利用基于CRISPR-Cas9 的基因编辑技术对miR-29a在GT1-7 细胞中进 行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。     

Sphingolipid Metabolism and Caveolin Expression in Gonadotropin-Releasing Hormone-Expressing GN11 and Gonadotropin-Releasing Hormone-Secreting GT1-7 Neuronal Cells

In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-³H]sphingosine, GN11 and GT1-7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1-³H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1-³H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Receptor-Mediated Desensitisation of Histamine H1 Receptor-Stimulated Inositol Phosphate Production and Calcium Mobilisation in GT1-7 Neuronal Cells Is Independent of Protein Kinase C

Abstract: GT1-7 cells, a clonal line derived from specific tumours of gonadotropin-releasing hormone-secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H₁ receptor-mediated desensitisation. GT1-7 cells contain H₁ receptors, acute stimulation of which leads to the desensitisation of histamine-mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12-myristate 13-acetate, can also ablate the histamine-stimulated calcium mobilisation. In addition, acute H₁-receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine-mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31-7549 or staurosporine. In contrast, the desensitisation of H₁-receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC-dependent mechanism and PKC-independent mechanism are involved in the H₁-receptor desensitisation cascade in GT1-7 cells and do not support the involvement of PKC in the receptor-mediated desensitisation of H₁ receptor-stimulated calcium and inositol phosphate responses.     

Hypothalamic cardiovascular effects of angiotensin-(1-7) in spontaneously hypertensive rats

The objective of the present work was to study the cardiovascular actions of the intrahypothalamic injection of Ang-(1-7) and its effects on the pressor response to Ang II in spontaneously hypertensive (SH) rats and Wistar Kyoto (WKY) animals. In anaesthetized SH and WKY rats, a carotid artery was cannulated for mean arterial pressure (MAP) measurement and a stainless-steel needle was inserted into the anterior hypothalamus for drug administration. The cardiovascular effects of the intrahypothalamic administration of Ang-(1-7) were determined in SH and WKY rats. In SH rats, the effect of irbesartan and D-Ala-Ang-(1-7) on Ang-(1-7) cardiovascular effect was also evaluated. Ang II was administered in the hypothalamus of SH and WKY rats and changes in blood pressure and heart rate were measured followed by the administration of Ang II, Ang II+Ang-(1-7) or Ang II+D-Ala-Ang-(1-7). Ang-(1-7) did not the change basal MAP in WKY rats, but induced a pressor response in SH animals. Whilst the co-administration of D-Ala-Ang-(1-7) did not affect the response to Ang-(1-7), the previous administration of irbesartan prevented the effect of the peptide. The intrahypothalamic injection of Ang II induced a significantly greater pressor response in SH animals compared to normotensive rats. The co-administration of Ang-(1-7) with Ang II did not affect the pressor response to Ang II in the WKY group. In SH rats, whilst the co-administration of Ang-(1-7) with Ang II reduced the pressor response to Ang II, the concomitant application of D-Ala-Ang-(1-7) with Ang II increased the pressor response to the octapeptide after 5 and 10 min of intrahypothalamic administration. In conclusion, our result demonstrated that the biologically active peptide Ang-(1-7) did not participate in the hypothalamic blood pressure regulation of WKY animals. In SH rats, Ang-(1-7) exerted pleiotropic effects on blood pressure regulation. High dose of the heptapeptide produced a pressor response because of an unspecific action by activation of AT1 receptors. The concomitant administration of lower doses of Ang-(1-7) with Ang II reduced the pressor response to the octapeptide. Finally, the effect of AT(1-7) antagonist on Ang II pressor response suggested that hypothalamic formed Ang-(1-7) are implicated in the regulation of the cardiovascular effects of Ang II.     

Srsf1敲低对GT1-7细胞基因表达谱的影响

目的:本研究通过RNA-seq技术分析Srsf1基因表达敲低的GT1-7细胞(knock down, KD)和野生型GT1-7细胞(wide type,WT)的表达谱和可变剪接事件,研究Srsf1敲低对GT1-7细胞基因表达谱的影响。方法:利用实验室现有的Srsf1基因表达敲低的GT1-7细胞(SRSF1-KD)和野生型GT1-7细胞分别抽提RNA,做RNA-seq数据分析,利用r MATS软件分析两株细胞内可变剪接事件的变化,确定Srsf1调控的下游关键基因。结果:结果显示共有875个基因表达存在显著性差异,对这些基因进行KEGG通路分析,发现γ-氨基丁酸能突触、PI3K-Akt信号通路、MAPK信号通路等与性发育相关的通路均受到影响。此外,P53通路也受到Srsf1敲低的影响。利用r MATS软件进行可变剪接事件分析,显示共发生839个可变剪接事件,涉及719个基因,进行KEGG通路分析发现与性发育相关的MAPK信号通路受到影响。结论:成功检测了GT1-7细胞和Srsf1基因敲低的GT1-7细胞表达谱,通过分析基因表达差异以及可变剪接事件,证明了Srsf1对于PI3K-Akt信号通路、MAPK信号通路、γ-氨基丁酸能突触信号通路、p53通路的调控作用,并且这些信号通路提示我们Srsf1可能更多的通过非可变剪接方式影响性发育相关基因的表达,而非可变剪接方式,从而为更深入的性发育研究提供了理论基础。     

Protein Kinase C Regulates the Expression of M1 Receptors and BDNF in Rat Retinal Cells

Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.     

Role of metalloproteinase-dependent EGF receptor activation in alpha-adrenoceptor-stimulated MAP kinase phosphorylation in GT1-7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.     

Nexilin,a Cardiomyopathy-Associated F-Actin Binding Protein,Binds and Regulates IRS1 Signaling in Skeletal Muscle Cells

Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Previous studies in L6 skeletal muscle cells have shown that insulin-induced activation and assembly of insulin receptor substrate 1 (IRS1) and p85α the regulatory subunit of the Type 1A phosphatidylinositol-3-kinase (PI3K), within remodeled actin-rich membrane structures is critical for downstream signalling mediating the translocation of GLUT4. The mechanism for localization within actin cytoskeletal scaffolds is not known, as direct interaction of IRS1 or p85α with F-actin has not been demonstrated. Here we show that nexilin, a F-actin binding protein implicated in the pathogenesis of familial dilated cardiomyopathies, preferentially binds to IRS1 over IRS2 to influence glucose transport in skeletal muscle cells. Nexilin stably associates with IRS1 under basal conditions in L6 myotubes and this complex is disassembled by insulin. Exposure of L6 myotubes to Latrunculin B disrupts the spatial patterning of nexilin and its transient association with IRS1. Functional silencing of nexilin has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, however it enhances recruitment of p85α to IRS1 resulting in increased PI-3, 4, 5-P₃ formation, coincident with enhanced AKT activation and glucose uptake. By contrast, overexpression of nexilin inhibits transmission of IRS1 signals to AKT. Based on these findings we propose that nexilin may tether IRS1 to actin-rich structures under basal conditions, confining IRS1 signaling to specific subcellular locations in the cell. Insulin-elicited release of this constraint may enhance the efficiency of IRS1/PI3K interaction and PI-3, 4, 5-P₃ production at localized sites. Moreover, the selective binding of nexilin to IRS1 and not IRS2 may contribute to the differential specificity of IRS isoforms in the modulation of GLUT4 trafficking in skeletal muscle cells.     

Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple "ozone-specific" oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca2 + levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3beta- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5beta, 6beta-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC50 = 21 +/- 2.4 microM) than did ChEpo (IC50 = 43 +/- 3.7 microM). When tested at their IC50 concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca2 + levels were not altered by either ChSeco or ChEpo. Methyl-beta-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.     

Expression and Functional Heterogeneity of Chemokine Receptors CXCR4 and CXCR7 in Primary Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of these tumors indicates a need to identify new therapeutic targets. Therapy resistance of GBMs is attributed to heterogeneity of the glioblastoma due to genetic alterations and functional subpopulations. Chemokine receptors CXCR4 and CXCR7 play important roles in progression of various cancers although the specific functions of the CXCL12−CXCR4−CXCR7 axis in GBM are less characterized. In this study we examined the expression and function of CXCR4 and CXCR7 in four primary patient-derived GBM cell lines of the proliferative subclass, investigating their roles in in vitro growth, migration, sphere and tube formation. CXCR4 and CXCR7 cell surface expression was heterogeneous both between and within each cell line examined, which was not reflected by RT-PCR analysis. Variable percentages of CXCR4+CXCR7− (CXCR4 single positive), CXCR4−CXCR7+ (CXCR7 single positive), CXCR4+CXCR7+ (double positive), and CXCR4−CXCR7− (double negative) subpopulations were evident across the lines examined. A subpopulation of slow cell cycling cells was enriched in CXCR4 and CXCR7. CXCR4+, CXCR7+, and CXCR4+/CXCR7+ subpopulations were able to initiate intracranial tumors in vivo. CXCL12 stimulated in vitro cell growth, migration, sphere formation and tube formation in some lines and, depending on the response, the effects were mediated by either CXCR4 or CXCR7. Collectively, our results indicate a high level of heterogeneity in both the surface expression and functions of CXCR4 and CXCR7 in primary human GBM cells of the proliferative subclass. Should targeting of CXCR4 and CXCR7 provide clinical benefits to GBM patients, a personalized treatment approach should be considered given the differential expression and functions of these receptors in GBM.     

Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple “ozone-specific” oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca^{2 +} levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3β- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5β, 6β-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC₅₀ = 21 ± 2.4 μM) than did ChEpo (IC₅₀ = 43 ± 3.7 μM). When tested at their IC₅₀ concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca^{2 +} levels were not altered by either ChSeco or ChEpo. Methyl-β-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.     

Estrogen modulates potassium currents and expression of the Kv4.2 subunit in GT1-7 cells

The proper maintenance of reproduction requires the pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is ensured by synchronized periodic firing of multiple GnRH neurons. Both hormone secretion and electrophysiological properties of GnRH cells are influenced by estrogen. The impact of 17beta-estradiol treatment on the function of voltage gated A- and K-type potassium channels, known modulators of firing rate, was therefore examined in our experiments using immortalized GnRH-producing GT1-7 neurons. Whole cell patch clamp recordings showed the absence of the A-type current in GT1-7 cells cultured in estrogen-free medium and after 8h 17beta-estradiol treatment. Exposure of the cells to 17beta-estradiol for 24 and 48 h, respectively, resulted in the appearance of the A-type current. The induction of the A-type current by 17beta-estradiol was dose-related (50 pM to 15 nM range). In contrast, the K-type potassium current was apparent in the estrogen-free environment and 17beta-estradiol administration significantly decreased its amplitude. Co-administration of 17beta-estradiol and estrogen receptor blocker, Faslodex (ICI 182,780; 1 microM) abolished the occurrence of the A-type current. Real-time PCR data demonstrated that expression of the Kv4.2 subunit of the A-type channel was low at 0, 0.5, 2 and 8h, peaked at 24h and diminished at 48 h 17beta-estradiol treatment (15 nM). These data indicate that potassium channels of GT1-7 neurons are regulated by estrogen a mechanism that might contribute to modulation of firing rate and hormone secretion in GnRH neurons.