Expression of β1 integrins in glomerular tissue of Streptozotocin-induced diabetic rats

1999 ◽  
Vol 77 (1) ◽  
pp. 71-78 ◽  
Author(s):  
Mari Regoli ◽  
Moïse Bendayan
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Plasma Membranes ◽  
Mesangial Cells ◽  
Diabetic Rats ◽  
Morphological Alteration ◽  
Western Blots ◽  
Extracellular Matrix Components

Based upon the importance of integrins as receptors for extracellular matrix components as well as transducers of extracellular signals, and since major alterations take place in the renal extracellular matrix during diabetes, it is important to study the role played by integrins in the development of the diabetic glomerulosclerosis. Expression of the β1 subunit by renal glomerular cells was evaluated by biochemical and morphological means in short- and long-term diabetic rats. Western blots of isolated rat renal glomeruli demonstrated that the expression of β1 increases along with age as well as with the hyperglycaemic state. These changes were significant as early as 6 weeks of hyperglycaemia. This was further demonstrated by immunocytochemistry, which revealed the presence of the β1 subunit at the level of the plasma membranes of endothelial, epithelial, and mesangial cells. Quantitation of the immunolabelings confirmed the increased expression of β1 under diabetic conditions. Further to this, expression of the focal adhesion kinase (FAK) was evaluated by immunoblotting showing little increase in diabetic conditions. On the other hand, testing the tyrosine phosphorylation of FAK, revealed significant increases in diabetes. To recover the fraction of FAK associated with the β1 subunit, immunoprecipitation of isolated glomeruli homogenates was carried out with the anti- β1 antibody. This demonstrated that the amounts of FAK co-precipitated with β1, as well as its tyrosine-phosphorylation, are in fact reduced in diabetic conditions. Since the changes reported were observed at time points prior to any morphological alteration of the renal extracellular matrix, it appears that modifications in integrins and in their intracellular relays constitute early events that precede the onset of the diabetic nephropathy and must then be associated with the hyperglycaemic condition.Key words: integrins, focal adhesion kinase, tyrosine phosphorylation, renal tissue, diabetes.

scholarly journals Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

1996 ◽  
Vol 7 (3) ◽  
pp. 415-423
Author(s):  
D A Troyer ◽  
A Bouton ◽  
R Bedolla ◽  
R Padilla
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Mesangial Cells ◽  
Close Contact ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Fiber Formation ◽  
Stress Fibers

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

scholarly journals Steel Factor Enhances Integrin-Mediated Tyrosine Phosphorylation of Focal Adhesion Kinase (pp125FAK) and Paxillin

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Hiroyuki Takahira ◽  
Akihiko Gotoh ◽  
Alec Ritchie ◽  
Hal E. Broxmeyer
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Direct Action ◽  
Human Growth ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Steel Factor ◽  
Extracellular Matrix Components ◽  
Dependent Cell

Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin β1 and combination of anti-α4 with anti-α5 antibodies, indicating α4β1 and α5β1 integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10−7 mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10−7 mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-α4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK through activation of integrin (“inside-out” signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.

scholarly journals Endothelin-1 stimulates tyrosine phosphorylation of p125 focal adhesion kinase in mesangial cells.

1995 ◽  
Vol 6 (5) ◽  
pp. 1504-1510
Author(s):  
M Haneda ◽  
R Kikkawa ◽  
D Koya ◽  
T Shikano ◽  
T Sugimoto ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Protein Tyrosine Kinase ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Protein Tyrosine ◽  
Endothelin 1

Endothelin-1 (ET-1) is known to induce the contraction and proliferation of glomerular mesangial cells. Because ET-1 was found to stimulate the tyrosine phosphorylation of unidentified cellular proteins in cultured mesangial cells, protein tyrosine kinase might serve as one of the important signals leading to various functions of ET-1. Focal adhesion kinase (p125FAK) is a newly identified cytoplasmic protein tyrosine kinase that is activated by the phosphorylation of its own tyrosine residue. Because p125FAK was found to play a role in the signal transduction of not only integrins but also various neurotransmitters, including bombesin, endothelin, and vasopressin in Swiss 3T3 cells and Rat-1 fibroblasts, whether ET-1 could stimulate the tyrosine phosphorylation of p125FAK in glomerular mesangial cells was examined. ET-1 stimulated the tyrosine phosphorylation of p125FAK by threefold to fourfold in cultured mesangial cells. This effect of ET-1 was detected at 1 min and reached a maximum within 5 min and was blocked by BQ-123, an antagonist for ETA receptor. A23187, a calcium ionophore, failed to stimulate the tyrosine phosphorylation of p125FAK, and ET-1 was able to stimulate the tyrosine phosphorylation of p125FAK, even in a calcium-free medium. The activation of protein kinase C (PKC) by phorbol 12, 13-dibutyrate resulted in a stimulation of the tyrosine phosphorylation of p125FAK, and an inhibition of PKC by calphostin C or staurosporine significantly reduced the effect of ET-1. Furthermore, prolonged treatment of the cells with phorbol 12, 13-dibutyrate markedly inhibited the ET-1-induced tyrosine phosphorylation of p125FAK. These results indicate that p125FAK might play a role in a signal transduction system of ET-1 in glomerular mesangial cells and that the ET-1-induced tyrosine phosphorylation of p125FAK is largely dependent on the PKC pathway.

scholarly journals Steel Factor Enhances Integrin-Mediated Tyrosine Phosphorylation of Focal Adhesion Kinase (pp125FAK) and Paxillin

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1574-1584 ◽  
Author(s):  
Hiroyuki Takahira ◽  
Akihiko Gotoh ◽  
Alec Ritchie ◽  
Hal E. Broxmeyer
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Direct Action ◽  
Human Growth ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Steel Factor ◽  
Extracellular Matrix Components ◽  
Dependent Cell

Abstract Integrin-mediated interaction of hematopoietic progenitor cells with bone marrow stromal extracellular matrix components is important in hematopoiesis. Focal adhesion kinase (pp125FAK) plays a central role in signal transduction through integrin receptors. We studied matrix-integrin interaction and subsequent signaling in human growth factor-dependent cell line, TF-1. Adherence of unstimulated TF-1 cells to fibronectin-coated wells was blocked by antiintegrin β1 and combination of anti-α4 with anti-α5 antibodies, indicating α4β1 and α5β1 integrin mediated adherence. Steel factor (SLF) increased TF-1 adhesion to fibronectin dose-dependently and 10−7 mol/L wortmannin suppressed SLF-induced adhesion. Immunoprecipitation and immunoblotting with antiphosphotyrosine antibody showed that adherence of TF-1 cells to fibronectin without cytokine caused tyrosine phosphorylation of several proteins identified as pp125FAK and paxillin. SLF induced spreading of adherent TF-1 cells and enhanced tyrosine phosphorylation of pp125FAK and paxillin in a dose-dependent manner. Treatment with SLF without plating on fibronectin did not induce tyrosine phosphorylation of pp125FAK. Wortmannin, at 10−7 mol/L, completely abolished SLF-induced enhancement of pp125FAK tyrosine phosphorylation, while c-kit autophosphorylation was not affected. This suggests that increase of pp125FAK tyrosine phosphorylation was mediated through a wortmannin sensitive pathway, rather than by direct action on c-kit tyrosine kinase. Treatment of adherent TF-1 cells with RGDS peptide plus anti-α4 antibody also inhibited SLF-induced enhancement of pp125FAK tyrosine phosphorylation without detachment of TF-1 cells. These data suggest that SLF enhances integrin-fibronectin-dependent tyrosine phosphorylation of pp125FAK through activation of integrin (“inside-out” signaling) and following integrin occupancy. This establishes a novel linkage between c-kit/SLF pathway and integrin fibronectin signaling.

scholarly journals Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D

1996 ◽  
Vol 318 (2) ◽  
pp. 609-614 ◽  
Author(s):  
D. Margriet OUWENS ◽  
Harald M. M. MIKKERS ◽  
Gerard C. M. van der ZON ◽  
Matthias STEIN-GERLACH ◽  
Axel ULLRICH ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Insulin Receptor ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Dominant Negative ◽  
Matrix Proteins ◽  
Dominant Negative Mutant ◽  
Phosphotyrosine Phosphatase ◽  
Insulin Receptor Substrates

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor β-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(l-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.

scholarly journals Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

1995 ◽  
Vol 15 (5) ◽  
pp. 2819-2827 ◽  
Author(s):  
B L Eide ◽  
C W Turck ◽  
J A Escobedo
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Consensus Sequence ◽  
Sh2 Domain ◽  
Protein Tyrosine ◽  
Adhesion Plaques

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.

scholarly journals Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of stress fibers

2000 ◽  
Vol 113 (2) ◽  
pp. 237-245 ◽  
Author(s):  
M.J. Wassler ◽  
B.D. Shur
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Basal Lamina ◽  
Stress Fibers ◽  
Intracellular Signal ◽  
Signal Cascades ◽  
Galactosyltransferase I ◽  
Actin Stress Fibers

It is well appreciated that clustering of receptors for the extracellular matrix, most notably the integrins, elicits intracellular signal cascades. One of the first indications that integrin-dependent signaling has occurred is by the activation of focal adhesion kinase (FAK). Another, although less well understood, receptor for the extracellular matrix is (beta)1, 4-galactosyltransferase I (GalT). GalT participates during lamellipodia formation and cell migration by recognizing terminal N-acetylglucosamine residues on basal lamina glycosides. In this study, we investigated whether GalT is also capable of eliciting intracellular signal cascades, specifically FAK activation, in response to ligand binding and/or aggregation. 3T3 fibroblasts were treated with two different reagents capable of aggregating GalT, either antibodies raised against recombinant GalT or multivalent polymers of N-acetylglucosamine, and the effects on tyrosine phosphorylation were analyzed. Both reagents induced an initial tyrosine phosphorylation (1-2 minutes) and subsequent dephosphorylation (5-10 minutes) of proteins with molecular mass 67 and 125 kDa. These proteins were identified as paxillin and FAK, respectively, by immunoprecipitation with anti-paxillin and anti-FAK antibodies. Preimmune IgG, anti-GalT Fab fragments, irrelevant polymers and monomeric N-acetylglucosamine had no effect. The ability of GalT aggregation to induce transient tyrosine phosphorylation was dependent upon cell density. In addition, FAK dephosphorylation was found to be sensitive to the phosphatase inhibitor, sodium pervanadate. Similar to the integrins, GalT requires association with the cytoskeleton in order to function as a matrix receptor. To determine if the transient tyrosine phosphorylation of FAK was dependent upon GalT binding to the cytoskeleton, stably transfected fibroblasts expressing different amounts of GalT were treated with polymeric N-acetylglucosamine. Cells expressing increased levels of GalT associated with the cytoskeleton showed increased levels of FAK tyrosine phosphorylation and prolonged dephosphorylation, relative to control cells. In contrast, cells in which a dominant negative form of GalT prevents association with the cytoskeleton showed no or weak response to polymeric N-acetylglucosamine. Concomitant with the GalT-stimulated dephosphorylation of FAK, cells treated with anti-GalT antibodies or polymeric N-acetylglucosamine showed a loss of actin stress fibers and focal adhesions. Pervanadate treatment inhibited the GalT-dependent loss of actin stress fibers. To confirm the requirement of GalT in transient FAK phosphorylation and stress fiber reorganization in this system, we created cells homozygous null for the GalT isoform that functions as a matrix receptor. These cells were incapable of phosphorylating FAK in response to GalT agonists and, interestingly, showed a lack of lamellar stress fibers when cultured on basal lamina matrices. These data suggest that GalT function as a basal lamina receptor involves transient activation of FAK and an associated reorganization of stress fibers.

Stretching Mesangial Cells Stimulates Tyrosine Phosphorylation of Focal Adhesion Kinase pp125FAK

1995 ◽  
Vol 212 (2) ◽  
pp. 544-549 ◽  
Author(s):  
K. Hamasaki ◽  
T. Mimura ◽  
H. Furuya ◽  
N. Morino ◽  
T. Yamazaki ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Mesangial Cells
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