Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Hypothalamic Regulation of Corticotropin-Releasing Factor under Stress and Stress Resilience.

This review addresses the molecular mechanisms of corticotropin-releasing factor (CRF) regulation in the hypothalamus under stress and stress resilience. CRF in the hypothalamus plays a central role in regulating the stress response. CRF stimulates adrenocorticotropic hormone (ACTH) release from the anterior pituitary. ACTH stimulates glucocorticoid secretion from the adrenal glands. Glucocorticoids are essential for stress coping, stress resilience, and homeostasis. The activated hypothalamic-pituitary-adrenal axis is suppressed by the negative feedback from glucocorticoids. Glucocorticoid-dependent repression of cAMP-stimulated Crf promoter activity is mediated by both the negative glucocorticoid response element and the serum response element. Conversely, the inducible cAMP-early repressor can suppress the stress response via inhibition of the cAMP-dependent Crf gene, as can the suppressor of cytokine signaling-3 in the hypothalamus. CRF receptor type 1 is mainly involved in a stress response, depression, anorexia, and seizure, while CRF receptor type 2 mediates "stress coping" mechanisms such as anxiolysis in the brain. Differential effects of FK506-binding immunophilins, FKBP4 and FKBP5, contribute to the efficiency of glucocorticoids under stress resilience. Together, a variety of factors contribute to stress resilience. All these factors would have the differential roles under stress resilience.

Effects of RXR Agonists on Cell Proliferation/Apoptosis and ACTH Secretion/Pomc Expression.

Various retinoid X receptor (RXR) agonists have recently been developed, and some of them have shown anti-tumor effects both in vivo and in vitro. However, there has been no report showing the effects of RXR agonists on Cushing's disease, which is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland. Therefore, we examined the effects of synthetic RXR pan-agonists HX630 and PA024 on the proliferation, apoptosis, ACTH secretion, and pro-opiomelanocortin (Pomc) gene expression of murine pituitary corticotroph tumor AtT20 cells. We demonstrated that both RXR agonists induced apoptosis dose-dependently in AtT20 cells, and inhibited their proliferation at their higher doses. Microarray analysis identified a significant gene network associated with caspase 3 induced by high dose HX630. On the other hand, HX630, but not PA024, inhibited Pomc transcription, Pomc mRNA expression, and ACTH secretion dose-dependently. Furthermore, we provide new evidence that HX630 negatively regulates the Pomc promoter activity at the transcriptional level due to the suppression of the transcription factor Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region. We also demonstrated that the HX630-mediated suppression of the Pomc gene expression was exerted via RXRα. Furthermore, HX630 inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, could be a new therapeutic candidate for Cushing's disease.

Stimulation of corticotropin-releasing factor gene expression by FosB in rat hypothalamic 4B cells.

The Fos- and Jun family proteins are immediate-early gene products, and the Fos/Jun heterodimer, activator protein-1 (AP-1), may be involved in the regulation of corticotropin-releasing factor (CRF) gene expression. FosB is a member of the Fos family proteins that is expressed in the paraventricular nucleus of the hypothalamus upon stress exposure, but it has not been clear whether FosB participates in the regulation of CRF gene expression. This study aimed to explore the effect of the FosB and cJun proteins on CRF gene expression in rat hypothalamic 4B cells. The levels of FosB mRNA and cJun mRNA increased following treatment with forskolin, phorbol-12-myristate-13-acetate (PMA), or A23187 in the hypothalamic cells. Overexpression of FosB or cJun potently increased CRF mRNA levels. Furthermore, downregulation of FosB or cJun suppressed the CRF gene expression induced by forskolin, PMA, or A23187. In addition, the basal CRF mRNA levels were partially reduced by cJun downregulation. These findings suggest that FosB, together with cJun, may mediate CRF gene expression in the hypothalamic cells.

Liver X receptor α is involved in the transcriptional regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The activity of 6-phosphofructo-1-kinase is strictly controlled by fructose-2,6-bisphosphate, the level of which is regulated by another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FBP2). PFK2/FBP2 is a bifunctional enzyme, having kinase and phosphatase activities, and regulates both glycolysis and gluconeogenesis. Here, we examined the hormonal regulation of the PFK2/FBP2 gene in vitro using the reporter assay, the electromobility shift assay (EMSA), and the chromatin immunoprecipitation (ChIP) assay in HuH7 cells and also using the mouse liver in vivo. We found that the transcriptional activity of the PFK2/FBP2 gene was stimulated by insulin and inhibited by cAMP and glucocorticoid. Liver X receptor (LXR) α showed a potent and specific stimulatory effect on PFK2/FBP2 gene transcription. Deletion and mutagenesis analyses identified the LXR response element (LXRE) in the 5'-promoter region of the PFK2/FBP2 gene. Binding of LXRα was confirmed by the EMSA and ChIP assay. Endogenous PFK2/FBP2 mRNA in the mouse liver was increased in the fasting/refeeding state compared with the fasting state. Altogether, PFK2/FBP2 gene transcription is found to be regulated in a way that is more similar to other glycolytic enzyme genes than to gluconeogenic genes. Furthermore, our data strongly suggest that LXRα is one of the key regulators of PFK2/FBP2 gene transcription.

Cytokines induce NF-κB, Nurr1 and corticotropin-releasing factor gene transcription in hypothalamic 4B cells.

OBJECTIVE: In the hypothalamus, corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. Cytokines are important mediators of the interaction between the neuroendocrine and immune systems, and are implicated in the regulation of CRF expression. Following inflammatory challenges, interleukin (IL)-1 or IL-6 stimulates the hypothalamic-pituitary-adrenal axis. CRF promoter contains multiple nuclear factor (NF)-kappaB and Nurr1 binding sites. In the present study, we determined the ability of the signaling pathways to activate the CRF gene in the hypothalamic paraventricular nucleus following inflammatory challenge. METHODS: Cytokine-induced changes in CRF gene expression were examined in the hypothalamic system. Luciferase assay and Western blotting were performed to assess transcriptional activity and the nuclear translocation of transcriptional factors. RESULTS: IL-1beta, IL-6 and tumor necrosis factor (TNF)-alpha stimulated the nuclear expression levels of NF-kappaB, NF-kappaB-dependent Nurr1 and c-Fos proteins. Direct stimulatory effects of TNF-alpha and IL-1beta, in addition to IL-6, were found on the transcriptional activity of the CRF gene in hypothalamic 4B cells. CONCLUSION: These cytokines are involved in the regulation of CRF gene activity in hypothalamic cells.

[Disorders in thyroid hormone metabolism].

The levels of the serum thyroid hormone (free T4 and free T3) are determined not only by thyroid hormone synthesis/secretion but also by their metabolism. Thyroid hormone metabolism is mediated by three selenoproteins, selenodeiodinase type 1, 2, and 3 (D1, D2, and D3), the expression and function of which are tightly regulated in a tissue-specific manner. Among them, D2 increases and D3 decreases the intracellular thyroid hormone levels, whereas D1 seems to play a role as a housekeeping/scavenger enzyme in general thyroid hormone metabolism. Although no mutation in either of the deiodinase enzyme genes has been reported, some related genes (SECISBP2, DEHAL1, and MCT8) can cause thyroid hormone-related inherited disorders. In addition, a variety of hormones, cytokines, and drugs can influence thyroid function through altered thyroid hormone metabolism.

Regulation and role of suppressor of cytokine signaling-3 in hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) plays a central role in regulating stress responses. In the hypothalamic paraventricular nucleus (PVN), CRF, produced in response to stress, stimulates the release of ACTH from the anterior pituitary. ACTH then stimulates the release of glucocorticoids from the adrenal glands; circulating glucocorticoids are critical for recovery from stress conditions. Cytokines are also implicated in the regulation of CRF expression. Among them, interleukin (IL)-6 plays a role in the regulation of CRF. Factors other than glucocorticoids are likely to be involved in limiting the stimulation of CRF during stress. Suppressor of cytokine signaling (SOCS)-3 acts as a potent negative regulator of cytokine signaling. Little is known about the ability of the inhibitory signaling pathways to limit activation of the CRF gene in parvocellular PVN neurons. Hypothalamic 4B cells are useful for exploring the mechanisms, because these cells show characteristics of the parvocellular neurons of the PVN. In the present study, we examined whether SOCS-3 is regulated by IL-6 and cAMP in hypothalamic 4B cells. We also explored the involvement of SOCS-3 in the regulation of CRF gene expression. SOCS-3 was found to be regulated by IL-6 and via the cAMP/protein kinase A pathway in the hypothalamic cells. SOCS-3 knockdown increased IL-6- or forskolin-induced CRF gene transcription and mRNA levels. Therefore, SOCS-3, induced by a cAMP stimulant and IL-6, would be involved in the negative regulation of CRF gene expression in hypothalamic cells.

CRH mRNA expression in the hypothalamic paraventricular nucleus is inhibited despite the activation of the hypothalamo-pituitary-adrenal axis during starvation.

Corticotropin-releasing hormone (CRH) is one of the anorexigenic neuropeptides, and indeed the expression of hypothalamic CRH is known to be inhibited by starvation. To clarify whether elevated plasma glucocorticoid during starvation is responsible for the CRH suppression, we examined the expression level of hypothalamic CRH mRNA after food deprivation in adrenalectomized, plasma corticosterone (B)-clamped animals. Male Wistar rats were divided into 2 groups: one group had adrenalectomy (ADX) and B pellet implantation (ADX+B, n=42), and the other group had only sham operation (sham, n=42). Rats were then treated with either ad libitum food supply or food deprivation for up to 96 h. The expression of CRH mRNA in the paraventricular nucleus (PVN) was estimated by in situ hybridization. After food deprivation, mean plasma B level was markedly elevated in sham group, but almost clamped in the ADX+B group. In this experimental condition, CRH mRNA in the PVN was significantly decreased in the sham group, whereas no change was obtained in the ADX+B group. Our data suggest the decrease in CRH mRNA seems to be related to the elevated glucocorticoid level during starvation. The status of hyperadrenocorticism without activation of CRH led us to speculate that adrenocortical function is predominant in the hypothalamic-pituitary-adrenal (HPA) axis during starvation.

Pituitary adenylate cyclase-activating polypeptide stimulates corticotropin-releasing factor, vasopressin and interleukin-6 gene transcription in hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN) in response to stress, is secreted into the pituitary portal circulation, resulting in the release of adrenocorticotropic hormone from the anterior pituitary. AVP is synthesized in the PVN and supraoptic nucleus by various stressors. Hypothalamic 4B cells coexpress CRF and AVP. In 4B cells transfected with either a CRF or an AVP promoter-luciferase construct, forskolin increased the transcriptional activity of CRF or AVP. In the present study, we tried to determine whether pituitary adenylate cyclase-activating polypeptide (PACAP) regulates both CRF and AVP genes in the hypothalamic cells, because receptors for PACAP were expressed in the hypothalamic cells. PACAP stimulated activity of both CRF and AVP promoter via protein kinase A pathway. PACAP stimulated interleukin (IL)-6 promoter activity and the levels of IL-6 mRNA and protein. IL-6 stimulated activity of both CRF and AVP promoter in a dose-dependent manner. Finally, we found that the stimulatory effects of PACAP on both activities were significantly inhibited by treatment with anti-IL-6 monoclonal antibody. These data suggest that PACAP is involved in regulating the synthesis of IL-6 mRNA and IL-6 protein, and that the increase in endogenous IL-6 also contributes to stimulate the expression of both CRF and AVP genes. Taken together, these findings indicate that PACAP stimulates the transcription of CRF, AVP, and IL-6 genes in hypothalamic 4B cells.

Transplantation of motoneuron-enriched neural cells derived from mouse embryonic stem cells improves motor function of hemiplegic mice.

Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cell-derived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an "island of the mature neuronal cells" of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.