Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for alpha-actinin, vinculin, paxillin and tensin, the integrin chains alpha1 and beta1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125(FAK) did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.
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PMID:Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix. 936 35

We previously reported that fully assembled basement membranes are nonpermissive to smooth muscle cell (SMC) replication and that perlecan (PN), a basement membrane heparan sulfate proteoglycan, is a dominant effector of this response. We report here that SMC adhesion to basement membranes, and perlecan in particular, up-regulate the expression of focal adhesion kinase-related nonkinase (FRNK), a SMC-specific endogenous inhibitor of FAK, which subsequently suppresses FAK-mediated, ERK1/2-dependent growth signals. Up-regulation of FRNK by perlecan is actively and continuously regulated. Relative to the matrix proteins studied, the effects are unique to perlecan, because plating of SMCs on several other basement membrane proteins is associated with low levels of FRNK and corresponding high levels of FAK and ERK1/2 phosphorylation and SMC growth. Perlecan supports SMC adhesion, although there is reduced cell spreading compared with fibronectin (FN), laminin (LN), or collagen type IV (IV). Despite the reduction in cell spreading, we report that perlecan-induced up-regulation of FRNK is independent of cell shape changes. Growth inhibition by perlecan was rescued by overexpressing a constitutively active FAK construct, but overexpressing kinase-inactivated mutant FAK or FRNK attenuated fibronectin-stimulated growth. These data indicate that perlecan functions as an endogenously produced inhibitor of SMC growth at least in part through the active regulation of FRNK expression. FRNK, in turn, may control SMC growth by downregulating FAK-dependent signaling events.
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PMID:Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signaling. 1280 66

We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.
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PMID:Perlecan-induced suppression of smooth muscle cell proliferation is mediated through increased activity of the tumor suppressor PTEN. 1465 29

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, alpha2beta1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and alpha2beta1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin.
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PMID:Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through alpha2beta1 integrin. 1524 May 72

Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing alpha2beta1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src(-/-)Fyn(-/-) fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an alpha2beta1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.
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PMID:Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation. 1688 56

Exposure of endothelial cells (ECs) to hypoxia leads to a decrease in EC proliferation. However, the mechanism by which hypoxia inhibits EC proliferation is unclear. Perlecan has been reported to play an important role in regulating EC proliferation. We hypothesized that perlecan was involved in the hypoxia-induced inhibition of EC proliferation. To test this hypothesis, rat cardiac microvascular ECs were cultured under normoxic or hypoxic conditions for 12 h and harvested for determination of perlecan mRNA expression using real-time reverse transcription-polymerase chain reaction (RT-PCR). The results showed that exposure of ECs to hypoxia for 12 h induced a decrease in perlecan mRNA expression (61.72%, P<0.05). Concomitantly, the down-regulation of endogenous perlecan induced by hypoxia or the neutralization of endogenous perlecan with anti-perlecan antibody significantly inhibited EC proliferation and responsiveness to basic fibroblast growth factor (bFGF), and decreased focal adhesion kinase (FAK) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. These data indicate that down-regulation of perlecan expression contributes to hypoxia-induced inhibition of rat cardiac microvascular EC proliferation by suppressing FAK-mediated and ERK1/2-dependent growth signals.
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PMID:Down-regulation of perlecan expression contributes to the inhibition of rat cardiac microvascular endothelial cell proliferation induced by hypoxia. 1743 47

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.
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PMID:A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation. 1799 86

Treatment of xerostomia would benefit from development of a functional implantable artificial salivary gland. Salivary gland tissue from surgical patients was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Ductal and acinar cells were identified in tissue and cultured cells from dispersed tissue. High levels of laminin and perlecan/HSPG2 (heparan sulfate proteoglycan 2) were noted in basement membranes, and perlecan also was secreted and organized by cultured acinar populations, which formed lobular structures that mimicked intact glands when cultured on Matrigel or a bioactive peptide derived from domain IV of perlecan. On either matrix, large acini-like lobular structures grew and formed connections between the lobes. alpha-Amylase secretion was confirmed by staining and activity assay. Biomarkers, including tight junction protein E-cadherin and water channel protein aquaporin 5 found in tissue, were expressed in cultured acinar cells. Cells cultured on Matrigel or domain IV of perlecan peptide organized stress fibers and activated focal adhesion kinase. We report a novel technique to isolate acinar cells from human salivary gland and identify a human peptide sequence in perlecan that triggers differentiation of salivary gland cells into self-assembling acini-like structures that express essential biomarkers and which secrete alpha-amylase.
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PMID:Perlecan domain IV peptide stimulates salivary gland cell assembly in vitro. 1938 72

NK4, a fragment of hepatocyte growth factor (HGF), exerts bifunctional action as a competitive antagonist against HGF and its receptor c-Met and an angiogenesis inhibitor. Here we studied the anti-angiogenic mechanism of NK4. In cultured human endothelial cells, NK4 inhibited DNA synthesis induced not only by HGF but also by either basic fibroblast growth factor or vascular endothelial growth factor. Even if c-Met expression was diminished by small interference RNA, NK4 inhibited basic fibroblast growth factor-induced DNA synthesis, indicating that anti-angiogenic action of NK4 is c-Met-independent. Affinity purification with NK4-immobilized beads revealed that NK4 binds to perlecan. Consistent with this, NK4 colocalized with perlecan in endothelial cells. Perlecan is a multidomain heparan sulfate proteoglycan that interacts with basement membrane components such as fibronectin. NK4 inhibited extracellular assembly of fibronectin, by which fibronectin-dependent endothelial cell spreading was inhibited by NK4. Knockdown of perlecan expression by small interference RNA significantly abrogated the inhibitory effect of NK4 on fibronectin assembly and cell spreading. In NK4-treated endothelial cells, tyrosine phosphorylation of focal adhesion kinase and Rac activation were reduced, whereas overexpression of activated Rac recovered the DNA synthesis in NK4-treated endothelial cells. These results indicate that the association between NK4 and perlecan impairs fibronectin assembly, thereby inhibiting anchorage-dependent signaling. The identified mechanism for angiostatic action provides further proof of significance for NK4 in the treatment of cancer and potentially for vascular regulation as well.
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PMID:Angioinhibitory action of NK4 involves impaired extracellular assembly of fibronectin mediated by perlecan-NK4 association. 1955

Because prostate cancer cells metastasize to bone and exhibit osteoblastic features (osteomimicry), the interrelationships between bone-specific microenvironment and prostate cancer cells at sites of bone metastasis are critical to disease progression. In this work the bone marrow microenvironment in vitro was recreated both by tailoring scaffolds physical properties and by functionalizing electrospun polymer fibers with a bioactive peptide derived from domain IV of perlecan heparan sulfate proteoglycan. Electrospun poly (epsilon-caprolactone) (PCL) fibers and PCL/gelatin composite scaffolds were modified covalently with perlecan domain IV (PlnDIV) peptide. The expression of tight junction protein (E-cadherin) and focal adhesion kinase (FAK) phosphorylation on tyrosine 397 also were investigated. The described bioactive motif significantly enhanced adherence and infiltration of the metastatic prostate cancer cells on all modified electrospun substrates by day 5 post-seeding. Cells cultured on PlnDIV-modified matrices organized stress fibers and increased proliferation at statistically significant rates. Additional findings suggest that presence of PlnDIV peptide in the matrix reduced expression of tight junction protein and binding to PlnDIV peptide was accompanied by increased focal adhesion kinase (FAK) phosphorylation on tyrosine 397. We conclude that PlnDIV peptide supports key signaling events leading to proliferation, survival, and migration of C4-2B cancer cells; hence its incorporation into electrospun matrix is a key improvement to create a successful three-dimensional (3-D) pharmacokinetic cancer model.
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PMID:Biofunctionalization of electrospun PCL-based scaffolds with perlecan domain IV peptide to create a 3-D pharmacokinetic cancer model. 2041 54


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