白血病相关的SHP2过表达对小鼠Lewis肺癌细胞增殖和侵袭及对MAPK信号通路的影响

目的研究超声造影剂介导SHP2（Src homology phosphatase 2）酪氨酸磷酸酶真核表达（pcDNA3.1SHP2）载体在小鼠Lewis肺癌（Lewis lung carcinoma,LLC）细胞中的转染,观察SHP2过表达对Lewis肺癌细胞增殖和侵袭的影响及分子机制初探。方法分别于接种小鼠Lewis肺癌细胞的6孔板中每孔加入200μL造影剂和5μL脂质体,以诊断超声剂量辐照60 s,细胞转染48 h后,Western-blot检测细胞转染前后SHP2及P38蛋白表达量的变化;MTT、划痕实验和Transwell系统分别检测细胞增殖、迁移和侵袭能力。结果 Western blotting显示转染组与未转染组比较,转染SHP2真核表达载体后,LLC中SHP2和磷酸化P38表达明显上调;LLC细胞增殖、迁移能力和侵袭能力均显著增强。结论 SHP2过表达可提高肺癌肿瘤细胞的增殖、迁移和侵袭能力;并初步证实SHP2过表达可能通过激活丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）信号转导系统促进肿瘤细胞的恶性进展。     

蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用     

蛋白酪氨酸磷酸酶SHP2在结直肠“炎-癌”中的作用

SHP2(SH2 domain-containing protein-tyrosine phosphatase-2)为蛋白酪氨酸磷酸酶,由人PTPN11基因编码,在生长因子、细胞因子的作用下激活ERK/MAPK通路。同时在不同的细胞类型中,SHP2也能够抑制JAK/STAT3通路的激活。SHP2通过调控这两条通路,发挥了不同甚至相反的角色,例如SHP2在乳腺癌、肺癌、头颈部肿瘤中发挥促癌功能,然而在肝癌中抑制了癌症的发生。本文针对SHP2在结直肠中的角色,综述了SHP2在结直肠上皮细胞分化、肠上皮细胞稳态、肠炎、结直肠癌中的功能,以及相关抗肿瘤药物及细胞因子对结直肠癌细胞中SHP2的调控作用。     

蛋白酪氨酸磷酸酶SHP2与呼吸系统疾病

蛋白酪氨酸磷酸酶SHP2是一种在人类细胞中广泛表达的含有SH2结构域的蛋白磷酸酶,与细胞增殖分化、死亡调控、免疫调节及肿瘤发生等密切相关。SHP2在一些细胞信号转导途径中发挥重要的调节作用,如RTK/Ras/MAPKJ、AK/STAT及PI3K/Akt等。本文简介SHP2的结构、功能、信号转导以及阐述它在相关呼吸系统疾病中的作用。对SHP2研究的进一步深入可能为一些疑难的呼吸系统疾病的诊治提供新的策略。     

铜藻多糖对H2O2诱导的HaCaT细胞氧化应激的保护作用

为探究铜藻多糖(Sargassum horneri polysaccharides, SHP)对H₂O₂诱导的人角质形成细胞(HaCaT)氧化应激损伤的保护作用,测定了SHP对总抗氧化能力(T-AOC)、DPPH自由基、羟自由基(·OH)和超氧阴离子自由基(O₂^·-)的清除作用,以评价SHP的体外抗氧化能力,并建立H₂O₂诱导HaCaT细胞氧化损伤模型;通过测定细胞存活率、细胞活性氧以及酶活,评价SHP对HaCaT细胞氧化损伤的保护作用。结果表明,当SHP为1 mg/mL时,DPPH的清除率为68%、·OH清除能力65.48 U/mL;在SHP为3 mg/mL时,O₂^·-清除能力为84.86 U/mL,T-AOC为33.55。SHP能显著提高H₂O₂诱导氧化损伤的HaCaT细胞活力,其中经100μg/mL SHP处理后,HaCaT细胞存活率由56.85%提高到80.57...     

支架蛋白Gab的结构与功能及其在肿瘤、炎症和心血管疾病发生中的作用北大核心CSCD

Gab家族蛋白(Grb2-associated binder family proteins)是生长因子受体结合蛋白2的结合蛋白。在哺乳动物中,Gab家族蛋白包括Gab1、Gab2和Gab3。该家族蛋白可通过介导膜受体与信号转导蛋白间的耦联及各信号分子间的相互作用参与信号转导,其主要是通过激活SHP2/RAS/ERK和PI3K/AKT两条经典的信号通路,进而参与一系列的生物应答。研究显示,Gab蛋白的表达或功能异常与肿瘤、炎症和心血管疾病的发生、发展密切相关。本文就Gab蛋白结构、参与信号转导的调节机制及其在肿瘤、炎症和心血管疾病发生、发展中的作用进行综述。     

支架蛋白Gab的结构与功能及其在肿瘤、炎症和心血管疾病发生中的作用

Gab家族蛋白(Grb2-associated binder family proteins)是生长因子受体结合蛋白2的结合蛋白。在哺乳动物中,Gab家族蛋白包括Gab1、Gab2和Gab3。该家族蛋白可通过介导膜受体与信号转导蛋白间的耦联及各信号分子间的相互作用参与信号转导,其主要是通过激活SHP2/RAS/ERK和PI3K/AKT两条经典的信号通路,进而参与一系列的生物应答。研究显示,Gab蛋白的表达或功能异常与肿瘤、炎症和心血管疾病的发生、发展密切相关。本文就Gab蛋白结构、参与信号转导的调节机制及其在肿瘤、炎症和心血管疾病发生、发展中的作用进行综述。     

七鳃鳗Lja-SHP2分子鉴定、重组表达及免疫学研究

高等脊椎动物的蛋白酪氨酸磷酸酶SHP2(SH2 domain-containing protein-tyrosine phosphatase-2)由ptpn11基因编码,催化酪氨酸残基去磷酸化,与其他能催化酪氨酸磷酸化的蛋白酪氨酸激酶共同调节机体内多种信号通路的信号传导。以往研究表明,SHP2在高等脊椎动物T细胞和B细胞的激活与信号转导过程中起着重要作用。为了研究无颌类脊椎动物日本七鳃鳗(Lampetra japonica)中与SHP2同源的分子——Lja-SHP2在免疫应答反应中的作用,本研究通过PCR扩增获取其Lja-SHP2开放阅读框序列,并构建到原核表达载体pET-32a中,成功在大肠杆菌中实现重组蛋白表达并制备了其兔源多克隆抗体。用混合菌免疫刺激日本七鳃鳗后,通过实时荧光定量PCR和免疫印迹方法检测了Lja-SHP2在日本七鳃鳗免疫相关组织中mRNA和蛋白水平表达谱。结果显示,混合菌免疫刺激后,Lja-SHP2 mRNA和蛋白表达在外周血白细胞和髓样小体中无显著变化,而在鳃组织中显著性上调(P<0.05),说明Lja-SHP2在混合菌刺激后主要参与了鳃组织的免疫应答反应。为了进一步探究Lja-SHP2与淋巴细胞亚群免疫应答反应的相关性,本研究分别使用B细胞有丝分裂原脂多糖(lipopolysaccharide,LPS)和T细胞的有丝分裂原植物凝集素(phytohemagglutinin,PHA)免疫刺激日本七鳃鳗。经LPS免疫刺激后,与对照组相比,白细胞中Lja-SHP2蛋白表达显著上调,鳃组织和髓样小体没有显著性差异表达;但经PHA免疫刺激后,与对照组相比,白细胞、鳃组织和髓样小体3种组织中Lja-SHP2均有上调,尤其在白细胞中上调最为显著,大约是对照组的2.5倍,说明Lja-SHP2参与了日本七鳃鳗由PHA介导的免疫应答反应。由于PHA能刺激日本七鳃鳗鳃组织中VLRA+淋巴细胞的活化,这表明Lja-SHP2可能参与了PHA介导的VLRA+淋巴细胞亚群的免疫应答反应。上述研究结果为进一步探索Lja-SHP2在七鳃鳗免疫应答过程中的功能奠定了基础,也为揭示SHP2分子家族的系统发生及探索高等脊椎动物适应性免疫系统的早期发生及其进化历程提供一定的线索。     

豆壳过氧化物酶的盐酸胍变性与化学修饰研究

研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。     

孤儿核受体SHP敲减抑制BMP9诱导C3H10T1/2细胞的成骨分化

孤儿核受体SHP(small heterodimer partner)是核受体超家族中的一员,具有LXXLL模体及配体结合域,但无经典的DNA结合域.它可与多种转录因子结合,调节细胞的增殖、分化和代谢等生物学过程.但目前关于SHP在BMP9诱导成骨分化中的确切作用却尚不清楚.本研究证明,SHP参与BMP9诱导的C3H10T1/2细胞成骨分化. RT-PCR结合Western印迹方法检测蛋白揭示,异位表达BMP9上调了SHP在C3H10T1/2细胞中的表达. 小干扰RNA敲减SHP基因在C3H10T1/2细胞的表达下调了成骨相关基因Runx2、Id1、Id2及CTGF的表达,而过表达BMP9则可上调这些基因的表达.碱性磷酸酶(ALP)活性测定/染色及茜素红染色显示,敲减核受体SHP基因可抑制BMP9的成骨分化作用,而过表达BMP9可部分消除SHP 敲减导致的成骨抑制作用.上述结果提示,核受体SHP为BMP9诱导的C3H10T1/2细胞成骨分化所必需. 究竟BMP9如何上调SHP基因表达,以及SHP究竟通过何种机制上调BMP9下游成骨分化相关基因的表达尚待进一步研究.     

SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130

Interleukin-6 is involved in the regulation of many biological activities such as gene expression, cell proliferation, and differentiation. The control of the termination of cytokine signaling is as important as the regulation of initiation of signal transduction pathways. Three families of proteins involved in the down-regulation of cytokine signaling have been described recently: (i) SH2 domain-containing protein-tyrosine phosphatases (SHP), (ii) suppressors of cytokine signaling (SOCS), and (iii) protein inhibitors of activated STATs (PIAS). We have analyzed the interplay of two inhibitors in the signal transduction pathway of interleukin-6 and demonstrate that the tyrosine phosphatase SHP2 and SOCS3 do not act independently but are functionally linked. The activation of one inhibitor modulates the activity of the other; Inhibition of SHP2 activation leads to increased SOCS3-mRNA levels, whereas increased expression of SOCS3 results in a reduction of SHP2 phosphorylation after activation of the interleukin-6 signal transduction pathway. Furthermore, we show that tyrosine 759 in gp130 is essential for both SHP2 and SOCS3 but not for SOCS1 to exert their inhibitory activities on interleukin-6 signal transduction. Besides SHP2, SOCS3 also interacts with the Tyr(P)-759 peptide of gp130. Taken together, our results suggest differences in the function of SOCS1 and SOCS3 and a link between SHP2 and SOCS3.     

SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.     

Flow activates ERK1/2 and endothelial nitric oxide synthase via a pathway involving PECAM1, SHP2, and Tie2

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.     

SHPS-1/SIRP1alpha contributes to interleukin-6 signalling

The transmembrane glycoprotein signal regulatory protein/SHP2-substrate (SIRP1alpha/SHPS-1) has been implicated in growth factor- and cell adhesion-induced signalling. Here we report on the contribution of SIRP1alpha to IL-6 type cytokine signalling. SIRP1alpha binds the protein tyrosine phosphatase SHP2 upon treatment with interleukin-6 in a stimulation-dependent manner. Mouse embryonic fibroblasts expressing a SIRP1alpha protein which lacks the intracellular part show enhanced SHP2 phosphorylation and ERK1/2 activation in response to IL-6, suggesting that SIRP1alpha affects IL-6-signalling through SHP2. Whereas SHP2 phosphorylation is enhanced in SIRP1alpha-deficient cells STAT3 activation is delayed and STAT3-dependent gene induction is reduced which correlates with reduced STAT3 serine phosphorylation. Our results indicate that SIRP1alpha contributes to IL-6 signalling by counteracting SHP2 phosphorylation which consequently affects ERK-activation and STAT3-dependent transactivation as well as target gene expression. Our observations will help to understand the tight balance of MAPK- and STAT3-activation in response to IL-6 which was found to be misbalanced in many autoimmune diseases, inflammatory proliferative diseases and cancer.     

Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase (SHP) 2 and p38 Regulate the Expression of Chemokine CXCL8 in Human Astrocytes

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.     

SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1

The epithelial-mesenchymal transition (EMT) is an essential process for embryogenesis. It also plays a critical role in the initiation of tumor metastasis. Src homology 2 (SH2)-domain containing protein-tyrosine phosphatase-2 (SHP2) is a ubiquitously expressed protein-tyrosine phosphatase and is mutated in many tumors. However, its functional role in tumor metastasis remains largely unknown. We found that TGFβ1-induced EMT in lung epithelial A549 cells was partially blocked when SHP2 was decreased by transfected siRNA. The constitutively active form (E76V) promoted EMT while the phosphatase-dead mutation (C459S) and the SHP2 inhibitor PHPS1 blocked EMT, which further demonstrated that the phosphatase activity of SHP2 was required for promoting TGFβ1-induced EMT. Using the protein-tyrosine phosphatase domain of SHP2 as bait, we identified a novel SHP2-interacting protein Hook1. Hook1 was down-regulated during EMT in A549 cells. Overexpression of Hook1 inhibited EMT while knockdown of Hook1 promoted EMT. Moreover, both the protein-tyrosine phosphatase domain and N-terminal SH2 domain of SHP2 directly interacted with Hook1. Down-regulation of Hook1 increased SHP2 activity. These results suggested that Hook1 was an endogenous negative regulator of SHP2 phosphatase activity. Our data showed that the protein-tyrosine phosphatase SHP2 was involved in the process of EMT and Hook1 repressed EMT by regulating the activation of SHP2. SHP2-Hook1 complex may play important roles in tumor metastases by regulating EMT in cancer cells.