Role of G-protein-coupled receptor kinases in cell migration and signal transduction

G-protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases. The investigators pay much attention to the roles of GRKs in the signal transduction through G-protein-coupled receptors (GPCRs) with arrestin ever since a long time ago. Due to the physiological and pathological observations with the methods of deletion or overexpression, GRKs are considered as new drug targets. The kinases play a role in the pathogenesis of hypertension and cell migration through GPCRs and Hedgehog signaling pathways. As the development of research techniques, especially bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), the special mechanism of GRKs for GPCRs is more evident. In this review, we discuss the recent achievement in the roles of GRKs signaling and the related newest research techniques.     

Structure,function and signal transduction of vascular endothelial growth factor receptor-2

Angiogenesis, or the formation of new blood vessels out of pre-existing capillaries, is very important in many physiologic and pathologic processes, such as embryonic development, cancer, retinopathies, etc. Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a key role in angiogenesis. In this review, we discussed the structure, function and signal transduction of vascular endothelial growth factor receptor-2. Understanding these should provide important insights into how new strategies can be devised to interfere in the physiologic and pathologic processes involved in angiogensis.     

PTEN and signal transduction and tumor

PTEN is a candidate tumor suppressor which has sequence homology withdual-specifici-ty phosphatase. PTEN is a multifunctional protein endowed with a phosphatase activity capable of dephosphory-lating both tyrosine phosphate, serine/threonine phosphate residues on proteins and phospholipids of the phosphati dylinositol pathway. PTEN appears to be mutated at considerable frequency in several types of humantumors, including those frombrain, breast, endometrium, and prostate. PTEN play an important role in pathogenesis of tumor, tumor cell invasion and metastasis. In this review, we will discuss the chemical structure of PTEN, its phosphatase activity, the ability of affecting signal transduction, and its mutational status in cancer.     

Recent progresses on the study of endotoxin-induced p38 MAPK signal transduction

The diseases caused by endotoxin have seriously affected human health. Previous studies have shown that p38 MAPK pathway is involved in the intracellular signal transduction induced by lipopolysaccharide (LPS), which plays an important role in the activation of inflammation-related cells to release inflammation mediator. Recently there have been some progresses in the isoforms distribution, substrate, molecular mechanism of regulating the release of inflammatory mediators, cellular specific activation and levels of p38 MAPK.     

IL-13Rα2 as a functional receptor mediates signal transduction

IL-13 is a pleiotropic cytokine mainly secreted by activated Th2 cells. It has 2 receptors, IL-13Rα1 and IL-13Rα2. The latter had been thought to serve exclusively as a decoy receptor for a long period of time due to its short cytoplasmic tail and lack of signal transduction structure. Since Fichtner-Feigl reported in Nature Medicine that IL-13 is involved in induction of TGF-β production and tissue fibrosis through IL-13Rα2-mediated signaling pathway, it was found that IL-13Rα2 has more sophisticated functions than just a simple decoy receptor as more and more researches have explored its signaling functions. This review combines the most advanced research results with previous investigation and discusses the gene structure, expression, production, distribution, subtype conversion and possible signal pathways mediated by this receptor. More importantly, the connection with human diseases and the applications in disease diagnosis and molecule targeted therapy for cancer are also discussed.     

Calcineurin signal transduction pathway involves in cardiomyocyte hypertrophy induced by prostaglandin F2α

AIM: To study the role of prostaglandin F₂α (PGF₂α) in cardiac hypertrophy and its relation with calcineurin (CaN) signal transduction pathway in vitro. METHODS: The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of PGF2α, and the hypertrophic response was assayed by measuring the cell diameter, protein content and atrial natriuretic factor (ANF) mRNA expression. For mechanism studies, the intracellular free calcium concentration (［Ca²⁺］_i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator. ANF and CaN mRNA expressions, and the expressions of CaN and its downstream effectors, NFAT₃ and GATA₄ proteins were assayed by RT-PCR and Western blotting, respectively.RESULTS: In cultured cardiomyocytes, PGF₂α induced profound hypertrophic morphology change, the significant increase in cell diameter and protein content in a concentration-dependent manner compared with those in vehicle control (P<0.01). The same result was found in measuring the ［Ca²⁺］_i in cardiomyocytes (P<0.01). PGF₂α at concentration of 10-7 mol/L significantly promoted ANF and CaN mRNA expressions and the protein expressions of CaN/NFAT₃/GATA₄ compared with those in the vehicle control (P<0.05). Cyclosporin A, a CaN inhibitor, markedly inhibited the myocyte hypertrophy (P<0.01), reduced the increased ［Ca²⁺］_i(P<0.01) and decreased the expressions of CaN mRNA and CaN/NFAT₃/GATA₄ proteins (P<0.05) compared with those of only PGF₂α　10^-7 mol/L treatment. CONCLUSION: Cardiomyocyte hypertrophy induced by PGF2α may be, at least in part, mediated by CaN signal transduction pathway activated by increasing ［Ca²⁺］_i.     

Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Inhibitory effects of Co-SZ eye drops on apoptosis of lens epithelial cells and their signal transduction mechanisms

AIM: To investigate the inhibitory effect of Co-SZ eye drops on apoptosis of lens epithelial cells (LEC) induced by H₂O₂ and to study the cellular and molecular mechanisms.METHODS: (1) All lenses of Sprague-Dawley rats were incubated with H₂O₂ and Co-SZ eye drops.The apoptosis rates of LEC were determined by TUNEL method.The changes of LEC ultrastructure and the formation of apoptotic body were observed by electron microscopy.(2) Bovine LEC were incubated with H₂O₂ and Co-SZ eye drops.The inhibitory of LEC apoptosis was detected by MTT after incubation.The changes of fractional DNA content in LEC were detected by flow cytometry (FCM).［Ca2+］i , cAMP and cGMP of LEC were determined by spectrofluoremeter and radioimmunoassay, respectively.RESULTS: The LEC apoptosis rates in Co-SZ eye drops group were decreased significantly compared with H₂O₂ group by TUNEL.The ultrastructure changes in LEC of Co-SZ eye drops group were lighter than that in H₂O₂ group.The LEC apoptosis rates of Co-SZ eye drops group were dose-dependently decreased significantly compared with H₂O₂ groups via MTT assay.LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops, and showing dose-dependent.The DNA contents in LEC of Co-SZ group were increased.The ［Ca2+］i and cAMP in Co-SZ group were decreased obviously.The cGMP was increased.CONCLUSION: The LEC apoptosis induced by H₂O₂ was inhibited by Co-SZ eye drops.The mechanism of apoptosis inhibition by Co-SZ eye drops maybe contribute to the increase in DNA content.The signal transduction mechanisms are related to the decrease in ［Ca2+］i and cAMP and the increase in cGMP.     

Change of Gαq/11-mediated signal transduction pathway in aorta of spontaneously hypertensive rats

AIM: To investigate the changes of Gαq/11and phospholipase C(PLC) of the aorta and to evaluate the role of signal transduction pathway mediated by Gαq/11in the pathogenesis of spontaneous hypertension. METHODS: Blood pressure was measured by intubation of carotid, and the level of plasma angiotension Ⅱ was measured by radioimmunoassay. In addition, Gαq/11and PLC contents in aorta were determined by Western blot analysis. RESULTS: Arterial blood pressure was not changed in 4-week-old spontaneously hypertensive rats(SHR), but it was increased markedly in12-week-old SHR. The Gαq/11expression of aorta in 4-week-old SHR was increased by 69.2%(P<0.05) The levels of PLCβ/₃ of aorta in 4-and12-week-old SHR were increased by 66.9% and 85.1%(P<0.05), respectively, compared with their age-matched WKY rats.CONCLUSION: The up-regulation of Gαq/11-mediated signal transduction pathway of aorta may contribute to the initiation and maintenance of spontaneous hypertension.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.     

Epoxyeicosatrienoic acid: the signal transduction

Cytochrome P450 monoxygenase converts arachionic acid to four epoxyeicosatrienoic acid regiosomes: 5, 6-EET (epoxyeicosatrienoic acid); 8, 9-EET; 11, 12-EET and 14, 15-EET. Recent studies show that EETs are involved in signal transduction. EETs open Ca²⁺-sensit ive K⁺ channel and inhibit Na⁺ channel, Ca²⁺-sensitive Cl^- channel and so on. What is more, EETs have been demonstrated to activate PP60^c-src and initiate a tyrosine kinase cascade that mediates mitogenic effects.     

Effects of angiotensin-(1-7) on calcification in vascular smooth muscle cells and its signal transduction pathway

AIM: To clarify the effects of angiotensin-(1-7) on the calcification in rat vascular smooth muscle cells(VSMCs) and its signal transduction.METHODS: Calcification of cultured rat VSMCs was prepared by incubation of VSMCs with β-glycerophosphate.Calcification was confirmed by Von Kossa staining.The cells were treated with angiotensin-(1-7).The calcium content,alkaline phosphatases activity,osteocalcin and Cbfa1 mRNA expression were also measured.RESULTS: Angiotensin-(1-7) inhibited the increases of calcium content,alkaline phosphatases activity（P>0.05）,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs（P<0.05）,and the effects of angiotensin II on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs were also inhibited （P<0.05）.Angiotensin-(1-7) increased cAMP concentration in calcified VSMCs（P<0.05）and selective PKA inhibitor blocked the effects of angiotensin-(1-7) on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression（P<0.05）.CONCLUSION: Angiotensin-(1-7) can inhibit beta-glycerophosphate-induced calcification in VSMCs through cAMP-PKA-Cbfa1 pathway and antagonize the effect of angiotensin II on calcification in VSMCs.     

Signal transduction pathway and blocking strategies of NF-κB

NF-κB is thought of as a genetic switch to control expressions of many target genes and directly participates in pathogenesis of infection, inflammation, stress, immunoresponse, cellular apoptosis, toxic shock and tumor as well as cell-cycle regulation and cell differentiation.The overactivation of NF-κB is intimately involved in many human diseases.Various therapeutic strategies against NF-κB, to date, in-clude anti-inflammatory drugs, antioxidants, immunosuppressive agents, inhibitors of protease and protea-some, prostaglandings, nitric oxide, IL-10, microbial products, synthetic inhibitors, antisense oligon cleotides and decoy deoxyoligonucleotides.Studies are underway to develop NF-κB member-specific and cell type-specific drugs that can inhibit the activation of NF-κB only in target cells and that may become a novel way to treat the human diseases.     

Preparation of m1AChR-G11 fusion protein and m4AChR-G16 fusion protein and effects of various ligands on them

AIM:To prepare m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein in Baculovirus-Sf9 cell system and detect the effects of various muscarinic ligands on the interaction between m₁AChR and G₁₁ and m₄AChR and G₁₆, and screen different kinds of ligands specific for m₁ and m₄. METHODS:To prepare fused DNA of m₁AChR-G₁₁and m₄AChR-G₁₆ in two PCR, then expressed in Sf9 cells and detect the pharmacological function of m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein by QNB and GTPγS binding experiments; To expore the way of the activation of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein by various ligands includingcetylcholine (ACh), Pilocarpine (Pilo), 4-hydroxy-2-butynyl-1-trimethylammonium-m-chloro-carbanilatechloride (McN-A-343), tetrandrine, pirenzepine (PZ), alcuronium, atropine, R-(+)-hyoscyamine and gallamine by displacement by GDP on GTPγS binding experiments. RESULTS:The expression levels of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein were (45.39±2.62) nmol·g^-1 protein, (47.04±1.58) nmol·g^-1 protein. The affinity of GDP to G₁₁ and G₁₆ partner changed in the presence of different muscarinic ligands. CONCLUSION: The m₁AChR-G₁₁ and m₄AChR-G16 showed the pharmacological specificity to m₁ and m₄ receptor and the efficient signaling of the two partners. Ligands of m₁AChR and m₄AchR mediated different signal transduction by changing the affinity of G₁₁/G₁₆ and GDP. So m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein can be taken as a tool to screen ligands specific for m₁AChR and m₄AChR.