (POMC) is synthesized in both the pituitary gland and the brain. Various peptide products of this precursor, namely beta-endorphin, ACTH and alpha-MSH are co-localized in the anterior lobe corticotrophs, all intermediate lobe cells and in hypothalamic neurons. Messenger RNA (mRNA) for POMC has further been shown to exist in these tissues. In this study, we have shown that POMC mRNA, and peptide accumulation as detected by in situ hybridization and immunocytochemistry, respectively, occur simultaneously within the rat pituitary gland during ontogeny and that their maturation occurs in parallel during prenatal and early postnatal development.