垂体腺苷酸环化酶激活肽基因的合成表达与活性研究

根据文献报道垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide, PACAP)的氨基酸序列,推导出其核苷酸序列并设计成部分互补的6条寡核苷酸片段,利用DNA合成仪人工合成、纯化这6条寡核苷酸片段,通过片段退火、连接、克隆及测序鉴定获得了PACAP基因.将PACAP基因克隆至pGEX-4T-3质粒中转化BL21进行表达分析,融合蛋白约占细胞总蛋白的30％,其中部分为可溶性,部分以包涵体形式存在.通过亲和层析纯化GST-PACAP融合蛋白,该蛋白质能促进PC12细胞轴突生长及脊髓神经元存活.     

垂体腺苷酸环化酶激活肽基因的合成表达与活性研究

根据文献报道垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide, PACAP)的氨基酸序列,推导出其核苷酸序列并设计成部分互补的6条寡核苷酸片段,利用DNA合成仪人工合成、纯化这6条寡核苷酸片段,通过片段退火、连接、克隆及测序鉴定获得了PACAP基因.将PACAP基因克隆至pGEX-4T-3质粒中转化BL21进行表达分析,融合蛋白约占细胞总蛋白的30％,其中部分为可溶性,部分以包涵体形式存在.通过亲和层析纯化GST-PACAP融合蛋白,该蛋白质能促进PC12细胞轴突生长及脊髓神经元存活.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.     

PACAP regulates immediate catecholamine release from adrenal chromaffin cells in an activity-dependent manner through a protein kinase C-dependent pathway

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic-chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential-dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP-evoked stimulation involves a phospholipase C and protein kinase C-dependant pathway to facilitate calcium influx through a Ni²⁺ and mibefradil-sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho-adrenal synapse under the stress response.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1–299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The β-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC₁-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.     

Epac1 regulates integrity of endothelial cell junctions through VE-cadherin

We have previously shown that Rap1 as well as its guanine nucleotide exchange factor Epac1 increases cell-cell junction formation. Here, we show that activation of Epac1 with the exchange protein directly activated by cAMP (Epac)-specific cAMP analog 8CPT-2'O-Me-cAMP (007) resulted in a tightening of the junctions and a decrease in the permeability of the endothelial cell monolayer. In addition, 007 treatment resulted in the breakdown of actin stress fibers and the formation of cortical actin. These effects were completely inhibited by siRNA against Epac1. In VE-cadherin knock-out cells Epac1 did not affect cell permeability, whereas in cells re-expressing VE-cadherin this effect was restored. Finally, the effect of Epac activation on the actin cytoskeleton was independent of junction formation. From these results we conclude that in human umbilical vein endothelial cells Epac1 controls VE-cadherin-mediated cell junction formation and induces reorganization of the actin cytoskeleton.     

Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.     

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well-established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.     

表皮生长因子受体(EGFR)的细胞内信号传导

表皮生长因子受体（EGFR）是一种存在于细胞表面的多功能跨膜蛋白分子,具有酪氨酸蛋白激酶活性,EGFR与配体结合后启动细胞内信号传导通路,不同的通路之间存在交叉对话（Cross-talks）共同完成细胞生理功能.对EGFR的深入研究,不仅可阐明细胞生长和发育等重要的生命过程,而且在医药和工业上也将有广泛的应用.     

Transforming growth factor-β inhibits cellular adenylate cyclase activity in cultured human arterial endothelial cells

Summary Stimulation of human arterial endothelial cells with heparin-binding growth factor-1 (HBGF-1) resulted in a 40% to 60% increase in the cellular adenylate cyclase activity and intracellular cAMP content. The stimulatory effect of HBGF-1 was effectively suppressed by pretreating the cells with transforming growth factor-β (TGF-β), an endothelial cell growth inhibitor. The inhibition of the adenylate cyclase activity precedes growth inhibition by at least 24 h. The half maximal inhibitory dose was calculated to be 0.2 ng/ml for the inhibition of both cyclase activity and cell growth. The possible role of the adenylate cyclase suppression in growth inhibition by TGF-β is discussed. This work was supported in part by grants from NCI (CA 37589), RJR Nabisco, Inc. and Kyowa Hakko Kogyo, Co., Ltd. Editor's Statement The observation that heparin-binding growth factor activates adenylate cyclase in endothelial cells and TGF beta lowers cAMP levels in endothelial cells treated with heparin-binding growth factor raises the possibility that growth control may be mediated, at least partially, through cyclic nucleotides in this system, as well as raising questions about relationships between activities of these peptide growth factors and G protein activation.     

In vivo antivascular endothelial growth factor treatment induces corneal endothelium apoptosis in rabbits through changes in p75NTR–proNGF pathway

Intravitreal injection (IVT) of antivascular endothelial growth factor (anti‐VEGF) agents is widely used for the treatment of retinal vascular diseases. Recently, the injection of anti‐VEGF agents in the ocular anterior chamber has been proposed for the treatment of neovascular glaucoma and potential side effects on the corneal structures have been investigated with contrasting results. Increasing evidence has demonstrated that VEGF inhibition is associated with cellular apoptotic changes and that this effect may be mediated by alterations in nerve growth factor (NGF) pathway. In this study, we demonstrated that anterior chamber injection (IC), but not IVT injection of two different anti‐VEGF agents, aflibercept and ranibizumab, affects rabbit corneal endothelium in terms of survival and apoptosis and is associated with changes in endothelial expression of NGF precursor (proNGF) and p75 neurotrophin receptor (p75NTR) receptor. We observed an increase in corneal endothelial cell incorporation of trypan blue and expression of cleaved‐caspase 3 (c‐Casp3), p75NTR, and RhoA after IC injection of both anti‐VEGF drugs when compared with the vehicle. Our results showed that apoptosis induction by aflibercept was more pronounced when compared with that of ranibizumab. Aflibercept also mediated a significant increase in endothelial expression of proNGF when compared with the vehicle. In line with these data, IC administration of both anti‐VEGF agents induced the activation of apoptotic signals in endothelial cells, including an increase in c‐Casp3, decrease in Bad Ser 112 phosphorylation, and unbalance of AKT phosphorylation. These results demonstrated that administration of anti‐VEGF in the anterior chamber of rabbit affects endothelial cell survival by inducing apoptosis through alteration of NGF pathway.     

Interaction of atrial natriuretic factor and endothelin-1 signals through receptor guanylate cyclase in pulmonary artery endothelial cells

The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [¹²⁵I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC₅₀ of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC₅₀ of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.     

Generation of novel monoclonal antibodies for the enrichment and characterization of human corneal endothelial cells (hCENC) necessary for the treatment of corneal endothelial blindness

Corneal transplantation is the primary treatment option to restore vision for patients with corneal endothelial blindness. Although the success rate of treatment is high, limited availability of transplant grade corneas is a major obstacle. Tissue-engineered corneal endothelial grafts constructed using cultivated human corneal endothelial cells (hCENC) isolated from cadaveric corneas may serve as a potential graft source. Currently, tools for the characterization of cultured hCENC and enrichment of hCENC from potential contaminating cells such as stromal fibroblasts are lacking. In this study, we describe the generation and characterization of novel cell surface monoclonal antibodies (mAbs) specific for hCENC. These mAbs could be used for enrichment and characterization of hCENC. Out of a total of 389 hybridomas, TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer by immunostaining of frozen tissue sections. Both mAbs were able to clearly identify hCENC with good ‘cobblestone-like’ morphology from multiple donors. The antigen targets for TAG-1A3 and TAG-2A12 were found to be CD166/ALCAM and Peroxiredoxin-6 (Prdx-6), respectively, both of which have not been previously described as markers of hCENC. Additionally, unlike other Prdx-6 mAbs, TAG-2A12 was found to specifically bind cell surface Prdx-6, which was only expressed on hCENC and not on other cell types screened such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC). From our studies, we conclude that TAG-1A3 and TAG-2A12 are promising tools to quantitatively assess hCENC quality. It is also noteworthy that the binding specificity of TAG-2A12 could be used for the enrichment of hCENC from cell mixtures of hCSF and hPSC.     

Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.