Regulation of articular chondrocyte phenotype by bone morphogenetic protein 7, interleukin 1, and cellular context is dependent on the cytoskeleton.

Bone morphogenetic proteins (BMPs) induce cartilage differentiation and morphogenesis. There are profound changes in the cytoskeletal architecture during the morphogenesis of cartilage. To investigate the possibility that morphogenetic signals such as BMPs may regulate chondrocyte phenotype by modulation of cytoskeletal protein expression, we determined whether the expression and distribution of cytoskeletal proteins in chondrocytes are regulated by bone morphogenetic protein 7 (BMP 7), interleukin 1 (IL-1), and cellular context. Addition of BMP 7, a morphogen that induces chondrogenesis, to primary cultures of bovine and murine chondrocytes induced increased expression of four cytoskeletal proteins: tensin, talin, paxillin, and focal adhesion kinase (FAK). The expression of cytoskeletal proteins is dependent on cellular context; compared to monolayer, chondrocytes in suspension exhibited increased expression of cytoskeletal components. Conversely, addition of IL-1, a catabolic cytokine, induced loss of chondrocyte phenotype and decreased the expression of these cytoskeletal components. Treatment of chondrocytes with cytochalasin D (an agent that disrupts the actin cytoskeleton) inhibited BMP 7-induced upregulation of tensin, talin, paxillin, and FAK, and blocked the effect of BMP 7 on chondrocyte phenotype. Taken together these data demonstrate that cytoskeletal components play a critical role in the response to morphogens and cytokines in the regulation of chondrocyte phenotype. (c)2001 Elsevier Science.     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

Protein Tyrosine Phosphatase LMW-PTP Exhibits Distinct Roles Between Vascular Endothelial and Smooth Muscle Cells

Abstract

The present study examined the cellular functions of low-molecular-weight protein tyrosine phosphatase (LMW-PTP), which consists of two active isoforms IF-1 and IF-2, in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), focusing on cell growth and migration. We transduced recombinant IF-1 and IF-2, and ribozyme targeting both isoforms using an adenovirus vector in these cells. We detected the expression of IF-1 and IF-2 in both types of cells. IF-1 as well as IF-2 inhibited PDGF-induced DNA synthesis and migration in VSMCs. In contrast, both isoforms enhanced lysophosphatidic acid-stimulated cell migration without change in DNA synthesis in ECs. Whereas there is a report indicating that reactive oxygen species-dependent inactivation of LMW-PTP regulates actin cytoskeleton reorganization during cell spreading and migration, the isoforms conversely suppressed the PDGF-induced H₂O₂ generation with subsequent decrease in the p38 activity in VSMCs. Catalytically inactive LMW-PTP exerted the opposite and similar effects to the wild type in ECs and in VSMCs, respectively, suggesting that substrates for the phosphatase differ between these cells. Moreover, high concentrations of glucose suppressed the expression of LMW-PTP in both cells. These data suggest that LMW-PTP negatively regulates the pathogenesis of atherosclerosis and that glucose-dependent suppression of LMW-PTP expression may promote the development of atherosclerosis in diabetics.     

LA－90细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究

ＬＡ－９０细胞在温度转化过程中蛋白质酪氨酸磷酸化作用研究夏英,高漫,颜卉君,吴国利（北京师范大学生物系生物化学及分子生物学研究室,１００８７５）关键词酪氨酸蛋白激酶;磷酸酪氨酸蛋白磷酸酶;细胞转化ｉｓ－ＲＳＶＬＡ－９０细胞是ＲＳＶ转染的小鼠３Ｔ３细胞...     

Altered Protein Tyrosine Phosphorylation in Alzheimer''s Disease

The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.     

Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Integrin adhesions

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation

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机械拉伸下人肺上皮细胞增殖及整联蛋白再分布

应用体外周期性拉伸装置研究机械拉伸对人肺上皮细胞A5 4 9增殖及其膜表面受体———整联蛋白α5、β1再分布的调控作用。结果表明 :在应变为 15 % ,频率为 2 0次 /min、4 0次 /min的拉伸刺激下 ,4 8h后 ,应用流式细胞技术检测细胞的增殖活性指数明显降低 ,A5 4 9细胞的DNA合成受到显著抑制。在 4 0次 /min的拉伸频率下 ,整联蛋白α5、β1的分布发生重组并向基底层转移 ,形成局部粘附连接。研究表明 :整联蛋白α5、β1可能在肺上皮细胞感应机械应力过程中起了重要的作用。     

Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Differential Regulation of Focal Adhesion Kinase and Mitogen-Activated Protein Kinase Tyrosine Phosphorylation During Insulin-Like Growth Factor-I-Mediated Cytoskeletal Reorganization

Abstract: In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.     

IgE Regulates the Expression of smMLCK in Human Airway Smooth Muscle Cells

Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.     

Protein Phosphorylation in Bovine Adrenal Medullary Chromaffin Cells: Histamine-Stimulated Phosphorylation of Tyrosine Hydroxylase

Histamine can cause the release of catecholamines from bovine adrenal medullary chromaffin cells by a mechanism distinct from that of the depolarizing agents nicotine or high K+ buffer. It was the aim of this study to determine the protein phosphorylation responses to histamine in these cells and to compare them with those induced by depolarization. A number of proteins showed increases in phosphorylation in response to histamine especially when analyzed on two-dimensional polyacrylamide gel electrophoresis or by phosphopeptide mapping; one protein of 20,000 daltons was markedly dephosphorylated. Emphasis was given to the effects of histamine on tyrosine hydroxylase (TOH) phosphorylation, because this protein showed the most prominent changes on one-dimensional gels. Histamine acted via H1 receptors to increase TOH phosphorylation; the response was blocked by the H1 antagonist mepyramine and could be mimicked by the H1 agonist thiazolylethylamine, but not by the H2 agonist dimaprit. The H3 agonist (R) alpha-methylhistamine increased TOH phosphorylation at high concentrations, but the response was blocked entirely by mepyramine. Histamine rapidly increased the phosphorylation of TOH, with a maximum reached within 5 s and maintained for at least 30 min. This was in marked contrast to nicotine-stimulated protein phosphorylation of TOH, which was rapidly desensitized. The initial phosphorylation response to histamine was independent of extracellular Ca2+ for at least 3 min, but the sustained response required extracellular Ca2+. This was in contrast to the situation with both nicotine and high K+ buffer, which under the conditions used here caused a response which was dependent on extracellular Ca2+ at all times investigated. In the presence of histamine, the phosphopeptide profiles for TOH were essentially the same with or without Ca2+, suggesting that the same protein kinases were involved, but at longer times there was evidence of new phosphorylation sites. The mechanism or mechanisms whereby histamine modulates TOH phosphorylation are discussed with emphasis on the differences from depolarizing agents.     

Mathematical Modelling of Ca2+ Oscillations in Airway Smooth Muscle Cells

The action of different agonists such as acetylcholine on the membrane of airway smooth muscle cells may induce cytosolic Ca²⁺ oscillations which can be a part of the Ca²⁺ signalling pathway, eventually leading to cell contraction. The aim of the present study is to present a mathematical model of the possible effect of the initial Ca²⁺ distribution within the cell on the form and frequency of induced Ca²⁺ oscillations. It takes into account intracellular Ca²⁺ stores such as sarcoplasmic reticulum and cytosolic proteins as well as Ca²⁺ exchange across the plasma membrane. We are able to demonstrate a closer agreement of model predictions with observed Ca²⁺ traces for a significantly wider range of parameter values, as was previously reported. We show also that the total cellular Ca²⁺ content is an important system parameter especially because of the content in sarcoplasmic reticulum. At a total Ca²⁺ increase of about 20%, the oscillation frequency increases by 25%; also, damped oscillations become sustained. Cases are indicated in which such a situation could occur.     

Tyrosine Phosphorylation of Microtubule-Associated Protein Kinase After Transient Ischemia in the Gerbil Brain

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.     

平滑肌细胞骨架结构及其信号调节途径

平滑肌细胞骨架是一个复杂的动态性网络,是细胞生命活动不可缺少的细胞结构。Rho通过活化其下游靶分子促进应力纤维的形成,其中Rho—associated coiled—coil kinase(ROCK)和Dial在该过程中起关键作用;PKC通过在细胞内不同定位的亚型使细胞骨架蛋白磷酸化,发挥其调节细胞骨架重构的作用。两条信号转导途径通过Src途径相互联系,共同参与细胞骨架动力学的调节。