BACKGROUND Regulator of G protein signaling(RGS)proteins participate in tumor formation and metastasis by acting on theα-subunit of heterotrimeric G proteins.The speci-fic effect of RGS,particularly RGS4,on the progr...BACKGROUND Regulator of G protein signaling(RGS)proteins participate in tumor formation and metastasis by acting on theα-subunit of heterotrimeric G proteins.The speci-fic effect of RGS,particularly RGS4,on the progression of gastric cancer(GC)is not yet clear.AIM To explore the role and underlying mechanisms of action of RGS4 in GC develop-ment.METHODS The prognostic significance of RGS4 in GC was analyzed using bioinformatics based public databases and verified by immunohistochemistry and quantitative polymerase chain reaction in 90 patients with GC.Function assays were employed to assess the carcinogenic impact of RGS4,and the mechanism of its possible influence was detected by western blot analysis.A nude mouse xenograft model was established to study the effects of RGS4 on GC growth in vitro.RESULTS RGS4 was highly expressed in GC tissues compared with matched adjacent normal tissues.Elevated RGS4 expression was correlated with increased tumor-node-metastasis stage,increased tumor grade as well as poorer overall survival in patients with GC.Cell experiments demonstrated that RGS4 knockdown suppressed GC cell proliferation,migration and invasion.Similarly,xenograft experiments confirmed that RGS4 silencing significantly inhibited tumor growth.Moreover,RGS4 knockdown resulted in reduced phosphorylation levels of focal adhesion kinase,phosphatidyl-inositol-3-kinase,and protein kinase B,decreased vimentin and N-cadherin,and elevated E-cadherin.CONCLUSION High RGS4 expression in GC indicates a worse prognosis and RGS4 is a prognostic marker.RGS4 influences tumor progression via the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.展开更多
BACKGROUND Inhibiting hepatic stellate cell(HSC)activation is a key therapeutic strategy in liver fibrosis(LF).During activation,aerobic glycolysis is upregulated to meet increased energy demands.Although focal adhesi...BACKGROUND Inhibiting hepatic stellate cell(HSC)activation is a key therapeutic strategy in liver fibrosis(LF).During activation,aerobic glycolysis is upregulated to meet increased energy demands.Although focal adhesion kinase(FAK)has been implicated in regulating HSC glycolysis,its precise role in activation remains unclear.AIM To investigate the effects of FAK and fructose-1,6-bisphosphatase 1(FBP1)on LF through the modulation of aerobic glycolysis in HSCs.METHODS Eighteen mice were randomly assigned to three groups:Control,carbon tetrachloride(CCl_(4))-induced LF,and CCl_(4)with FAK inhibitor treatment.Liver tissues were analyzed using transcriptomic and proteomic sequencing.Differential gene expression,Mfuzz clustering,and protein interaction network analyses identified key regulatory factors.Immunohistochemistry(IHC)and Western blot(WB)analysis were used to assess FAK and FBP1 expression,along with glycolysis-related enzymes.The migratory behavior of HSCs was evaluated using Transwell migration and scratch assays.RESULTS Transcriptomic and proteomic analyses revealed significantly reduced FBP1 expression in CCl_(4)-induced fibrosis,which was restored upon FAK inhibition.Histological staining(hematoxylin and eosin,Masson’s trichrome,Sirius red)confirmed reduced fibrosis following FAK inhibition.WB analysis demonstrated suppression of glycolysisrelated enzymes.In LX-2 cells,FAK inhibition attenuated HSC activation and glycolysis while upregulating FBP1.Exogenous recombinant FBP1 inhibited HSC activation and glycolysis.Transwell and scratch assays showed that FBP1 significantly impaired HSC migration.In addition,WB and IHC analyses confirmed lower FBP1 expression in fibrotic liver tissues from patients compared to healthy controls.CONCLUSION FAK inhibitors and increased FBP1 expression inhibit aerobic glycolysis in HSCs,thereby improving LF.Thus,FAK and FBP1 may be potential targets for LF treatment.展开更多
Although the etiology of inflammatory bowel disease (IBD) remains unclear,compromised epithelial barrier integrity is believed to promote susceptibility toIBD and be associated with disease severity, suggesting that i...Although the etiology of inflammatory bowel disease (IBD) remains unclear,compromised epithelial barrier integrity is believed to promote susceptibility toIBD and be associated with disease severity, suggesting that improving gut barrierintegrity may palliate or treat IBD. Such a notion gets support from the clinicalfindings that mucosal healing in IBD patients is associated with improvedprognosis, and reduced risk of relapse or colitis-associated cancer. It thereforebecomes critical to understand the intracellular signals that regulate mucosalhealing and gut barrier integrity. Focal adhesion kinase (FAK) is a non-receptortyrosine kinase that critically modulates epithelial cell growth and mobility andhas been associated with carcinogenesis. However, studies also suggest that FAKactivation may promote mucosal healing under conditions of colitis, which shouldreduce the risk of colitis-associated cancer. These findings highlight a potentiallytransformative role for FAK in the context of IBD. Understanding the molecularmechanisms by which FAK influences gut barrier repair and mucosal integritycould offer novel therapeutic avenues for treating IBD and preventing its longtermcomplications. This review focuses on the potential role of FAK in promotingcolitis-associated mucosal healing and the underlying molecular mechanismsdriving these processes, offering critical insights into IBD pathogenesis and therapy.展开更多
Objective To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism.Methods Mouse models of radiation pneumonitis induced by thoracic radiation exposu...Objective To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism.Methods Mouse models of radiation pneumonitis induced by thoracic radiation exposure were given intravenous injections of 100μL normal mouse serum or normal saline immediately after the exposure followed by injections once every other day for a total of 8 injections.On the 15th day after irradiation,histopathological changes of the lungs of the mice were examined using HE staining,the levels of TNF-α,TGF-β,IL-1αand IL-6 in the lung tissue and serum were detected using ELISA,and the percentages of lymphocytes in the lung tissue were analyzed with flow cytometry.High-throughput sequencing of exosome miRNA was carried out to explore the changes in the signaling pathways.The mRNA expression levels of the immune-related genes were detected by qRT-PCR,and the protein expressions of talin-1,tensin2,FAK,vinculin,α-actinin and paxillin in the focal adhesion signaling pathway were detected with Western blotting.Results In the mouse models of radiation pneumonitis,injections of normal mouse serum significantly decreased the lung organ coefficient,lowered the levels of TNF-α,TGF-β,IL-1αand IL-6 in the serum and lung tissues,and ameliorated infiltration of CD45^(+),CD4^(+)and Treg lymphocytes in the lung tissue(all P<0.05).The expression levels of Egfr and Pik3cd genes at both the mRNA and protein levels and the protein expressions of talin-1,tensin2,FAK,vinculin,α‑actinin and paxillin were all significantly down‑regulated in the mouse models after normal mouse serum treatment.Conclusion Normal mouse serum ameliorates radiation pneumonitis in mice by inhibiting the expressions of key proteins in the Focal adhesion signaling pathway.展开更多
BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic gl...BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.展开更多
Focal adhesion kinase(FAK)is a 125-kDa non-receptor protein tyrosine.Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein ty...Focal adhesion kinase(FAK)is a 125-kDa non-receptor protein tyrosine.Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases,resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation.FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract.FAK also plays an important role in the restitution,cell survival and apoptosis and carcinogenesis of the gastrointestinal tract.FAK is over-expressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells.FAK has been proposed as a potential target in cancer therapy.Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells,indicating a high potential for application in cancer therapy.展开更多
AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells ...AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.展开更多
OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carci...OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carcinoma (HCC) nude mice. METHODS: Forty male nude mice were randomly divided into 4 groups. Human HCC tissue was implanted in the livers of three groups. After 24 h, the three groups were treated respectively with JPJDF (37.5 g/kg), saline (20 mL/kg) and Tegafur (FT-207, 160 mg/kg) once a day for 10 weeks. The control group without implanting the tissue was concurrently treated with saline (20 mL/kg). The survival data and body weight of all mice were recorded, and expression levels of PTEN, PI3K and FAK in normal tissue and cancer tissue of the livers were eval-uated with immunohistochemical method. RESULTS: The cumulative survival rate of the mice in the JPJDF group was higher than those of the other groups. The rate of weight loss was the lowest in JPJDF group. The survivability and weight loss rate in FT-207 group were the poorest in all groups. The expression intensity of PTEN was higher in normal tissues than in cancer tissues, and lower in the normal tissues of HCC models than in that of mice without HCC. The PTEN expression intensity in normal tissue and cancer tissue from mice treated with FT-207 were lower than that from the mice treated with JPJDF or saline.The expression intensity of PI3K was higher in cancer tissue than in normal tissue. The PI3K expression intensity was the lowest in normal tissue and cancer tissue from mice treated with JPJDF, and the intensity from mice treated with FT-207 was the highest. In mice treated with JPJDF, the expression intensity of FAK was higher in the normal tissue and lower in the cancer tissue than those of the other treatment groups. CONCLUSION: The mechanism accounting for the prolonged survival of HCC-bearing mice treated with JPJDF might be related to the reduction in weight loss and the benign regulation of PTEN, PI3K, and FAK.展开更多
Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has al...Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.展开更多
Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21...Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.展开更多
Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kin...Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kinase, which is recruited to focal adhesions and plays a key role in cell migration, proliferation and survival, could reflect the invasive capacity of bladder carcinomas. The aim of this study was to evaluate the FAK expression in cancer ceils as an important prognostic factor of the evolution of bladder carcinomas. Tumor and paired peritumoral biopsies were obtained during transurethral endoscopic resection or cystectomy of bladder tumors in 280 patients at the Urology Unit of the Mustapha Hospital of Algiers and the Hospital of Tizi-Ouzou （Algeria）. The authors studied FAK expression in samples from bladder carcinomas at different stages of malignant transformation by western blot analysis using a specific anti-FAK antibody. Western blot is one of the most common laboratory techniques; it is used to detect the presence of a specific protein in a complex mixture extracted from cells. A weak increase in FAK expression was observed in tumors of grade 1 and 2 （1.65; 2.99） as compared to healthy tissues; it became particularly important in grade 3 tumors; the authors show that FAK levels significantly increased gradually according to the tumor stage.展开更多
Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the clust...Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the cluster often became unstable under fluctuated forces. However, an unusual case was also reported, where the stability of the cluster might be substantially enhanced by a fluctuated force with a relatively low fluctuation frequency and high fluctuation amplitude.Such an observation cannot be explained by the conventional fracture theory of fatigue. Here, we intensively investigate this intriguing observation by carrying out systematic parametric studies. Our intensive simulation results indicate that stability enhancement of this kind is in fact quite robust, which can be affected by the stochastic features of a single bond and the profile of the fluctuated forces such as the average value of bond force. We then suggest that the fluctuation of traction force within a focal adhesion might enhance its stability in a certain way.展开更多
Chen et al demonstrated that regulator of G protein signaling(RGS)4 promotes gastric cancer(GC)progression by activating the focal adhesion kinase/phospha-tidyl-inositol-3-kinase/protein kinase B pathway and inducing ...Chen et al demonstrated that regulator of G protein signaling(RGS)4 promotes gastric cancer(GC)progression by activating the focal adhesion kinase/phospha-tidyl-inositol-3-kinase/protein kinase B pathway and inducing epithelial-mesen-chymal transition.Although their multilevel approach integrating clinical data,functional assays,and xenograft models demonstrated a key role for RGS4 in GC pathogenesis,several limitations should be considered.The mechanism of the RGS4-focal adhesion kinase interaction remains unclear,specifically whether it involves direct binding or intermediaries.The clinical analysis of 90 patients lacks stratification by GC subtypes or immune features,potentially limiting generaliz-ability.Furthermore,fully validating RGS4’s oncogenic role requires additional studies,including functional assays in chemotherapy-resistant and metastatic cell lines,metastasis models including orthotopic implantation and tail vein injection,and comparison with other RGS family members.Addressing these via targeted mechanistic studies and expanded clinical validation could strengthen RGS4’s po-tential as a therapeutic target in GC.展开更多
Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer.It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors ...Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer.It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors in vivo.However,the role of extracellular matrix(ECM)stiffness in colon cancer cell(CCCs)invasion and metastasis remains unclear.Here,bioinformatical analysis suggested that a high expression level of yes associated protein 1(YAP1)was significantly associated with metastasis and poor prognosis in colon cancer patients.We further investigated the effects of polyacrylamide hydrogels with different stiffnesses(3,20,and 38 kPa),which were simulated as ECM,on the mechanophenotype(F-actin cytoskeleton organization,electrophoretic rate,membrane fluidity,and Young's modulus)of CCCs.The results showed that a stiffer ECM could induce the maturation of focal adhesions and formation of stress fibers in CCCs,regulate their mechanophenotypes,and promote cell motility.We also demonstrated that the expression levels of YAP1 and paxillin were positively correlated in patients with colon cancer.YAP1 knockdown reduces paxillin clustering and cell motility and alters the cellular mechanophenotypes of CCCs.This is of great significance for an in-depth understanding of the invasion and metastatic mechanisms of colon cancer and for the optimization of clinical therapy from the perspective of mechanobiology.展开更多
Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants.However,it remains challenging to identify physiologically relevant and tissue-selective pr...Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants.However,it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing.Hence,we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail.By using a combination of in silico target prediction and in-cell validation,we identified several focal adhesion proteins as alternative splicing targets of Rbfox1.We focused on the alternative splicing patterns of vinculin(metavinculin isoform)and paxillin(extended paxillin isoform)and identified both as potential Rbfox1 targets.Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns.Focal adhesions play an important role in the cardiac muscle context,since they mainly influence cell shape,cytoskeletal organization,and cell–matrix association.Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology,cytoskeletal organization,and multinuclearity after differentiation,which might be due to changes in alternative splicing of focal adhesion proteins.Hence,our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells,which might contribute to heart disease progression,where downregulation of Rbfox1 is frequently observed.展开更多
OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stim...OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively. RESULTS: FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P展开更多
To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin Methods Adhesion and migration of cultured SMCs were stimulated by ...To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin Methods Adhesion and migration of cultured SMCs were stimulated by different concentrati ons of fibronectin (FN), FAK and its phosphorylation were detected by immunoprec ipitation and Western blot FAK antisense oligodeoxynucleotides (ODNs) were tra nsfected into SMCs by cationic lipid to investigate its modulatory effects on ty rosine phosphorylation SMCs adhesion and migration were also measured by morph ological enumeration and modified Boyden Chambers, respectively Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN FAK phosphorylation were detected only at 20 ?μg/ml FN or more FAK antisense ODNs were transfected efficiently by cationi c lipid and FAK phosphorylation was inhibited substantially The SMCs migration rate in the 5-60?μg/ml FN groups was reduced by 17 89%-27 67% Cell migrat ion stimulated by FN at 10, 20, 40 and 60?μg/ml were reduced by 23 26%, 21 6 3%, 19 31% and 17 88%, respectively ( P 【0 05) Conclusions FAK phosphorylation and FAK mediated signal transduction play important roles i n SMCs adhesion and migration stimulated by ECM The process can be inhibited e ffectively by FAK antisense ODNs展开更多
Background Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an esse...Background Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion. Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase （FAK） as a target of miR-7. The levels of miR-7, matrix metalloproteinases （MMP）-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase （AKT）, and extracellular signal-regulated kinase （ERK） 1/2 were measured by Western blotting analysis. Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3＇UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues. Conclusion To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.展开更多
The skeletal system,which contains bones,joints,tendons,ligaments and other elements,plays a wide variety of roles in body shaping,support and movement,protection of internal organs,production of blood cells and regul...The skeletal system,which contains bones,joints,tendons,ligaments and other elements,plays a wide variety of roles in body shaping,support and movement,protection of internal organs,production of blood cells and regulation of calcium and phosphate metabolism.The prevalence of skeletal diseases and disorders,such as osteoporosis and bone fracture,osteoarthritis,rheumatoid arthritis,and intervertebral disc degeneration,increases with age,causing pain and loss of mobility and creating a huge social and economic burden globally.Focal adhesions(FAs)are macromolecular assemblies that are composed of the extracellular matrix(ECM),integrins,intracellular cytoskeleton and other proteins,including kindlin,talin,vinculin,paxillin,pinch,Src,focal adhesion kinase(FAK)and integrin-linked protein kinase(ILK)and other proteins.FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell–environment communications and modulates important processes,such as cell attachment,spreading,migration,differentiation and mechanotransduction,in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways.This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.展开更多
Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear ...Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis展开更多
基金Supported by the Fundamental Research Program of Shanxi Province,No.202203021222418Research Program of Shanxi Provincial Health Commission,No.2023061+2 种基金Fundamental Research Cooperation Program of Beijing-Tianjin-Hebei Region of Natural Science Foundation of Tianjin,No.22JCZXJC00140Tianjin Major Science and Technology Project,No.21ZXJBSY00110Tianjin Health and Science and Technology Project,No.TJWJ2024ZK001.
文摘BACKGROUND Regulator of G protein signaling(RGS)proteins participate in tumor formation and metastasis by acting on theα-subunit of heterotrimeric G proteins.The speci-fic effect of RGS,particularly RGS4,on the progression of gastric cancer(GC)is not yet clear.AIM To explore the role and underlying mechanisms of action of RGS4 in GC develop-ment.METHODS The prognostic significance of RGS4 in GC was analyzed using bioinformatics based public databases and verified by immunohistochemistry and quantitative polymerase chain reaction in 90 patients with GC.Function assays were employed to assess the carcinogenic impact of RGS4,and the mechanism of its possible influence was detected by western blot analysis.A nude mouse xenograft model was established to study the effects of RGS4 on GC growth in vitro.RESULTS RGS4 was highly expressed in GC tissues compared with matched adjacent normal tissues.Elevated RGS4 expression was correlated with increased tumor-node-metastasis stage,increased tumor grade as well as poorer overall survival in patients with GC.Cell experiments demonstrated that RGS4 knockdown suppressed GC cell proliferation,migration and invasion.Similarly,xenograft experiments confirmed that RGS4 silencing significantly inhibited tumor growth.Moreover,RGS4 knockdown resulted in reduced phosphorylation levels of focal adhesion kinase,phosphatidyl-inositol-3-kinase,and protein kinase B,decreased vimentin and N-cadherin,and elevated E-cadherin.CONCLUSION High RGS4 expression in GC indicates a worse prognosis and RGS4 is a prognostic marker.RGS4 influences tumor progression via the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition.
基金Supported by the Science and Technology Program of the Guizhou Province,No.[2021]094National Natural Science Foundation of China,No.82060116 and No.82260129+1 种基金Guizhou Provincial Science and Technology Program,No.QKHJC-ZK[2023]214Doctoral Research Start-up Fund Project of Guizhou Medical University,No.gyfybsky[2021]63.
文摘BACKGROUND Inhibiting hepatic stellate cell(HSC)activation is a key therapeutic strategy in liver fibrosis(LF).During activation,aerobic glycolysis is upregulated to meet increased energy demands.Although focal adhesion kinase(FAK)has been implicated in regulating HSC glycolysis,its precise role in activation remains unclear.AIM To investigate the effects of FAK and fructose-1,6-bisphosphatase 1(FBP1)on LF through the modulation of aerobic glycolysis in HSCs.METHODS Eighteen mice were randomly assigned to three groups:Control,carbon tetrachloride(CCl_(4))-induced LF,and CCl_(4)with FAK inhibitor treatment.Liver tissues were analyzed using transcriptomic and proteomic sequencing.Differential gene expression,Mfuzz clustering,and protein interaction network analyses identified key regulatory factors.Immunohistochemistry(IHC)and Western blot(WB)analysis were used to assess FAK and FBP1 expression,along with glycolysis-related enzymes.The migratory behavior of HSCs was evaluated using Transwell migration and scratch assays.RESULTS Transcriptomic and proteomic analyses revealed significantly reduced FBP1 expression in CCl_(4)-induced fibrosis,which was restored upon FAK inhibition.Histological staining(hematoxylin and eosin,Masson’s trichrome,Sirius red)confirmed reduced fibrosis following FAK inhibition.WB analysis demonstrated suppression of glycolysisrelated enzymes.In LX-2 cells,FAK inhibition attenuated HSC activation and glycolysis while upregulating FBP1.Exogenous recombinant FBP1 inhibited HSC activation and glycolysis.Transwell and scratch assays showed that FBP1 significantly impaired HSC migration.In addition,WB and IHC analyses confirmed lower FBP1 expression in fibrotic liver tissues from patients compared to healthy controls.CONCLUSION FAK inhibitors and increased FBP1 expression inhibit aerobic glycolysis in HSCs,thereby improving LF.Thus,FAK and FBP1 may be potential targets for LF treatment.
文摘Although the etiology of inflammatory bowel disease (IBD) remains unclear,compromised epithelial barrier integrity is believed to promote susceptibility toIBD and be associated with disease severity, suggesting that improving gut barrierintegrity may palliate or treat IBD. Such a notion gets support from the clinicalfindings that mucosal healing in IBD patients is associated with improvedprognosis, and reduced risk of relapse or colitis-associated cancer. It thereforebecomes critical to understand the intracellular signals that regulate mucosalhealing and gut barrier integrity. Focal adhesion kinase (FAK) is a non-receptortyrosine kinase that critically modulates epithelial cell growth and mobility andhas been associated with carcinogenesis. However, studies also suggest that FAKactivation may promote mucosal healing under conditions of colitis, which shouldreduce the risk of colitis-associated cancer. These findings highlight a potentiallytransformative role for FAK in the context of IBD. Understanding the molecularmechanisms by which FAK influences gut barrier repair and mucosal integritycould offer novel therapeutic avenues for treating IBD and preventing its longtermcomplications. This review focuses on the potential role of FAK in promotingcolitis-associated mucosal healing and the underlying molecular mechanismsdriving these processes, offering critical insights into IBD pathogenesis and therapy.
文摘Objective To evaluate the therapeutic effect of normal mouse serum on radiation pneumonitis in mice and explore the possible mechanism.Methods Mouse models of radiation pneumonitis induced by thoracic radiation exposure were given intravenous injections of 100μL normal mouse serum or normal saline immediately after the exposure followed by injections once every other day for a total of 8 injections.On the 15th day after irradiation,histopathological changes of the lungs of the mice were examined using HE staining,the levels of TNF-α,TGF-β,IL-1αand IL-6 in the lung tissue and serum were detected using ELISA,and the percentages of lymphocytes in the lung tissue were analyzed with flow cytometry.High-throughput sequencing of exosome miRNA was carried out to explore the changes in the signaling pathways.The mRNA expression levels of the immune-related genes were detected by qRT-PCR,and the protein expressions of talin-1,tensin2,FAK,vinculin,α-actinin and paxillin in the focal adhesion signaling pathway were detected with Western blotting.Results In the mouse models of radiation pneumonitis,injections of normal mouse serum significantly decreased the lung organ coefficient,lowered the levels of TNF-α,TGF-β,IL-1αand IL-6 in the serum and lung tissues,and ameliorated infiltration of CD45^(+),CD4^(+)and Treg lymphocytes in the lung tissue(all P<0.05).The expression levels of Egfr and Pik3cd genes at both the mRNA and protein levels and the protein expressions of talin-1,tensin2,FAK,vinculin,α‑actinin and paxillin were all significantly down‑regulated in the mouse models after normal mouse serum treatment.Conclusion Normal mouse serum ameliorates radiation pneumonitis in mice by inhibiting the expressions of key proteins in the Focal adhesion signaling pathway.
基金the National Natural Science Foundation of China,No.81860115,No.82060116 and No.81960118the Science and Technology Support Project of Guizhou Province,No.[2021]094.
文摘BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.
文摘Focal adhesion kinase(FAK)is a 125-kDa non-receptor protein tyrosine.Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases,resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation.FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract.FAK also plays an important role in the restitution,cell survival and apoptosis and carcinogenesis of the gastrointestinal tract.FAK is over-expressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells.FAK has been proposed as a potential target in cancer therapy.Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells,indicating a high potential for application in cancer therapy.
基金Supported by the Royal Golden Jubilee PhD Program of the Thailand Research Fund (RGJ/PHD/0112/2542)
文摘AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.
基金Supported by The National Natural Science Foundation of China(No. 81072806)The National Natural Science Foundation of China for Young Scholars(No. 811102581)The Chinese Medicines Agency Project of Guangdong Province(No.2010098)
文摘OBJECTIVE: To investigate the effect of Jianpijiedu Fang (JPJDF) on phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and focal adhesion kinase (FAK), and on the survival of hepatocellular carcinoma (HCC) nude mice. METHODS: Forty male nude mice were randomly divided into 4 groups. Human HCC tissue was implanted in the livers of three groups. After 24 h, the three groups were treated respectively with JPJDF (37.5 g/kg), saline (20 mL/kg) and Tegafur (FT-207, 160 mg/kg) once a day for 10 weeks. The control group without implanting the tissue was concurrently treated with saline (20 mL/kg). The survival data and body weight of all mice were recorded, and expression levels of PTEN, PI3K and FAK in normal tissue and cancer tissue of the livers were eval-uated with immunohistochemical method. RESULTS: The cumulative survival rate of the mice in the JPJDF group was higher than those of the other groups. The rate of weight loss was the lowest in JPJDF group. The survivability and weight loss rate in FT-207 group were the poorest in all groups. The expression intensity of PTEN was higher in normal tissues than in cancer tissues, and lower in the normal tissues of HCC models than in that of mice without HCC. The PTEN expression intensity in normal tissue and cancer tissue from mice treated with FT-207 were lower than that from the mice treated with JPJDF or saline.The expression intensity of PI3K was higher in cancer tissue than in normal tissue. The PI3K expression intensity was the lowest in normal tissue and cancer tissue from mice treated with JPJDF, and the intensity from mice treated with FT-207 was the highest. In mice treated with JPJDF, the expression intensity of FAK was higher in the normal tissue and lower in the cancer tissue than those of the other treatment groups. CONCLUSION: The mechanism accounting for the prolonged survival of HCC-bearing mice treated with JPJDF might be related to the reduction in weight loss and the benign regulation of PTEN, PI3K, and FAK.
文摘Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.
基金Supported by grants from National Science Foundation of China （No. 30873038） and Young Research Foundation of Health Department of Hubei Province, China （No. QJX2008-3）. We are grateful to Dr. Zhaokang Hu （North Carolina State University） for helpful discussion.
文摘Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.
文摘Malignant transformation of normal cells involves important structural and functional changes, particularly in cell adhesion. In this study, we wanted to assess whether changes in the expression of FAK, a tyrosine kinase, which is recruited to focal adhesions and plays a key role in cell migration, proliferation and survival, could reflect the invasive capacity of bladder carcinomas. The aim of this study was to evaluate the FAK expression in cancer ceils as an important prognostic factor of the evolution of bladder carcinomas. Tumor and paired peritumoral biopsies were obtained during transurethral endoscopic resection or cystectomy of bladder tumors in 280 patients at the Urology Unit of the Mustapha Hospital of Algiers and the Hospital of Tizi-Ouzou （Algeria）. The authors studied FAK expression in samples from bladder carcinomas at different stages of malignant transformation by western blot analysis using a specific anti-FAK antibody. Western blot is one of the most common laboratory techniques; it is used to detect the presence of a specific protein in a complex mixture extracted from cells. A weak increase in FAK expression was observed in tumors of grade 1 and 2 （1.65; 2.99） as compared to healthy tissues; it became particularly important in grade 3 tumors; the authors show that FAK levels significantly increased gradually according to the tumor stage.
基金supported by the National Natural Science Foundation of China(Grant No.11372279)
文摘Cells actively sense and respond to mechanical signals from the extracellular matrix through focal adhesions. By representing a single focal adhesion as a cluster of slip bonds, it has been demonstrated that the cluster often became unstable under fluctuated forces. However, an unusual case was also reported, where the stability of the cluster might be substantially enhanced by a fluctuated force with a relatively low fluctuation frequency and high fluctuation amplitude.Such an observation cannot be explained by the conventional fracture theory of fatigue. Here, we intensively investigate this intriguing observation by carrying out systematic parametric studies. Our intensive simulation results indicate that stability enhancement of this kind is in fact quite robust, which can be affected by the stochastic features of a single bond and the profile of the fluctuated forces such as the average value of bond force. We then suggest that the fluctuation of traction force within a focal adhesion might enhance its stability in a certain way.
文摘Chen et al demonstrated that regulator of G protein signaling(RGS)4 promotes gastric cancer(GC)progression by activating the focal adhesion kinase/phospha-tidyl-inositol-3-kinase/protein kinase B pathway and inducing epithelial-mesen-chymal transition.Although their multilevel approach integrating clinical data,functional assays,and xenograft models demonstrated a key role for RGS4 in GC pathogenesis,several limitations should be considered.The mechanism of the RGS4-focal adhesion kinase interaction remains unclear,specifically whether it involves direct binding or intermediaries.The clinical analysis of 90 patients lacks stratification by GC subtypes or immune features,potentially limiting generaliz-ability.Furthermore,fully validating RGS4’s oncogenic role requires additional studies,including functional assays in chemotherapy-resistant and metastatic cell lines,metastasis models including orthotopic implantation and tail vein injection,and comparison with other RGS family members.Addressing these via targeted mechanistic studies and expanded clinical validation could strengthen RGS4’s po-tential as a therapeutic target in GC.
基金supported by the National Natural Science Foundation of China(grant numbers 12132006,32371373,31771014,and 82060555).
文摘Distal metastasis is the main cause of clinical treatment failure in patients with colon cancer.It is now known that the invasion and metastasis of cancer cells is precisely regulated by chemical and physical factors in vivo.However,the role of extracellular matrix(ECM)stiffness in colon cancer cell(CCCs)invasion and metastasis remains unclear.Here,bioinformatical analysis suggested that a high expression level of yes associated protein 1(YAP1)was significantly associated with metastasis and poor prognosis in colon cancer patients.We further investigated the effects of polyacrylamide hydrogels with different stiffnesses(3,20,and 38 kPa),which were simulated as ECM,on the mechanophenotype(F-actin cytoskeleton organization,electrophoretic rate,membrane fluidity,and Young's modulus)of CCCs.The results showed that a stiffer ECM could induce the maturation of focal adhesions and formation of stress fibers in CCCs,regulate their mechanophenotypes,and promote cell motility.We also demonstrated that the expression levels of YAP1 and paxillin were positively correlated in patients with colon cancer.YAP1 knockdown reduces paxillin clustering and cell motility and alters the cellular mechanophenotypes of CCCs.This is of great significance for an in-depth understanding of the invasion and metastatic mechanisms of colon cancer and for the optimization of clinical therapy from the perspective of mechanobiology.
基金supported by grants from the Deutsche Forschungsgemeinschaft[DFG,German Research FoundationRTG 2467,project number 391498659‘Intrinsically disordered proteins—molecular principles,cellular functions,and diseases’+1 种基金FOR 5433,project number 468534282‘RNA in focus(RIF):from mechanisms to novel therapeutic strategies in cancer treatment’]the Wilhelm-Roux-Program(Medical Faculty,Martin-Luther-University Halle–Wittenberg).
文摘Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants.However,it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing.Hence,we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail.By using a combination of in silico target prediction and in-cell validation,we identified several focal adhesion proteins as alternative splicing targets of Rbfox1.We focused on the alternative splicing patterns of vinculin(metavinculin isoform)and paxillin(extended paxillin isoform)and identified both as potential Rbfox1 targets.Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns.Focal adhesions play an important role in the cardiac muscle context,since they mainly influence cell shape,cytoskeletal organization,and cell–matrix association.Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology,cytoskeletal organization,and multinuclearity after differentiation,which might be due to changes in alternative splicing of focal adhesion proteins.Hence,our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells,which might contribute to heart disease progression,where downregulation of Rbfox1 is frequently observed.
文摘OBJECTIVE: To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth muscle cells (SMCs) adhesion and migration stimulated by fibronectin. METHODS: Adhesion and migration of cultured SMCs were stimulated by different concentrations of fibronectin (FN), FAK and its phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation. SMCs adhesion and migration were also measured by morphological enumeration and modified Boyden Chambers, respectively. RESULTS: FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 microg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5 - 60 microg/ml FN groups was reduced by 17.89% - 27.67%. Cell migration stimulated by FN at 10, 20, 40 and 60 microg/ml were reduced by 23.26%, 21.63%, 19.31% and 17.88%, respectively (P
文摘To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin Methods Adhesion and migration of cultured SMCs were stimulated by different concentrati ons of fibronectin (FN), FAK and its phosphorylation were detected by immunoprec ipitation and Western blot FAK antisense oligodeoxynucleotides (ODNs) were tra nsfected into SMCs by cationic lipid to investigate its modulatory effects on ty rosine phosphorylation SMCs adhesion and migration were also measured by morph ological enumeration and modified Boyden Chambers, respectively Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN FAK phosphorylation were detected only at 20 ?μg/ml FN or more FAK antisense ODNs were transfected efficiently by cationi c lipid and FAK phosphorylation was inhibited substantially The SMCs migration rate in the 5-60?μg/ml FN groups was reduced by 17 89%-27 67% Cell migrat ion stimulated by FN at 10, 20, 40 and 60?μg/ml were reduced by 23 26%, 21 6 3%, 19 31% and 17 88%, respectively ( P 【0 05) Conclusions FAK phosphorylation and FAK mediated signal transduction play important roles i n SMCs adhesion and migration stimulated by ECM The process can be inhibited e ffectively by FAK antisense ODNs
基金This work was supported by grants from the National Natural Scientific Foundation of China （No. 81072078 and No. 30872657）, Jiangsu Province＇s Natural Science Foundation （No. BK2008475, No. 2009444 and No. 2010580）, the Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU, and Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Background Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion. Methods Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase （FAK） as a target of miR-7. The levels of miR-7, matrix metalloproteinases （MMP）-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase （AKT）, and extracellular signal-regulated kinase （ERK） 1/2 were measured by Western blotting analysis. Results Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3＇UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues. Conclusion To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.
基金supported,in part,by the National Key Research and Development Program of China Grants(2019YFA0906004)the National Natural Science Foundation of China Grants(82230081,82250710175,81991513 and 81870532)+1 种基金the Guangdong Provincial Science and Technology Innovation Council Grant(2017B030301018,China)the Shenzhen Municipal Science and Technology Innovation Council Grant(20200925150409001,China).
文摘The skeletal system,which contains bones,joints,tendons,ligaments and other elements,plays a wide variety of roles in body shaping,support and movement,protection of internal organs,production of blood cells and regulation of calcium and phosphate metabolism.The prevalence of skeletal diseases and disorders,such as osteoporosis and bone fracture,osteoarthritis,rheumatoid arthritis,and intervertebral disc degeneration,increases with age,causing pain and loss of mobility and creating a huge social and economic burden globally.Focal adhesions(FAs)are macromolecular assemblies that are composed of the extracellular matrix(ECM),integrins,intracellular cytoskeleton and other proteins,including kindlin,talin,vinculin,paxillin,pinch,Src,focal adhesion kinase(FAK)and integrin-linked protein kinase(ILK)and other proteins.FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell–environment communications and modulates important processes,such as cell attachment,spreading,migration,differentiation and mechanotransduction,in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways.This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.
文摘Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling At present, the mechanisms related to proliferation of PASMCs are not clear Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs) We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein Methods Cultured HPASMCs stimulated by fibronectin (40 μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense FAK respectively Expression of FAK, Jun NH2 terminal kinase (JNK), cyclin dependent kinase 2 (CDK 2) and caspase 3 proteins were detected by immunoprecipitation and Western blots Cell cycle and cell apoptosis were analysed by flow cytometry In addition, cytoplasmic FAK expression was detected by immunocytochemical staining Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense FAK ODNs group and increased in sense FAK ODNs group significantly Caspase 3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs When compared with mismatch sense ODNs group, the proportion of cells at G 1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly In contrast, compared with mismatch sense ODNs group, the proportion of cells at G 1 phase was increased significantly in antisense FAK ODNs group The level of cell apoptosis in antisense FAK group was higher than in the mismatch sense group and the latter was higher than sense FAK group In addition, the sense FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense FAK ODNs group was weakly stained Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase 3 inhibits HPASMCs apoptosis
