SHP-2通过ERK和JNK通路调节PC12细胞应对NGF的细胞生存和凋亡

目的：探讨蛋白酪氨酸磷酸酶SHP-2在神经生长因子(NGF)作用下大鼠嗜铬细胞瘤PC12细胞生存及NGF撤除后细胞凋亡的过程中的作用及机制。方法：SHP-2 抑制剂NSC87877作用于PC12细胞, MTT测定PC12细胞存活率;流式细胞仪检测细胞凋亡率。将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体通过脂质体方法转染PC12细胞;加入NGF作用１h和撤除NGF 5 h后分别用Western blotting方法检测细胞外信号调节蛋白激酶(ERK)、磷酸化ERK（p-ERK）、c-Jun氨基末端激酶(JNK)及磷酸化JNK (p-JNK)表达变化。结果：MTT和流式细胞仪检测表明SHP-2 可以促进PC12细胞生存,抑制细胞凋亡。Western blotting结果显示无SHP-2抑制剂组和转染pIRES-SHP-2野生型组p-ERK表达在加入NGF的过程中升高;撤除NGF后,各组p-ERK表达均降低,pIRES-GFP-SHP-2C459S突变体组和pIRES-GFP组p-ERK表达明显低于pIRES-GFP-SHP-2野生型组, NGF去除+ SHP-2抑制剂组的表达水平明显低于NGF去除对照组; NGF去除+SHP-2抑制剂组p-JNK表达高于NGF去除对照组;pIRES-GFP-SHP-2C459S突变体组高于pIRES-GFP空载体组,pIRES-GFP-SHP-2野生型组低于pIRES-GFP空载体组。结论：SHP-2可能通过对ERK的正向激活,抑制JNK的激活,增强NGF作用下PC12细胞生存及抑制NGF撤除后所致细胞凋亡,从而在NGF的信号转导中起到一个中心环节的作用。     

Steroid receptors in blood vessels of the rhesus macaque endometrium: a review

Estradiol (E) and progesterone (P) act on the primate endometrium to induce dramatic changes in the vascular system during the menstrual cycle. These changes include vessel breakdown and bleeding during menses, heightened angiogenesis during the early proliferative phase, and extensive growth of the spiral arteries in the luteal phase of the cycle. Because steroid hormone action is dependent upon the presence of specific nuclear receptors in target tissues, we used immunocytochemistry with receptor-specific monoclonal antibodies to characterize the spatial and temporal expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), progesterone receptor PR and androgen (A) receptor (AR) in the endometrial vessels of rhesus macaques (Macaca mulatta). The only sex steroid receptor that was present in the endothelium and smooth muscle walls of endometrial vessels was ERbeta. ERalpha, PR, and AR were not detectable in either the endothelium or vascular smooth muscle cells of primate endometrial vessels. However, all of these receptors were strongly expressed by the perivascular stroma, and in these cells, all were modulated by the changes in levels of E and P during the cycle. We concluded that any direct effects of E on endometrial vessels would be mediated by ERbeta, and that the actions of P and A, and possibly some of E, were indirectly mediated through perivascular stromal cells.     

Expression of progesterone receptor isoforms in corpora lutea of human subjects: correlation with serum oestrogen and progesterone concentrations

To understand the biological significance of progesterone receptor forms A (PR-A) and B (PR-B) in human corpus luteum, the expression of mRNA and serum steroid hormone concentrations were determined simultaneously in the luteal stages. The expression of PR-A mRNA predominated over PR-B mRNA in all samples analysed. Total PR (PR-AB) and PR-B mRNA concentrations at the late secretory phase were significantly (P < 0.01) lower than those at the early and mid secretory phases of the menstrual cycle. The ratio of PR-B to PR-AB mRNA concentration showed no significant change during the secretory phase. In the early and mid secretory phases, there was a negative correlation between PR-B mRNA concentration and serum progesterone concentration, and between the ratio of PR-B to PR-AB mRNA concentrations and serum progesterone concentration (P < 0.01). These findings suggest that human corpus luteum might intracellularly synthesize PR-A and PR-B, and thus be involved in the steroid functional regulation of the corpus luteum, especially at the early and mid secretory phases, and that progesterone might regulate the synthesis of PR-A and PR-B.      

Involvement of P2Y13 receptor in suppression of neuronal differentiation

We examined the receptor-mediated effects of extracellular ATP on neuronal differentiation of PC12 cells, Neuro2a cells and MEB5 cells by using a series of receptor antagonists. The P2Y13 receptor antagonist MRS2211 significantly accelerated neurite outgrowth in all cases. Treatment with nerve growth factor (NGF) alone activated ERK1/2 in PC12 cells, and the activation was further increased by MRS2211. These results suggest involvement of P2Y13 receptor in suppression of neuronal differentiation. Thus, P2Y13 receptor antagonists might be candidates for treatment of neurodegenerative diseases.     

A novel organotypic culture model for normal human endometrium: regulation of epithelial cell proliferation by estradiol and medroxyprogesterone acetate

BACKGROUND: A novel organotypic culture system was established for modelling the hormonal responses of the normal human endometrium in vitro. METHODS: Endometrial epithelial cells were cultured as glandular organoids within reconstituted extracellular matrix (Matrigel) in tissue culture inserts and stromal cells on plastic below the epithelial compartment. The effects of estradiol (E2) and E2 together with medroxyprogesterone acetate (MPA) on cell proliferation and the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) were studied in 10 epithelial-stromal co-cultures and in three parallel monocultures of epithelial organoids. RESULTS: In co-cultures, E2 was shown to increase the percentage of Ki67-positive cells by approximately 2-fold relative to untreated controls. In the presence of MPA, a significant decrease in cell proliferation was detected. Similar results were obtained when the corresponding percentages of Ki67-positive organoids were calculated instead of individual cells. In the absence of stromal fibroblasts, Ki67 epithelial labelling remained below the control value after both hormonal treatments. Epithelial organoids retained their capacity to express estrogen and progesterone receptors in culture. E2 was shown to markedly increase and MPA to down-regulate the expression of PR. The expression of ERalpha was only slightly affected by either hormonal treatment. CONCLUSIONS: The present organotypic model provides a novel in vitro system in which to study the effects of steroids in the normal human endometrium both in terms of cell proliferation and gene expression. The culture system holds promise as a useful method to screen novel steroid compounds and may help to circumvent problems related to the use of animal models.     

A synthetic analog of verbenachalcone potentiates NGF-induced neurite outgrowth and enhances cell survival in neuronal cell models

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2 ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2 μM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.     

Urea enhances the nerve growth factor-induced neuroprotective effect on cholinergic neurons in organotypic rat brain slices

Cholinergic neurons degenerate in Alzheimer's disease and dementia and neuroprotective substances are of high interest to counteract this cell death. The aim of the present study was to test the effect of urea and the nitric oxide synthetase inhibitor l-thiocitrulline on the survival of cholinergic neurons. Organotypic brain slices of the basal nucleus of Meynert were cultured for 2 weeks in the presence of 1-100 microM urea with or without NGF or other growth factors or with or without 1-10 microM of the NOS inhibitor L-thiocitrulline. A high number of cholinergic neurons survived in the presence of 0.1-100 ng/ml NGF. Urea or L-thiocitrulline alone did not exhibit neuroprotective activity; however, when brain slices were incubated with urea or L-thiocitrulline together with NGF there was a significant potentiating survival effect. Incubation of brain slices with NGF + urea + L-thiocitrulline did not further enhance the number of cholinergic neurons. NGF as well as urea did not stimulate expression of the enzyme choline acetyltransferase pointing to survival promoting effects. Urea did not modulate the NGF binding in PC12 cells indicating that this effect was indirect. It is concluded that urea may play a role as an indirect survival promoting molecule possibly involving the nitric oxide pathway.     

Enhanced gene delivery to PC12 cells by a cationic polypeptide

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.     

Term myometrium is characterized by increased activating epigenetic modifications at the progesterone receptor-A promoter

Term human myometrial expression of progesterone receptor (PR)-A is increased relative to PR-B, and as PR-A is a repressor of progesterone action mediated through PR-B, this increase may mediate the withdrawal of progesterone action and precipitate the onset of labour. PR-A and PR-B expression is regulated by two separate promoters of the PR gene. We hypothesized that epigenetic histone modifications at the two promoters contribute to the labour-associated regulation of PR-A and PR-B expression in term myometrium. PR total, PR-B and PR-A mRNA levels were determined using quantitative real-time PCR, and chromatin immunoprecipitation was used to determine the levels of activating and repressive histone modifications at the PR-A and PR-B promoters in human myometrial samples not in labour (n = 4) and in labour (n = 4). Chromatin extracts were immunoprecipitated with antibodies against activating (histone H3 and H4 acetylation and histone H3 lysine 4 trimethylation), and repressive (histone H3 lysine 9 trimethylation, histone H3 lysine 27 trimethylation and asymmetrical histone H3 arginine 2 dimethylation) histone modifications. PR-A mRNA levels increased during labour, while PR-B mRNA levels remained constant resulting in an increase of PR-A/PR-B mRNA ratio, as expected. Regardless of labour status, significantly higher levels of the activating histone modifications were found at the PR-A promoter compared with the PR-B promoter (P <0.001). H3K4me3 increased significantly at both promoters with labour onset (P =0.001). Low levels of the repressive histone modifications were also present at both promoters, with no labour-associated changes observed. Our data indicate that the PR-A promoter is epigenetically marked for activation in term myometrium more extensively than the PR-B promoter, and that labour is associated with an increase in H3K4me3 activating modification, consistent with the previously described increase in PR protein at this time.     

Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor P75

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within 10 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-phosphate (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) 1/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of sphingosine-kinase blocked the neuroprotective effect of NGF, suggesting that sphingosine-1-phosphate was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.     

Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells

It is well known that the smooth muscle of the human myometrium is a target for the steroid hormones progesterone (P4) and estrogen. Progesterone is believed to participate in the maintenance of pregnancy, while estrogen is possibly involved in the process of parturition by promoting cervical dilatation. We examined the combined effects of P4 and 17beta-estradiol (E2) on components of signalling pathways in human myometrial cells in vitro by immunoblotting. Long-term treatment of myometrial cells with a series of concentrations of P4 and E2 in combination caused a change in the phosphorylation status of p42/44 mitogen-activated protein kinase and of c-Jun N-terminal kinase (SAPK/JNK). P4 and E2 caused a decrease in protein expression of Gqalpha, Gzalpha, Gi1/2alpha and, to a lesser extent, G0alpha. The two steroids caused a decrease in the expression of the two small GSalpha isoforms. Cyclo-oxygenase-2 expression was increased by 2.5-fold after steroid treatment, while proliferating cell nuclear antigen expression levels remained unchanged. These observations show that the combination of P4 and E2 influences intracellular and membrane-bound components of signal transduction pathways in human myometrial cells. The implications of the two steroid hormones on intracellular signalling pathways in the human myometrium merits further investigation.     

Sphingomyelinase but not ceramide induces nitric oxide synthase expression in rat brain microglia

We have asked whether treatment of PC12 cells with cyclic adenosine monophosphate (cAMP) and epidermal growth factor (EGF) results, like treatment with cAMP and nerve growth factor (NGF), in irreversible neuronal differentiation characterized by irreversible neurite extension, loss of serum-dependence, and death by apoptosis after trophic factor withdrawal. Although EGF alone, unlike NGF, did not cause morphological differentiation or prevent cell death, synergy between a cAMP-mediated signal transduction pathway and a pathway activated by the EGF receptor tyrosine kinase resulted in the same irreversible differentiation. EGF/cAMP-differentiated cells required cAMP to survive, but NGF, through a TrkA-dependent mechanism, could substitute for cAMP. The cyclin-dependent kinase inhibitors olomoucine and roscovitine also promoted survival of the irreversibly differentiated cells, by a mechanism that must be determined, since cell death was not associated with nuclear (3)H-thymidine accumulation, an index of mitotic activity.     

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.     

Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal

In humans, progesterone levels are sustained before the onset of labour. Therefore, the mechanism for parturition that has been proposed for humans is 'functional' progesterone withdrawal. Immunohistochemical staining for the progesterone receptor (PR) was positive in the decidua with a decline after contractions began. Western blot analysis revealed a number of PR isoforms expressed in the decidua, with the PR-B form being dominant. After contractions began, all PR isoforms decreased sharply. PR-B and PR-A decreased by 85.8% +/- 6.7 and 78.2% +/- 7.1, respectively (P < 0.001). Incubation of decidua with Prostaglandin F2alpha 1.0 microg/ml decreased the expression of all forms of PR isoforms. PR-B was reduced by 64% +/- 6.09 (P < 0.01); PR-A was reduced by 77% +/- 5.9 (P < 0.05), while PR-C was reduced by 80% +/- 7.24 (P < 0.05). Progesterone (80 microg/ml) increased the PR-B, PR-C the 45 and 36 kDa isoforms to 150% +/- 7.89, 210% +/- 12.4, 270% +/- 9.7 and 216% +/- 13.5, respectively (P < 0.05). In immunohistochemical studies, the PR was not identified in the amnion or in the chorion, regardless of the presence or absence of contractions. Western blot analysis demonstrated that PR-C (60 kDa) and the 36 kDa isoforms were dominant in the amnion. After contractions began, PR-A decreased significantly by 61.9% +/- 7.1 (P < 0.001).     

ErbB-4 activation inhibits apoptosis in PC12 cells.

Neuregulins, a large family of polypeptide growth factors, exert various distinctive effects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced differentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)alpha treatment. The neuregulin-induced survival is comparable to the effect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNF alpha treatment. Moreover, neuregulin like NGF induces the survival of neuronal differentiated PC12-ErbB-4 cells. The survival effect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulin-induced survival effect.Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks.