Differential regulation of corticotropin-releasing factor receptor type 1 (CRF1 receptor) mRNA via protein kinase A and mitogen-activated protein kinase pathways in rat anterior pituitary cells.

Corticotropin-releasing factor (CRF) receptor type 1 (CRF(1) receptor) mRNA levels are down-regulated by CRF via the cyclic AMP-protein kinase A (PKA) pathway. In this study, we focused on the involvement of both the mitogen-activated protein (MAP) kinase pathway and PKA in this regulation. Real-time PCR (RT-PCR) revealed that a MAP kinase, extracellular signal-regulated kinases 1/2, pathway was also involved in the down-regulation of CRF(1) receptor mRNA levels by CRF in the rat anterior pituitary (AP). Down-regulation of CRF(1) receptor mRNA levels was caused by a post-transcriptional system such as mRNA degradation, as incubation with CRF significantly decreased the half-life of CRF(1) receptor mRNA. Furthermore, pre-treatment with a PKA inhibitor completely blocked CRF-induced CRF(1) receptor mRNA destabilization, while pre-treatment with an extracellular signal-regulated kinases 1/2 inhibitor had no inhibitory effect. These results suggested that in the rat AP, down-regulation of CRF(1) receptor mRNA levels is caused by mRNA degradation via PKA, but not by the MAP kinase pathway.

Effects of corticotropin-releasing hormone (CRH) on the synthesis and secretion of proopiomelanocortin-related peptides in the anterior pituitary: a study using CRH-deficient mice.

Corticotropin-releasing hormone (CRH) mainly regulates the synthesis and secretion of adrenocorticotropin (ACTH) in the anterior pituitary (AP). By using CRH-deficient mice (CRH KO), we investigated the role of CRH in the processing of proopiomelanocortin (POMC), a precursor of ACTH, beta-lipotropic hormone, and beta-endorphin (EP). In the basal condition, the plasma ACTH level was similar in CRH KO and wild-type mice (WT), while its response to pain stress in CRH KO was smaller than that in WT. Immunoreactive (ir) beta-EP contents in the AP of CRH KO were not significantly different from those of WT. In order to determine the different molecule profile of POMC-related peptides between WT and CRH KO, ir beta-EP contents extracted from AP of WT and CRH KO were assayed by gel filtration chromatography. The gel filtration analyses revealed that a higher molecular weight form of ir beta-EP, putative POMC, was increased in CRH KO, but the beta-EP peak level was small and similar between two groups. These results suggest that CRH has little influence on the basal release of ACTH and prohormone convertase-2 processing enzyme. On the other hand, it is essential for ACTH secretion under stress conditions, and CRH would affect on the prohormone convertase-1/3 processing enzyme in AP.

Human growth hormone induces SOCS3 and CIS mRNA increase in the hypothalamic neurons of hypophysectomized rats.

The activation of the growth hormone (GH) receptor is followed by activation of the JAK2-STAT system in peripheral tissues, which in turn induces the expression of suppressors of cytokine signaling (SOCS) and/or cytokine-inducible SH2 protein (CIS) to achieve the attenuation of the signaling. To examine whether GH involves the SOCS/CIS system as intracellular negative regulators in the hypothalamus, we observed the effects of human GH on the gene expression of SOCS/CIS in the rat hypothalamus. The mRNAs of CIS, SOCS2, and SOCS3 in the hypothalamus of hypophysectomized male rats were examined by Northern analysis following the intravenous administration of recombinant human GH (hGH), 50 microg/100 g BW. The SOCS3 and CIS mRNAs were increased transiently with maximum expression at 1 h after hGH administration. The intravenous hGH did not induce SOCS2 mRNA expression in the hypothalamus. In situ hybridization demonstrated the increase of SOCS3 and CIS mRNAs in the arcuate nucleus after hGH administration, and the increase of SOCS3 mRNA in the periventricular nucleus. The hGH applied to primary cultured hypothalamic neurons at 500 ng/ml induced transient increase of SOCS3 and CIS mRNAs, but not SOCS2 mRNA. The results show that hGH acts directly on the neurons in the hypothalamus, and increases SOCS3 and CIS mRNAs, suggesting that these negative regulators may be involved in the mechanism that turns off the hGH action in the hypothalamic neurons.

Regulation of corticotropin-releasing factor (CRF) type-1 receptor gene expression by CRF in the hypothalamus.

We reported previously that acute stress and intracerebroventricular (i.c.v.) injection of corticotropin-releasing factor (CRF) increased neuronal activation and CRF type-1 receptor (CRFR-1) mRNA expression in the CRF-producing neurons of the parvocellular paraventricular nucleus (PVN) of the hypothalamus. In this study, to determine whether CRF can act directly on hypothalamic CRF neurons, thereby increasing CRFR-1 expression, microinjection of CRF into PVN neurons in vivo and primary cultures of dispersed rat fetal hypothalami in vitro were performed. Microinjection of 0.1 microg of CRF into the PVN significantly increased c-fos and CRFR-1 mRNA expression in the CRF-producing parvocellular PVN, 30 min or 180 min after injection, respectively. This effect was blocked by a CRF antagonist, alpha-helical CRF. CRF, when injected into the lateral ventricle at the same dose, increased neither CRFR-1 nor c-fos mRNA levels in the PVN. Primary culture of hypothalamic neurons revealed that CRFR-1 like immunoreactivity was located in CRF-containing neurons, and that the CRFR-1 mRNA level was significantly increased 4 h after incubation with 10(-8) M CRF. These results demonstrate that CRF directly affects hypothalamic neurons to increase CRFR-1 mRNA expression, providing evidence of a direct role for CRF in the regulation of CRFR-1 expression of hypothalamic neurons.

Regulation of corticotropin releasing hormone receptor (CRH-R) in the rat anterior pituitary as assessed by radioimmunoassay.

Since the cloning of corticotropin releasing hormone receptor type 1 (CRH-R1), an essential component of the hypothalamo-pituitary-adrenal (HPA) axis, numerous studies have been conducted to monitor its changes in transcription levels under various conditions. However, the precise dynamics at the protein levels are yet to be elucidated. In the present study we aimed at establishing an RIA system for CRH-R1 protein, with an antiserum against the C-terminal fragment of human/rat CRH-R1. The generated antiserum showed a moderate cross-reactivity with CRH-R2. We examined the in vivo effect of adrenalectomy (ADX) on immunoreactive CRH-R (irCRH-R) levels in the rat AP, and the in vitro profile of irCRH-R levels in cultured rat AP cells after administration of CRH. The irCRH-R in the AP membrane of intact rats was 51.8 +/- 6.8 fmol/mg protein, which is comparable to those reported in binding studies. ADX elicited a significant decrease of irCRH-R to approximately 50% of the control level one day after ADX, which returned to the baseline level the following day. Addition of CRH to cultured AP cells resulted in a significant decrease of irCRH-R in the membrane fraction to 18% of the control level at 4 h, and it returned rapidly to 70% at 8 h. These experiments together with our previous study implicate that irCRH-R makes a different profile, with an earlier recovery than that of mRNA. Although this system cannot precisely discriminate between CRH-R1 and CRH-R2, our findings may serve to demonstrate differing CRH receptor regulations at the synthesis level and at the protein level in the rat AP.

Microinjection of dihydrotestosterone into the medial preoptic area produces male-like pattern of growth hormone secretion in ovariectomized female rats.

The pattern of growth hormone (GH) secretion is sexually dimorphic in rats. We have previously shown that the secretory pattern in adult ovariectomized (OVX) female rats is masculinized by the administration of a single dose of dihydrotestosterone (DHT), a nonaromatizable androgen. To investigate the primary site of action of DHT in the brain, a small amount of DHT was injected directly into a defined area of the brain, and the blood GH profile was observed for 18 h in conscious adult OVX female rats. The bilateral direct injection of 1 microg DHT into the medial preoptic area (MPA) produced a male-like secretory pattern of GH in OVX rats. The masculinizing effects became apparent at 9 h after injection, from which time the episodic GH secretion was produced regularly at intervals of about 150 min, the amplitude of the peak increased and baseline levels were lowered. These parameters, analyzed during 9-18 h after DHT injection, were not different from those in adult male rats. On the contrary, microinjection of DHT into the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, or the hypothalamic arcuate-ventromedial nucleus did not affect the secretory pattern of GH. The data indicate that DHT primarily acts on cells in the MPA through androgen receptors and modulates the secretion of somatostatin and/or GH-releasing hormone secondarily to masculinize the GH secretory pattern in OVX rats.