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PubMed2005

COX-2 inhibitors suppress integrin alpha5 expression in human lung carcinoma cells through activation of Erk: involvement of Sp1 and AP-1 sites.

Tumor cell expression of COX-2 has been implicated in the progression of murine and human lung cancer. Inhibition of COX-2 by nonsteroidal antiinflammatory drugs reduces the risk of cancer development in humans and suppresses tumor growth in animal models. However, the underlying mechanisms for this beneficial effect are not fully understood. Here we explore the potential link between the anticancer effects of COX-2 inhibitors and the expression of the integrin alpha5beta1. Expression of this integrin in carcinoma cells is associated with invasiveness and malignant progression. This, together with our studies showing that fibronectin, the ligand of alpha5beta1, stimulates the growth of human lung carcinoma cells, and that this effect is mediated through alpha5beta1-dependent signals, has prompted us to examine the effects of COX-2 inhibitors on alpha5beta1 expression in human non small cell lung carcinoma (NSCLC) cells. We found that the selective COX-2 inhibitors NS398 and Nimesulide decreased mRNA expression and protein production of the integrin alpha5 subunit. This effect was associated with inhibition of NSCLC cell adhesion to fibronectin. The COX-2 inhibitors triggered the phosphorylation of extracellular signal-regulated kinase (Erk) in a time-dependent manner, and the inhibitor of Mek-1/Erk PD98095 prevented their inhibitory effects on integrin alpha5 expression. Transient transfection assays showed that the COX-2 inhibitors affected integrin alpha5 gene transcription by acting between -92 to -41 bp of the human integrin alpha5 gene promoter. Gel mobility shift assays showed that the COX-2 inhibitors increased Sp1 DNA binding, but decreased that of AP-1. These effects were accompanied by an increase in Sp1 protein and a decrease in c-Jun protein expression, as well as inhibition of SAPK/JNK phosphorylation. The Sp1 inhibitor, Mithramycin A, also blocked the inhibitory effect of the COX-2 inhibitors on alpha5 expression and promoter activity. Overall, these findings suggest that COX-2 inhibitors suppress alpha5beta1 integrin expression in NSCLC through effects on integrin alpha5 gene transcription mediated by Erk activation, increased Sp1, decreased AP-1 DNA binding and inactivation of SAPK/JNK signals. Our observations unveil a new mechanism of action against NSCLC for COX-2 inhibitors that relates to regulation of integrin alpha5 gene expression and, consequently, recognition of extracellular matrices (i.e., fibronectin) by tumor cells. (c) 2005 Wiley-Liss, Inc.

Carcinoma, Non-Small-Cell Lung↗
PubMed2006

The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity.

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.

ADAM Proteins↗
PubMed2003

CAR is a cell-cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFalpha, and TGFbeta.

The coxsackie adenovirus receptor (CAR) has become of interest for gene therapy due to its crucial function in adenoviral cell entry. In clinical trials with adenoviral vectors, dexamethasone is applied to reduce side effects such as inflammatory reactions or emesis. By using a beta-galactosidase-expressing adenovirus (AdGal), we observed that dexamethasone treatment resulted in decreased adenoviral gene transfer into human cancer cells. Expression of CAR and integrin alpha5beta1 was transcriptionally downregulated by dexamethasone as shown for HeLa cervical cancer cells and U87MG glioblastoma cells. TNFalpha increased CAR expression in HeLa and ovarian cancer cells but decreased CAR expression in U87MG cells. In all tested cancer cell lines, TNFalpha induced a significant increase in the expression of adenovirus-binding integrins alpha5beta1, alphavbeta3 and alphavbeta5. Pretreatment with TNFalpha increased AdGal gene transfer into cancer cells and enhanced the cytotoxic effect of a p53-expressing adenovirus. In contrast, TGFbeta reduced CAR expression level and adenoviral gene transfer into OV-UL-2 ovarian cancer cells. Confocal immunofluorescence analysis revealed localization of CAR at cell-cell adhesions in several human cancer cell lines and disruption of cell-cell contacts increased adenoviral gene transfer into human cancer cells. In clinical cancer gene therapy, efficiency of adenoviral gene delivery could be altered by cell adhesion, TNFalpha, TGFbeta, and dexamethasone.

Adenoviridae↗
PubMed2000

A novel RGD-independent fibronectin assembly pathway initiated by alpha4beta1 integrin binding to the alternatively spliced V region.

Fibronectin (FN) matrix assembly is a multi-step process that involves binding to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton. Ultimately, FN is converted into stable matrix fibrils that are detergent-insoluble. RGD-binding integrins such as alpha5beta1 play a major role in the assembly of fibrillar FN. Here we show that alpha4beta1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of alpha4beta1 with exogenous agents such as Mn(2+) or a beta1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)alpha4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25/CS-1 segment. Previously, we have characterized an activated recombinant FN (FN III(1-7)) that rapidly forms detergent-insoluble multimers upon binding to alpha5beta1 integrin. Alpha4beta1 also formed FNdeltaIII(1-7) multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin. In contrast to assembly by alpha5beta1, actin filaments remained largely cortical and no change in cell growth rate was observed with alpha4beta1-mediated assembly. These results show that binding sites on FN other than the RGD sequence/synergy site and distant from the cell binding domain can promote FN assembly. Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.

Alternative Splicing↗
PubMed2005

Quantitatively distinct requirements for signaling-competent cell spreading on engineered versus natural adhesion ligands.

To design synthetic microenvironments that elicit desired cell behaviors, we must better understand the molecular mechanisms by which cells interact with candidate biomaterials. Using cell lines with distinct alpha5beta1 integrin expression profiles, we demonstrate that this integrin mediates cell spreading on substrata coated with genetically engineered artificial extracellular matrix (aECM) proteins containing the RGD sequence (RGD-containing aECM protein [aRGD]) but lacking the PHSRN synergy site. Furthermore, aRGD-mediated adhesion stimulates an intracellular focal adhesion kinase (FAK) signal that is indicative of integrin tethering. Although both aRGD and the natural ECM protein fibronectin (FN) support alpha5beta1 integrin-mediated cell spreading, quantitative single-cell analysis revealed that aRGD-mediated spreading requires ten-fold greater threshold amount of integrin expression than FN-mediated spreading. Our analysis demonstrates that aRGD-based substrata mediate both biophysical (cell spreading) and biochemical (FAK signaling) events via the alpha5beta1 integrin, albeit with efficacy quantitatively distinct from that of natural ECM proteins that possess the full spectrum of adhesion and synergy domains.

Amino Acid Sequence↗
PubMed1997

The 80-kD fibronectin fragment increases the production of fibronectin and tumour necrosis factor-alpha (TNF-alpha) in cultured mesangial cells.

The presence of fibronectin (FN) fragments has been demonstrated in several inflammatory processes, and they have been implicated in the recruitment of mononuclear cells. However, the interaction of these FN fragments with resident cells has hardly been studied. We have hypothesized that the 80-kD FN fragment, which includes the RGD cell binding domain of FN, could contribute to the pathogenesis of glomerular damage through the interaction with mesangial cells (MC) via alpha5beta1 integrin. Since an increase in the glomerular deposit of matrix components, particularly FN, is frequently observed in progressive glomerulonephritis, we studied whether its synthesis is modulated by the 80-kD FN fragment and the native FN molecule. While the 80-kD FN fragment stimulated FN in a dose-dependent manner, both at the mRNA and protein level, the whole FN molecule exerted a dual effect. High doses produced FN inhibition, while low doses elicited a certain increase. This stimulation was abrogated by the presence of Sam-1, a MoAb against the alpha-subunit of the alpha5beta1 integrin. Since cytokines play a fundamental role in glomerular injury, we studied the production of TNF-alpha, one of the most powerful mediators of inflammation. TNF-alpha synthesis was induced by the 80-kD FN fragment, in a dose-dependent manner, but not by native FN. When MC were incubated with the 29- and 31-kD FN fragments, which lack the RGD cell binding domain, TNF-alpha secretion was not detected. These results strongly suggest that in cultured MC, the 80-kD FN fragment induces the synthesis of matrix proteins such as FN, and cytokines such as TNF-alpha, via alpha5beta1 integrin. This mechanism could contribute to the perpetuation of renal injury.

Animals↗
PubMed1998

Role of integrins in mouse eyelid development: studies in normal embryos and embryos in which there is a failure of eyelid fusion.

Eyelid fusion normally occurs between E15.5 and E16.5 of mouse embryonic development and results from the migration of a population of periderm-derived epithelial cells over the corneal surface. Cell migration is known to depend on extracellular matrix receptors of the integrin family and to be regulated by growth factors. We were therefore interested that a failure of eyelid fusion has been reported in mice that are homozygous null for the transforming growth factor alpha (TGF-alpha) gene and in mice (invalpha5beta1) in which a transgenic alpha5beta1 integrin under the control of the involucrin promoter is misexpressed in differentiating keratinocytes. We examined expression of the alpha2beta1, alpha3beta1, alpha5beta1 and alpha6beta4 integrins during eyelid fusion in wild-type embryos and found selective upregulation of the alpha5beta1 integrin and its ligand, fibronectin, in the migrating eyelid tip cells. In TGF-alpha null embryos, the failure of eyelid fusion was correlated with a failure to upregulate the alpha5beta1 integrin and fibronectin in the tip cells. Using beta-galactosidase as a reporter gene in transgenic mice, we observed specific activity of the involucrin promoter in the eyelid tip cells. In invalpha5beta1 mice the transgenic human integrin was overexpressed not only in the tip cells but throughout the eyelid epidermis. In contrast, the endogenous, murine, alpha5beta1 integrin was only weakly expressed in the tip cells. We speculate that selective and coordinated expression of the alpha5beta1 integrin and fibronectin in eyelid tip cells is required for eyelid fusion and may be under the control of growth factors that include TGF-alpha.

Animals↗
PubMed2005

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.

Animals↗
PubMed2002

Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding.

Integrins represent the primary mechanism of cell-extracellular matrix interactions and control cell morphology, proliferation, and differentiation. We have previously shown that substrate-dependent modulation of adsorbed fibronectin (Fn) conformation alters alpha5beta1 integrin binding to Fn and directs C2C12 myoblast proliferation and differentiation (Mol. Biol. Cell 10 (1999) 785). The model substrates used in these experiments were bacteriological (untreated) polystyrene (B), tissue culture polystyrene (T), and type-I collagen-coated T (C). In the present study, we examined MC3T3-EI osteoblast-like cell differentiation on Fn-coated B, T, and C substrates. Immunofluorescence staining revealed substrate-dependent differences in integrin alpha5beta1 binding and clustering into focal adhesions (C > T > B), consistent with our previous integrin binding analysis. Alkaline phosphatase activity and matrix mineralization showed substrate-dependent differences (C > T > B, p < 0.05). Similar trends were observed for alkaline phosphatase, osteocalcin, and bone sialoprotein gene expression. Blocking experiments with antibodies directed against Fn completely inhibited matrix mineralization on Fn-coated C, indicating that Fn is critical to expression of the osteoblastic phenotype on this extracellular matrix component. These substrate-dependent differences in osteoblast differentiation correlated with differences in alpha5beta1 binding, suggesting that these differences arise from substrate modulation of integrin-matrix interactions. Substrate-dependent modulation of cell function may provide a versatile mechanism to control cell responses in numerous biomedical applications.

Alkaline Phosphatase↗
PubMed2003

Skeletal muscle LIM protein 1 (SLIM1/FHL1) induces alpha 5 beta 1-integrin-dependent myocyte elongation.

Skeletal muscle LIM protein 1 (SLIM1/FHL1) contains four and a half LIM domains and is highly expressed in skeletal and cardiac muscle. Elevated SLIM1 mRNA expression has been associated with postnatal skeletal muscle growth and stretch-induced muscle hypertrophy in mice. Conversely, SLIM1 mRNA levels decrease during muscle atrophy. Together, these observations suggest a link between skeletal muscle growth and increased SLIM1 expression. However, the precise function of SLIM1 in skeletal muscle, specifically the role of SLIM1 during skeletal muscle differentiation, is not known. This study investigated the effect of increased SLIM1 expression during skeletal muscle differentiation. Western blot analysis showed an initial decrease followed by an increase in SLIM1 expression during differentiation. Overexpression of SLIM1 in Sol8 or C2C12 skeletal muscle cell lines, at levels observed during hypertrophy, induced distinct effects in differentiating myocytes and undifferentiated reserve cells, which were distinguished by differential staining for two markers of differentiation, MyoD and myogenin. In differentiating skeletal myocytes, SLIM1 overexpression induced hyperelongation, which, by either plating cells on poly-l-lysine or using a series of peptide blockade experiments, was shown to be specifically dependent on ligand binding to the alpha5beta1-integrin, whereas in reserve cells, SLIM1 overexpression induced the formation of multiple cytoplasmic protrusions (branching), which was also integrin mediated. These results suggest that SLIM1 may play an important role during the early stages of skeletal muscle differentiation, specifically in alpha5beta1-integrin-mediated signaling pathways.

Animals↗
PubMed1997

Activation of beta1 integrins on CML progenitors reveals cooperation between beta1 integrins and CD44 in the regulation of adhesion and proliferation.

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the alpha4beta1 and alpha5beta1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of alpha4, alpha5, and beta1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that beta1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the beta1 integrin activating antibody, 8A2, and demonstrated a role for the alpha5beta1 integrin in this phenomenon. Since the integrin alpha4beta1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between beta1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN but also to alpha4beta1, alpha5beta1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the alpha5, alpha4, or beta1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of beta1 integrins on CML Ph+ CFC not only results in upregulation of beta1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between beta1 integrins and CD44. These studies suggest that decreased beta1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML.

Antigens, CD↗
PubMed2004

Inhibition of alpha5 integrin decreases PI3K activation and cell adhesion of human colon cancers.

BACKGROUND: Phosphatidylinositol 3-kinase (PI3K), acting through the downstream kinase AKT, regulates a number of cellular processes such as cell growth and survival. Poorly differentiated colon cancers are characterized by increased integrin-mediated extracellular matrix interactions; disruption of this integrin-mediated adhesion leads to apoptosis and involves reduced PI3K activity. The purpose of this study was to determine: (1) the role of alpha5beta1 integrin expression on PI3K activity, and (2) whether blockade of alpha5beta1 integrin decreases colon cancer cell adhesion and increases apoptosis. METHODS: Human colon cancer cells were treated with anti-alpha5 integrin antibody, anti-beta1 integrin antibody, the PI3K inhibitor LY294002 (20 micromol/L), or wortmannin (400 nmol/L). Expression and function of alpha5 were quantitated by FACS analysis and cell adhesion assay, respectively. DNA fragmentation was measured to assess apoptosis. Protein was extracted to determine phosphorylated AKT (pAKT) expression as a measure of PI3K activity. RESULTS: Increased pAKT and alpha5 integrin expression were noted in the colon cancer cells; blockade of alpha5 integrin decreased alpha5 integrin and pAKT expression and decreased cell adhesion. Moreover, PI3K inhibition resulted in decreased alpha5 integrin expression. In contrast, treatment with anti-beta1 integrin antibody produced no change in pAKT expression or cell number. CONCLUSION: Our results are the first to show that blockade of cell surface alpha5 integrin expression decreases PI3K activity, inhibits colon cancer cell attachment, and induces apoptosis. These findings suggest that agents which selectively target alpha5 integrin subunit expression may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.

Apoptosis↗
PubMed2006

Evidence for a differential expression of fibronectin splice forms ED-A and ED-B in Crohn's disease (CD) mucosa.

BACKGROUND AND AIMS: Fibronectin (FN) is an essential factor for the induction of migration of primary colonic lamina propria fibroblasts (CLPF). The FN isoform ED-A is an important inducer of migration. Recently, we have shown that CLPF isolated from inflamed Crohn's disease (CD) mucosa migrated significantly less than control CLPF. We, therefore, investigated changes in FN or integrin expression that could be relevant for CLPF migration. MATERIALS AND METHODS: mRNA of control-CLPF and CLPF isolated from fibrotic mucosa of CD patients was subtractively hybridized. Expression of FN, ED-A, and ED-B in frozen sections from intestinal mucosa was determined by immunohistochemistry. The mRNA expression of the FN isoforms in control, CD, and fibrosis biopsies was quantified by real-time polymerase chain reaction (PCR). Integrin alpha5beta1 protein and mRNA expression was analyzed by fluorescence activated cell sorting (FACS) and PCR, respectively. RESULTS: Subtractive hybridization indicated differential regulation of FN isoform expression in CD. The immunohistochemical analysis of FN protein revealed a reduction of FN isoforms in inflamed CD mucosa compared to control mucosa. In CD fistulae, the ED-A and ED-B isoforms were virtually absent. In fibrotic mucosa, both proteins were increased. Real-time PCR showed a decrease of FN and ED-A expression during mucosal inflammation in CD in contrast to UC and a significant increase of FN and isoforms in CD fibrosis. No difference was found for protein and mRNA of integrin alpha5beta1 in control, CD, and fibrosis CLPF by FACS and PCR. CONCLUSION: Downregulated expression of migration-inducing FN-isoforms in contrast to unchanged FN receptor expression may contribute to the observed alterations of CD CLPF migration.

Adult↗
PubMed1999

Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin.

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins.

Antibodies↗
PubMed2004

The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK.

Nischarin, a novel intracellular protein, was originally identified as a binding partner for the alpha5beta1 integrin. Here we show that Nischarin also interacts with members of the PAK family of kinases. The amino terminus of Nischarin preferentially binds to the carboxy-terminal domain of PAK1 when the kinase is in its activated conformation. Nischarin binding to PAK1 is enhanced by active Rac, with the three proteins forming a complex, while expression of the alpha5beta1 integrin also increases the Nischarin/PAK1 association. Interaction with Nischarin strongly inhibits the ability of PAK1 to phosphorylate substrates. This effect on PAK kinase activity closely parallels Nischarin's ability to inhibit cell migration. Conversely, reduction of endogenous levels of Nischarin by RNA interference promotes cell migration. In addition, PAK1 and Nischarin colocalize in membrane ruffles, structures known to be involved in cell motility. Thus, Nischarin may regulate cell migration by forming inhibitory complexes with PAK family kinases.

Animals↗
PubMed1998

Prevention of arterial structural alterations with verapamil and trandolapril and consequences for mechanical properties in spontaneously hypertensive rats.

We compared the chronic effects in spontaneously hypertensive rats (SHR) of low doses of an angiotensin converting enzyme inhibitor, trandolapril, a Ca2+ channel antagonist, verapamil, and their combination (trandolapril-verapamil), on arterial mechanical properties, arterial wall hypertrophy and extracellular matrix proteins. Four-week-old SHR were randomly allocated to oral treatment with verapamil (50 mg kg(-1) day(-1)), trandolapril (0.3 mg kg(-1) day(-1)), the combination of verapamil (50 mg kg(-1) day(-1)) plus trandolapril (0.3 mg kg(-1) day(-1)), or placebo for 4 months. A group of Wistar Kyoto (WKY) control rats received placebo for the same period of time. At the end of the treatment, mean blood pressure was lower in verapamil-trandolapril than in trandolapril SHR, but remained higher than in WKY. Verapamil had no effects on blood pressure. Equivalent reduction in aortic wall hypertrophy was obtained in all treated SHR. Trandolapril and verapamil-trandolapril combination produced a significant reduction of aortic collagen density compared with placebo SHR. Carotid total fibronectin, EIIIA fibronectin isoform and alpha5beta1 integrin, were higher in the media of placebo SHR than in WKY. EIIIA fibronectin isoform and alpha5beta1 integrin were reduced in verapamil-SHR compared with placebo-SHR and normalized in trandolapril and verapamil-trandolapril-SHR compared with WKY. SHR-placebo and SHR treated with either verapamil or trandolapril as single-drug treatment showed a 4-fold increase in total fibronectin compared to the WKY. Only SHR treated with verapamil-trandolapril combination had total fibronectin not significantly different from that of WKY. Carotid arterial distensibility increased only in verapamil-trandolapril treated rats. Multivariate analysis showed arterial distensibility to be negatively correlated to mean blood pressure (P < 0.0001) and total fibronectin (P < 0.01). In conclusion, chronic treatment with the verapamil-trandolapril combination significantly improved in vivo arterial distensibility in SHR. The most important effects of the combination on arterial mechanics compared to those of verapamil or trandolapril alone may have been the consequence of its stronger action on arterial pressure, arterial wall hypertrophy and total fibronectin density. However we suggest that, in addition to the structural effects, complete normalization of blood pressure is necessary to obtain normal arterial distensibility.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2006

The microtubule binding drug laulimalide inhibits vascular endothelial growth factor-induced human endothelial cell migration and is synergistic when combined with docetaxel (taxotere).

Laulimalide, a natural product from marine sponges, is a microtubule-stabilizing agent that binds to tubulin at a site distinct from that of the taxoids. In the present study, we found that laulimalide inhibited human umbilical vein endothelial cell (HUVEC) tubule formation and vascular endothelial growth factor (VEGF)-induced HUVEC migration, key components of the angiogenic process. These occurred at concentrations substantially lower than that which inhibited HUVEC proliferation. When combined, laulimalide and docetaxel (Taxotere) synergistically inhibited migration and tubule formation, but their combined effect on proliferation was antagonistic. Possible mechanism(s) by which laulimalide inhibited VEGF-induced HUVEC migration were explored. Similar to docetaxel, laulimalide had no effect on the VEGF-induced tyrosine phosphorylation of the VEGF receptor Flk-1/KDR (VEGFR-2). Low concentrations of laulimalide substantially blocked subsequent VEGFR-2 downstream events, as did docetaxel, including the phosphorylation of the Tyr397 and Tyr407 residues of focal adhesion kinase (FAK), the association of VEGFR-2 with FAK and Hsp90, and the Tyr31 phosphorylation of paxillin. Laulimalide inhibited integrin activation; however, compared with docetaxel, it had a weaker inhibitory effect on the VEGF-induced association of VEGFR-2 with the alpha5beta1 integrin. Compared with docetaxel, laulimalide more potently caused a reduction in the constitutive levels (i.e., in the absence of VEGF) of phosphorylated paxillin and more potently inhibited the association of RhoA with the alpha5beta1 integrin. In conclusion, although both docetaxel and laulimalide inhibited integrin-associated signaling pathways that mediated VEGF-induced cell migration, their actions on the signaling cascade seemed not to be identical. These complementary actions could account for their synergistic effects on HUVEC.

Cell Movement↗
PubMed2002

Regulation of fibronectin fibrillogenesis by protein kinases in cultured rat osteoblasts.

Fibronectin (Fn) plays an important role in the regulation of adhesion, migration, and maturation of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, Fn synthesis and assembly were examined in cultured osteoblasts. Osteoblasts assembled the endogenously released soluble Fn into immobilized form on the substratum in a time-dependent manner. Both 12-O-tetradecanoylphorbol-13 acetate (TPA) and forskolin increased the synthesis of Fn. However, the extracellular assembly of Fn fibril from both endogenously released and exogenously applied soluble Fn was increased by TPA but decreased by forskolin. Protein kinase C (PKC) inhibitors, such as H7, Ro 318220, and Gö 6976, inhibited Fn fibrillogenesis. These results suggest that the dynamic of Fn fibrillogenesis is differentially regulated by the activation of PKC and protein kinase A (PKA). Both classic and novel isoforms of PKC are involved in the action of TPA in osteoblasts. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. Immunocytochemistry and flow cytometry showed that TPA and forskolin increased and inhibited, respectively, the clustering and surface expression of alpha5 integrins. TPA and forskolin did not affect protein levels of alpha5 integrins. The Western blot and reverse transcriptase-polymerase chain reaction showed that protein and mRNA levels of beta1 integrins also were not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the surface expression of alpha5beta1 integrins. cAMP response element-binding protein phosphorylation is involved in the action of forskolin but not that of TPA. Our results suggest that PKC activation enhanced Fn fibrillogenesis, whereas PKA activation inhibited extracellular Fn fibrillogenesis in primary cultured osteoblasts. Cytosolic Fn synthesis and extracellular Fn assembly may be differentially regulated by the activation of PKA.

Animals↗