Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

Evolutionary conservation of an atypical glucocorticoid‐responsive element in the human tyrosine hydroxylase gene

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at ?7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein‐1 (AP‐1)‐like motif in the rat TH gene. We cloned this hTH‐CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH‐CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5′‐TGACTAA at ?7243 bp completely abolished the Dex‐stimulated Luc activity of hTH‐CRII construct. The AP‐1 agonist, tetradeconoyl‐12,13‐phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP‐1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid‐responsive element in a 7 bp AP‐1‐like motif in the promoter region at ?7.24 kb of the human TH gene.     

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

海马cAMP反应元件结合蛋白与抗抑郁治疗

随着对抑郁症发病机制研究的不断深入,当前的重点已从单胺递质调节机制转向抑郁症的病理生理基础研究和抗抑郁治疗的长期作用机制。以脑内cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)为交汇点的细胞内信号转导通路正受到越来越多的关注,尤其是海马CREB的改变。明确海马CERB与抗抑郁治疗的关系,对深入理解抑郁症的病理生理基础和抗抑郁治疗的长期作用机制有重要的意义。因此,本文对近年来海马CREB与抗抑郁治疗的相关研究进行综述,主要涉及CREB的结构和海马内的分布、海马CREB的上游信号通路与抗抑郁治疗、调节海马CREB发挥抗抑郁作用的可能机制。     

Relevance of c-fos proto-oncogene induction for the steroidogenic response to ACTH,dcAMP and phorbol ester in adrenocortical cells

We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells.Here we show that PMA induces c-fos with similar kinetics when compared with ACTH (0.5–1 h peak) but reaches only 60% of the maximal ACTH induction and dcAMP is a weak c-fos inducer (15% of ACTH). However, combination of PMA and dcAMP has a synergistic effect leading to maximal c-fos induction. c-fos expression may play a role in the RNA synthesis-dependent corticosteroidogenesis response and/or growth regulation by ACTH.We also show that, in contrast to dcAMP, PMA is a poor steroidogenesis stimulator (15 to 17% of maximum ACTH-stimulated level), its activity being completely dependent on RNA synthesis. Combination of dcAMP and PMA yields an additive steroidogenesis stimulation, an effect that is also dependent on RNA synthesis. Although no strict correlation was found between c-fos induction and early steroidogenesis stimulation, particularly with respect to cAMP derivatives, the results suggest that a PKC pathway is likely to cooperate with the classical cAMP-PKA pathway in adrenal cells' RNA-dependent steroidogenesis.Abbreviations ACTH Adrenocorticotropic Hormone - PMA Phorbol-12-Myrystate-13-Acetate - dcAMP dibutyryl cyclic AMP - DME Dulbecco's Modified Eagle's minimal medium - FCS Fetal Calf Serum     

BDNF heightens the sensitivity of motor neurons to excitotoxic insults through activation of TrkB

The survival promoting and neuroprotective actions of brain-derived neurotrophic factor (BDNF) are well known but under certain circumstances this growth factor can also exacerbate excitotoxic insults to neurons. Prior exploration of the receptor through which BDNF exerts this action on motor neurons deflects attention away from p75. Here we investigated the possibility that BDNF acts through the receptor tyrosine kinase, TrkB, to confer on motor neurons sensitivity to excitotoxic challenge. We blocked BDNF activation of TrkB using a dominant negative TrkB mutant or a TrkB function blocking antibody, and found that this protected motor neurons against excitotoxic insult in cultures of mixed spinal cord neurons. Addition of a function blocking antibody to BDNF to mixed spinal cord neuron cultures is also neuroprotective indicating that endogenously produced BDNF participates in vulnerability to excitotoxicity. We next examined the intracellular signaling cascades that are engaged upon TrkB activation. Previously we found that inhibition of the phosphatidylinositide-3'-kinase (PI3'K) pathway blocks BDNF-induced excitotoxic sensitivity. Here we show that expression of a constitutively active catalytic subunit of PI3'K, p110, confers excitotoxic sensitivity (ES) upon motor neurons not incubated with BDNF. Parallel studies with purified motor neurons confirm that these events are likely to be occuring specifically within motor neurons. The abrogation of BDNF's capacity to accentuate excitotoxic insults may make it a more attractive neuroprotective agent.     

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

CREB与其相关的非编码RNA在肿瘤发生发展中的作用

cAMP反应元件结合蛋白（cAMP responsive element binding protein, CREB）是亮氨酸拉链家族转录因子。新近研究发现,其在肿瘤组织中的表达显著高于癌旁,被认为是体内的原癌基因之一。非编码RNA（non-coding RNA, ncRNA）是生物体内不能翻译成蛋白质的RNA,主要包括微小RNA（microRNA, miRNA）和长链非编码RNA（long non-coding RNA, lncRNA）等,其异常表达与肿瘤的发生发展密切相关,是目前肿瘤研究的热点。研究表明,CREB与ncRNA之间存在互动效应,并且二者之间的相互作用影响肿瘤的发生发展,然而miRNA和lncRNA的作用机制却不相同。肿瘤细胞内高表达的CREB在影响下游靶基因表达时能够正调控miRNA,而对lncRNA则有促进和抑制两方面的作用。反之,肿瘤细胞中一些低表达的miRNA能促进CREB的表达;有趣的是,高表达的lncRNA能够促进CREB的表达和诱导其活性增强。在影响下游靶基因表达时miRNA仅仅发挥抑制作用,而lncRNA则分别具有促进和抑制作用。本文结合我们的系列报道和最新的研究结果,对ncRNA与CREB的互动效应及其与肿瘤的发生发展之间的关系作一综述。     

Involvement of the cAMP response element binding protein, CREB, and cyclin D1 in LPA-induced proliferation of P19 embryonic carcinoma cells

Lysophosphatidic acid (LPA) is a lipid growth factor that induces proliferation of fibroblasts by activating the cAMP response element binding protein (CREB). Here, we further investigated whether LPA induces proliferation of P19 cells, a line of pluripotent embryonic carcinoma cells. 5′-Bromo-2-deoxyuridine incorporation and cell viability assays showed that LPA stimulated proliferation of P19 cells. Immunoblot experiments with P19 cells revealed that the mitogen activated protein kinases, including p-ERK, p38, pAKT, glycogen synthase kinase 3β, and CREB were phosphorylated by treatment with 10 μM LPA. LPA-induced phosphorylation of CREB was efficiently blocked by U0126 and H89, inhibitors of the MAP kinases ERK1/2 and mitogen- and stress-activated protein kinase 1, respectively. Involvement of cyclin D1 in LPA-induced P19 cell proliferation was verified by immunoblot analysis in combination with pharmacological inhibitor treatment. Furthermore, LPA up-regulated CRE-harboring cyclin D1 promoter activity, suggesting that CREB and cyclin D1 play significant roles in LPA-induced proliferation of P19 embryonic carcinoma cells.     

Induction of the collagenase phorbol ester response element by staurosporine

The indol alkaloid staurosporine is a potent inhibitor of protein kinase C, but has also been shown to have certain effects paradoxically similar to those of protein kinase C–activating phorbol esters. We show here that collagenase mRNA expression is stimulated by 10 nM staurosporine in normal and ras-oncogene–transformed rat fibroblasts. The kinetics of collagenase mRNA induction by staurosporine were slow compared to induction by phorbol ester. Staurosporine induction of the collagenase promoter appeared to be mediated via the TPA response element (TRE). Induction did not involve any increase in jun mRNA expression and did not require expression of c-Jun. Prolonged treatment with phorbol ester to deplete protein kinase C did not inhibit stimulation of the collagenase promoter by staurosporine. Instead, involvement of cAMP-dependent protein kinase (PKA) was indicated by inhibition of staurosporine induction by the PKA inhibitor H-89. In addition, raised levels of cAMP were observed during the first hour of staurosporine treatment. Altogether, our data indicate that staurosporine induces a PKA-dependent pathway leading to c-Jun–independent activation of the collagenase TRE element. © 1994 Wiley-Liss, Inc.