Integrin-Induced Protein Kinase C and C Translocation to Focal Adhesions Mediates Vascular Smooth Muscle Cell Spreading

From the Franz Volhard Clinic and the Max Delbrück Center for Molecular Medicine, Virchow Klinikum, Humboldt University of Berlin (Germany).

Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail haller{at}orion.rz.mdc-berlin.de

	Abstract

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Abstract—The extracellular matrix influences the cellular spreading of vascular smooth muscle cells (VSMCs) via integrin receptors. However, the intracellular signaling mechanisms are still incompletely understood. We investigated the hypothesis that VSMCs binding to fibronectin activates the protein kinase C (PKC) pathway, causes differential intracellular PKC isoform translocation, and mediates cell spreading. VSMCs binding to poly-L-lysine or preincubated with Arg-Gly-Asp (RGD) peptides were used as controls. Diacylglycerol (DAG) and phospholipase D (PLD) activity were measured by thin-layer chromatography. Intracellular distribution of PKC isoforms was assessed by confocal microscopy. VSMCs binding to fibronectin induced focal adhesions and cell spreading within 30 minutes. Fibronectin induced a rapid increase in DAG content, peaking at 10 minutes with a sustained response for <1 hour. In contrast, PLD activity was not influenced by specific binding to fibronectin. PKC isoforms , , , and were assessed by confocal microscopy. Fibronectin induced a PKC isoform translocation to the cell nucleus and to focal adhesions within minutes. The nuclear PKC immunoreactivity was transiently increased. PKC isoforms and were both translocated to focal adhesions. The intracellular distributions of other PKC isoforms were not influenced by fibronectin. The effects of fibronectin on DAG generation, the translocation of PKC and PKC, and cell spreading were all abolished by the incubation with RGD peptides. Downregulation of PKC isoforms and with specific antisense oligodinucleotides resulted in a significant inhibition of cell spreading. Our results show that integrins induce intracellular signaling in VSMCs via DAG and PKC. PKC isoform is translocated to the nucleus, whereas PKC isoforms and are translocated to focal adhesions. Both isoforms seem to play a role in inside-out integrin signaling and cell spreading.

Key Words: matrix protein • fibronectin • protein kinase C • vascular smooth muscle cell • integrin

	Introduction

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The local cellular environment determines differentiation, proliferation, and migration of VSMCs. The extracellular matrix plays an important role in mediating cell spreading and migration.¹ VSMCs bind to the matrix via membrane-bound integrins. In addition to establishing the cell-matrix interaction, integrins are cell surface receptors that can mediate the bidirectional transfer of information from the outside to the inside of the cell.^{2 3 4 5 6 7} Several signaling pathways are stimulated after integrin occupation. Integrin-initiated signals stimulate the tyrosine phosphorylation of several cellular proteins, such as GTP-bound p21 ras,⁸ pp125FAK, and others.⁹ Ca²⁺-dependent signaling pathways are also activated by integrins. McNamee et al¹⁰ have shown that adhesion to fibronectin stimulates inositol-lipid synthesis and inositol trisphosphate generation. Integrin occupation also leads to increased [Ca²⁺]_i in different cell types.^{11 12 13 14 15 16 17 18 19 20} The activation of the Ca²⁺ messenger system appears to be due to stimulation of phospholipase C.^{12 13} These intracellular events lead to DAG formation and activation of PKC. DAG formation by integrin occupation has been shown in leukocytes and pancreatic acinar cells.^{13 20} Moreover, translocation of PKC from the cytosol to the membrane fraction on binding to extracellular matrix has been demonstrated.^{21 22}

PKC activation may influence several intracellular events, including cell spreading. PKC is activated before integrin-mediated cell spreading, and inhibition of PKC prevents cell spreading.^{21 22} PKC seems also to play a role in the formation of focal adhesions.²³ However, which of the PKC isoforms is influenced by integrin-mediated PKC activation is unclear. PKC consists of a family of isoforms²⁴ that are targeted to different cellular compartments on cell stimulation by growth factors and cytokines.^{25 26 27 28 29 30 31 32} These isoforms are expressed in a tissue-dependent fashion. In VSMCs, PKC isoforms , ß1, , , and have been identified.²⁶ Jaken, Leach, and colleagues^{27 28} studied fibroblasts and showed that specific PKC isoforms may associate with nuclear structures or focal adhesions. PKC isoforms also bind to cytoskeletal proteins and to other subcellular compartments.²⁹ We recently demonstrated a rapid translocation of PKC to the nucleus on cell stimulation by extracellular hormones and growth factors.³³ Some investigators have recently reported that PKC isoforms are translocated from a cytosolic to a membrane fraction after integrin occupation.³⁴

Which PKC isoforms are induced by integrin occupation in VSMCs is unclear. Whether or not binding to extracellular matrix leads to a differential distribution of PKC isoforms in VSMCs is not defined. Finally, the functional consequences of these PKC-mediated events are unknown. We investigated the DAG-PKC system in VSMCs after binding to fibronectin. We demonstrate (1) that binding to fibronectin induces DAG generation in a biphasic fashion, (2) that fibronectin induces a rapid, short-lasting translocation of PKC to the nucleus, (3) that PKC and PKC are translocated to focal adhesions, and (4) that inhibition of PKC and PKC by antisense ODNs inhibited cell spreading. Our findings suggest that PKC may play a role in integrin-induced gene expression and that PKC and PKC mediate the effects of PKC activation in cell spreading.

	Materials and Methods

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Materials
Fibronectin was purchased from Boehringer-Mannheim Biochemicals. The RGD peptide, as Gly-Arg-Gly-Asp-Asn-Pro, was obtained from Biomol.

VSMC Isolation and Culture
Rat aortic VSMCs from the fourth to eighth passages were separated according to the modified method of Chamley-Campbell described elsewhere.^{26 35} Briefly, the rats (12 to 14 weeks old) were killed instantly, and their thoracic aortas were excised. After adherent fat and connective tissue were removed, the aortas were cut longitudinally, and the endothelial cells were removed by gentle scraping with fine forceps. The aortas were then minced into small pieces and incubated at 37°C for 2 hours in PBS without Ca²⁺ but with 1 mg/mL collagenase (type I, 150 IU/mg, Worthington Biochemical Corp), 0.5 mg/mL elastase (type III, 40 IU/mg, Sigma), and 0.5 mg/mL trypsin inhibitor (Sigma). After 2 hours, DMEM/F-12 containing 10% FBS (GIBCO) was added to the suspension to inactivate enzymes. The cells were then centrifuged at 120g for 10 minutes, and the pellet was resuspended in DMEM/F-12 with 10% FBS. The cells were then seeded at a density of 3 to 5x10⁵/cm² and were cultured in medium 199 (Seromed) containing 10% FBS, 2 mmol/L glutamine, and 100 U/mL penicillin-streptomycin at 37°C in 95% air/5% CO₂. Cells from passages 2 to 4 were used in all experiments. The phenotype of the cultured VSMCs was determined by staining the cells for -actin and desmin. Antibodies for muscle specific -actin and desmin were obtained from Boehringer-Mannheim.

Twenty-four hours before the examinations, cells were put into 0.1% FBS for quiescence. On the next day, cells were trypsinized and washed, and 1 million cells per well were plated either on fibronectin (final concentration, 50 µg/mL)–coated or poly-L-lysine (200 µg/mL)–coated dishes. In addition, the specificity of integrin binding of fibronectin was determined by the addition of an RGD peptide in a concentration of 400 µL/mL before plating. At 0, 10, 30, 60, and 120 minutes, the experiments were stopped by aspirating the medium in a time-dependent manner. The cells were immediately washed with PBS and scraped for lipid extraction.

Lipid Extraction
Lipid extraction was performed by the method of Bligh-Dyer as described earlier.³⁶ Briefly, the cells were solubilized with 0.4 mL of 0.2% SDS for 5 minutes and scraped into glass tubes. Culture wells were washed with 1 mL of ice-cold methanol, which was added to the cells. Chloroform and 0.2 mol/L NaCl were added to yield a final concentration of 1:1:0.9 (chloroform/methanol/water [vol/vol]). After centrifugation, the lower organic phase was separated from the upper phase and dried under nitrogen. Lipids were dissolved in chloroform and kept at -20°C until further processing.

DAG Kinase Assay
The DAG content in the lipid extracts was assayed using established methods.³⁶ Briefly, DAG-containing lipids were solubilized in a octyl ß-glucoside/cardiolipin solution and incubated with DAG kinase from Escherichia coli in the presence of [-³²P]ATP to quantitatively convert DAG into [³²P]phosphatidic acid. Lipids were extracted as described above, and phosphatidic acid was separated from other lipids by thin-layer chromatography, using the solvent system chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5 [vol/vol]). Phosphatidic acid spots were identified using an authentic DAG standard and autoradiography, cut from the thin-layer chromatographic plate, and counted in a scintillation counter.

Immunocytochemistry
The techniques for confocal microscopy have been described previously.^{26 35 37} The cells were fixed with 4% paraformaldehyde and permeabilized with 80% methanol at -20°C at the described time points. After incubation with 3% skimmed milk in phosphate-buffered solution (SM/PBS) for 60 minutes, the preparation was incubated for 1 hour at room temperature with the PKC antibodies. We used highly specific affinity-purified polyclonal antibodies directed against peptide sequences of PKC that reacted specifically with the , , and subspecies of PKC (antibodies were from GIBCO; the antibody against PKC was a monoclonal from UBI). Specificity was demonstrated by using specific oligopeptides that prevent binding of the antibodies to the isoforms. This characterization of the antibodies has been recently published.^{33 35} Antibodies were then diluted in PBS with 0.1% BSA (1:80). After they were washed with the primary antibodies, cells were washed three times with PBS and exposed to the secondary antibody (FITC-conjugated anti-rabbit or anti-mouse IgG at 1:100, 1% BSA/PBS, Pierce Chemicals) for 60 minutes. The preparation was mounted with 50% glycerol under a glass coverslip on a Nikon-Diaphot microscope. An MRC 600 confocal imaging system (Bio-Rad Laboratories) with an argon/krypton laser was used. At least 50 to 80 cells from each of at least seven experiments were examined under each experimental condition. The results were reproduced by two separate investigators, and multiple experiments were performed. The observers were unaware of the experimental design and antibodies used.

Quantification in nuclear, cytoplasmic, and periplasmic membrane regions was done with histogram/area functions in the MRC-Comos software. The subcellular regions were outlined manually, and the calculated mean fluorescence intensity was obtained for the delineated regions. Data are presented as the ratio of the mean fluorescence intensity in the respective regions to the mean fluorescence intensity of the whole cell area.

Western Blotting
Western blot analysis was carried out as described previously.^{26 35} After the experiments, the cultured endothelial cells were treated with ice-cold homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 250 mmol/L sucrose, 3 mmol/L EGTA, 10 mmol/L mercaptoethanol, 1 mmol/L phenylmethylsulfonyl fluoride, and 50 µmol/L leupeptin) and homogenized. The homogenate was resuspended in buffer containing 1.0% Triton X-100 and centrifuged at 100 000 rpm for 10 minutes. The supernatant underwent chromatography using 10% SDS-polyacrylamide gels. Ten to 40 µg of protein was loaded into each lane. The fractions were then electroblotted by the semidry technique onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). The membranes were successively incubated, first with blocking buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 10% nonfat dry milk powder (Merck), 0.2% (vol/vol) Tween 20, and 0.02% NaN₃ for 120 minutes at room temperature. The next incubation was conducted in affinity-purified isoenzyme-specific antibody diluted in incubation buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 1% BSA at room temperature. PKC antibodies used are described above. A final incubation was carried out in Tris-buffered saline with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Pierce Chemicals). The membranes were thoroughly washed after each incubation with a buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 0.2% (vol/vol) Tween-20. Visualization was achieved by chemiluminescence (Renaissance, DuPont).

Oligonucleotides
Phosphorothioate ODNs were purchased (Tib Molbiol). We selected an antisense ODN (ISIS 3521) against the human 3' untranslated region derived from the human PKC sequence (European Molecular Biology Laboratories database).³⁸ The antisense sequence used for PKC was 5'GTT CTC GCT GGT GAG TTT CA3'. The sense ODN sequence (5'TG AAA CTC ACC AGC GAG AAC3'), a reverse ODN sequence (5'AC TTT GAG TGG TCG CTC TTG3'), and a scrambled version (5'GAG TTG CTT GCT TAT CGG TC3') were used as controls. The antisense sequence used for PKC against the human AUG start codon was (5'GCC ATT GAA CAC TAC CAT3'). The sense ODN sequence (5'ATG GTA GTG TTC AAT GGC3') was used as a control. We used a cationic lipid solution (Lipofectin, GIBCO BAL, Life Technologies) to enhance ODN uptake. For transfection, the cells were incubated with lipofectin (10 µg/mL) and ODN (1 µmol/L) at 37°C for 4 hours, washed 2 times with medium, and then incubated with medium and ODN (1 µmol/L) for another 4 hours.

Statistics
Statistical analysis was carried out on a Macintosh computer (Apple Inc) using a commercially available statistical program (Statview, Cricket Software Inc). We used nonparametric sign tests and Mann-Whitney tests to analyze the data from the 7 to 10 separate experiments. A value of P<.05 was accepted as significant. References to increases or decreases in the following section are only so stated if statistically significant.

	Results

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We first analyzed the effects of fibronectin on the spreading of cultured VSMCs at 10, 30 and 60 minutes after cell seeding. Plates coated with fibronectin, poly-L-lysine, or fibronectin plus incubation with RGD peptides were used. VSMCs on fibronectin showed slight spreading after 10 minutes compared with VSMCs on poly-L-lysine or fibronectin+RGD. The cell spreading increased after 30 and 60 minutes on fibronectin. On fibronectin, 76% of VSMCs showed spreading after 60 minutes compared with 17% of VSMCs on poly-L-lysine. Preincubation with RGD peptides inhibited cell spreading on fibronectin to a similar degree. Cells on poly-L-lysine or fibronectin+RGD became slowly unrounded after 60 minutes (data not shown). We then asked whether the binding to fibronectin leads to an increase in DAG generation in VSMCs. DAG content in VSMCs was measured at 1, 10, 30, 60, and 120 minutes after seeding. Adherence to fibronectin induced a rapid increase in DAG content, whereas binding of the cells to poly-L-lysine had no effect on DAG generation. The fibronectin-induced DAG generation showed a biphasic response, with an initial peak at 10 minutes (2.17±0.67 versus 1.18±0.07 nmol/L per well, P.05, n=4) and a decrease toward basal levels thereafter. DAG content in VSMCs on fibronectin remained slightly elevated for <60 minutes (1.53±0.05 versus 1.23±0.2 nmol/L per well, n=4). This fibronectin effect was integrin dependent, because preincubation of fibronectin-coated culture dishes with RGD peptides for 10 minutes before VSMC seeding prevented the fibronectin-induced increase in DAG almost completely (data not shown).

DAG can be generated by activation of phospholipase C, by polyinositol breakdown, or by phosphatidic acid and the activation of DAG kinase. Because the latter pathway can be induced by PKC directly, we asked whether fibronectin binding induced the formation of phosphatidic acid. Binding to fibronectin induced a rapid increase in phosphatidic acid; however, this effect was not fibronectin specific, since the binding of VSMCs to poly-L-lysine also led an increase in phosphatidic acid (data not shown). To further define possible intracellular signals that may be involved in the formation of DAG, we used the kinase inhibitors staurosporine (5x10^-8) and genistein (10^-5 mol/L). The tyrosine kinase inhibitor genistein inhibited the fibronectin-induced DAG production by 54±16% (P<.05). In contrast, the PKC inhibitor staurosporine had a smaller, but nonsignificant, effect (18±7%) (data not shown).

We then analyzed the effects of fibronectin binding on the intracellular distribution of PKC isoforms , , , and . Using confocal microscopy, we assessed intracellular PKC immunoreactivity with two sections on different cell levels. The first section (Fig 1A, top micrographs) was aligned approximately at the level of the cell nucleus (5 µm above the basolateral cell surface), and the second section (Fig 1A, bottom micrographs) was placed 0.5 µm above the cell surface, in order to assess PKC immunoreactivity close to the basolateral cell surface. Fibronectin binding (5 minutes) induced an intense staining both of the perinuclear and nuclear area (top). These effects were not present in cells bound to poly-L-lysine and could be prevented by preincubation of the VSMCs with RGD peptides. A semiquantification of the fluorescence signal from the nuclear area and the cytoplasmic area (n=60) showed a significant increase in nuclear immunoreactivity for PKC after binding to fibronectin compared with binding to poly-L-lysine and preincubation with RGD peptides (P.05) (Fig 1A). The fibronectin-induced increase in PKC immunoreactivity was transient and disappeared within 15 to 20 minutes. The increase in nuclear immunoreactivity was also observed when the relation between cytosolic and nuclear immunoreactivity was assessed (Fig 1B). The comparison between the two cellular compartments suggests a fibronectin-induced translocation of PKC from the cytosol into the nucleus.

View larger version (36K): [in this window] [in a new window]   Figure 1. A, Effect of fibronectin (left), poly-L-lysine (middle), or fibronectin with RGD peptide (right) on the intracellular distribution of the PKC isoform in VSMCs 10 minutes after seeding. Shown are confocal micrographs of VSMCs dissected at 5 µm (top micrographs) or 0.5 µm (bottom micrographs) above the basolateral surface of the cells. Compared with the other two conditions, the presence of fibronectin induced an increase in nuclear immunoreactivity (top micrograph) and focal adhesions. The white bar represent 5 µm. B and C, Semiquantitative analysis of the effect of fibronectin, fibronectin with RGD peptide, or poly-L-lysine on the cytosolic/nuclear distribution of the PKC isoform in VSMCs 5, 15, and 30 minutes after seeding. Fibronectin induced a transient increase in nuclear immunoreactivity at 5 minutes (B). This effect is especially obvious when a cytoplasmic/nuclear ratio is formed (C).

We then assessed the effects of fibronectin on PKC immunoreactivity near the basolateral surface of the cells. The bottom micrographs of Fig 1A show the effects of fibronectin binding on the basolateral distribution of PKC. Fibronectin induced an increase in PKC immunoreactivity in distinct focal spots. These focal spots were only rarely present on poly-L-lysine and were almost completely abolished by preincubation with RGD peptides. Quantification of the focal spots (n=60) confirmed this observation and showed a significant difference between fibronectin and controls. The increase of PKC immunoreactivity in focal spots was only transient and had disappeared at 15 minutes (Fig 2A). In order to further demonstrate that fibronectin induced translocation of PKC to focal adhesions, we used double stains for FAK and PKC after the binding of VSMCs to fibronectin. A representative photomicrograph of these experiments is shown in Fig 2B, which clearly demonstrates colocalization of both proteins. This finding indicates that fibronectin induces translocation of PKC to focal adhesions.

View larger version (80K): [in this window] [in a new window]   Figure 2. A, Semiquantitative analysis of the effect of fibronectin, fibronectin with RGD peptide, or poly-L-lysine on the basolateral distribution of the PKC isoform in VSMCs 5, 15, and 30 minutes after seeding. Fibronectin induced a focal increase in immunoreactivity at 5 minutes. B, Confocal photomicrograph showing colocalization of PKC immunoreactivity and pp125FAK immunoreactivity after binding to fibronectin at 30 minutes. A horizontal sectioning of a single VSMC stained for PKC (left, green) and pp125FAK (middle, red) is shown. The colocalization of PKC and 125FAK is shown at the right (yellow). Displayed is a section obtained at a 0.5-µm step above the basolateral membrane, as described in "Materials and Methods."

We then analyzed the effects of fibronectin adhesion on PKC isoform . In contrast to the observed changes in nuclear immunoreactivity with PKC, PKC immunoreactivity did not increase in the nuclear area (data not shown). However, fibronectin induced a PKC increase in the basolateral area. Fig 3 shows the effects of fibronectin binding on the basolateral distribution of PKC. Fibronectin induced a focal distribution of PKC immunoreactivity (Fig 3A). These focal spots were only rarely present on poly-L-lysine and were almost completely abolished by preincubation with RGD peptides. The semiquantification of these results confirmed this observation (Fig 3B, n=60). However, the number of spots per cell was smaller compared with the experiments with PKC. In contrast to PKC, the time course of translocation to focal spots was still significantly increased at 15 minutes. As in the previous experiments, we used colocalization experiments with FAK and could confirm the fibronectin-induced translocation of PKC to focal adhesions (Fig 3C).

View larger version (63K): [in this window] [in a new window]   Figure 3. A, Effect of fibronectin, fibronectin with RGD peptide, or poly-L-lysine on the intracellular distribution of the PKC isoform in VSMCs. Shown are confocal micrographs of cells dissected at 0.5 µm above the basolateral surface of the cells. Compared with the other two conditions, the presence of fibronectin induced an increase in focal immunoreactivity. B, Semiquantitative analysis of the effect of fibronectin, fibronectin with RGD peptide, or poly-L-lysine on the basolateral distribution of the PKC isoform in VSMCs 5, 15, and 30 minutes after seeding. Fibronectin induced a focal increase in immunoreactivity at 5 and 15 minutes. C, Confocal photomicrograph showing colocalization of PKC immunoreactivity and pp125FAK immunoreactivity after binding to fibronectin at 5 minutes. The figure shows a horizontal sectioning of a single VSMC stained for PKC (left, green) and pp125FAK (middle, red). The colocalization of PKC and 125FAK is shown at the right (yellow). Displayed is a section obtained at a 0.5-µm step above the basolateral membrane, as described in "Materials and Methods."

We further analyzed the intracellular distribution of PKC and PKC. The first isoform was found to be located in the cytosol and appeared to be associated with cytoskeletal proteins. Fibronectin binding had no specific effect on the intracellular distribution of PKC and showed no difference compared with poly-L-lysine binding or preincubation with RGD peptides (data not shown). PKC showed weak speckled immunoreactivity mostly localized in the cytosolic area. We did not observe a significant fibronectin effect on the intracellular distribution of this PKC isoform (data not shown).

Finally, we investigated the hypothesis that the PKC isoforms and play a functional role in the mediation of cell spreading. For specific inhibition of PKC isoforms and , we used antisense ODNs directed against these PKC isoforms. Antisense ODNs against the 3' untranslated region of PKC and PKC were prepared as described in "Materials and Methods." We first investigated the effect of ODNs on the expression of the respective PKC isoforms in VSMCs. Fig 4A shows a Western blot analysis of PKC and PKC after 24 hours of exposure to antisense ODN against PKC compared with a sense ODN control. The cells were incubated with 1 µmol/L ODN together with lipofectin (10 µg/mL). Antisense ODN led to a downregulation of PKC to 44±11% compared with control. In contrast, protein levels of PKC were only slightly affected by exposure of VSMCs to antisense ODN against PKC. Lipofectin alone had no effect on PKC expression levels (data not shown). We then used an antisense ODN against the AUG start codon of PKC and investigated its effect on the expression of this PKC isoform. Fig 4A (right) shows a Western blot analysis of PKC after 24 hours of exposure to antisense ODN compared with a sense ODN control. The cells were incubated with 1 µmol/L ODN together with lipofectin (10 µg/mL). Antisense ODN led to a downregulation of PKC to 61±14% compared with control. In contrast, protein levels of PKC were not affected by exposure to antisense ODN against PKC. For the control experiments, sense and scrambled ODNs were used. These ODNs were also tested with respect to their effects on protein expression of the respective PKC isoform. None of the control ODNs reduced protein expression significantly.

View larger version (49K): [in this window] [in a new window]   Figure 4. A, Western blot analysis of antisense ODN against PKC and PKC as described in "Materials and Methods." Western blots were stained with PKC-specific antibodies as indicated. Left, Compared with sense-treated cells (control), antisense ODNs against PKC led to a significant downregulation of PKC. Protein levels of PKC were not significantly influenced by antisense ODNs against PKC. Right, Compared with control or sense-treated cells, antisense ODNs against PKC led to a significant downregulation of PKC. PKC protein levels were not influenced by antisense ODNs against PKC (n=4). B, VSMC spreading on fibronectin after downregulation of PKC isoforms by antisense ODNs against PKC, PKC, or both. Shown is the time course of cell spreading 10, 30, and 60 minutes after VSMC seeding. The initial cell spreading (10 minutes) on fibronectin was partially inhibited by downregulation of PKC and PKC. Combined treatment with antisense ODNs for both PKC isoforms almost completely inhibited cell spreading. A similar effect was observed after 60 minutes. Control experiments with sense and scrambled ODNs for both isoforms had no significant effect on fibronectin-induced cell spreading (n=4).

We then examined whether the specific downregulation of PKC and PKC with antisense ODN influenced VSMC spreading on fibronectin. The results of these experiments are shown in Fig 4B. VSMCs were incubated with lipofectin (10 µg/mL) and antisense ODN or sense ODN (control) against PKC, PKC, or both 24 hours before seeding on fibronectin. As an additional control experiment, antisense ODN against another PKC isoform, PKC, was used (data not shown). Antisense ODN for PKC decreased the fibronectin-induced cell spreading by 50%. Both the initial spreading and the later response to fibronectin were reduced. Antisense ODN against PKC also reduced the fibronectin-induced cell spreading significantly. Control experiments with sense and scrambled ODNs for both isoforms had no significant effect on fibronectin-induced cell spreading. The downregulation of both PKC isoforms had a combined effect on cell spreading and reduced the percentage of spread cells at 60 minutes to 22% compared with 68% in the control cells.

	Discussion

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The important finding in the present study is that fibronectin binding of VSMCs induces a rapid translocation of PKC isoforms and to focal adhesions and that these PKC isoforms mediate cell spreading. Thus, PKC and PKC may play a role in inside-out integrin signaling. These results suggest a positive-feedback mechanism, whereby integrin-induced generation of DAG and activation of distinct PKC isoforms influence focal adhesions and further enhance cell-matrix contact. In addition, we observed that integrin binding leads to a transient translocation of PKC into the nucleus. PKC and PKC were not affected by the binding to fibronectin. We also present evidence that the integrin-induced increase in DAG is not linked to activation of PLD but seems to be partially dependent on tyrosine phosphorylation.

We first observed that VSMC binding led to increased DAG production. Most likely, this increase in DAG is due to the previously described activation of phospholipase C and the breakdown of inositol phospholipids by integrin occupation. Our findings are in agreement with observations made in other cell types, suggesting that the occupation of ß₁ integrins leads to the generation of DAG.^{13 20 39 40} However, the exact mechanism whereby integrins mediate phospholipid breakdown is not clear.⁶ We could rule out the possibility that the DAG production is mediated via PLD and that PKC acts in a positive-feedback loop mechanism. We observed that an inhibition of tyrosine phosphorylation decreased DAG production. This observation implies that activation of a tyrosine kinase through one of the integrin receptors stimulates phospholipase C and increases DAG. The actions exerted by a tyrosine kinase inhibitor suggest that tyrosine phosphorylation also plays a role in the integrin-induced generation of DAG.²⁰ These results support a model whereby tyrosine kinases are recruited to focal adhesions after integrin-induced autophosphorylation. Elucidating the interactions of the signal transduction molecules with each other and with the integrin cytoplasmic domains will be key to understanding the initial events of signal transduction through the integrins.

Fibronectin binding induced a translocation of PKC to the nucleus. A nuclear translocation of PKC by growth factors and hormones has been described earlier (for review see References 41 and 42^{41 42} ). Several investigators observed that PKC is associated with nuclear membranes.^{25 27 28 30 33 37 39 43 44} Nuclear substrates of PKC have also been identified.^{32 42} These substrates include proteins implicated in maintaining chromatin structure and in the replication or repair of DNA, such as topoisomerase II.⁴⁵ These observations support the notion that PKC may perform important tasks within the cell nucleus.^{42 46} Since binding to fibronectin is associated with increased gene expression, it is conceivable that integrin-induced translocation of PKC plays a role in integrin-mediated gene expression. This hypothesis is presently under investigation. Which signal directs PKC isoforms to the nucleus is presently unclear. The presence of a nuclear localization sequence in the regulatory domain of the enzyme has been suggested.⁴⁷ However, this motif is absent in the nonconventional PKC isoforms. Alternatively, PKC itself may be directed to the nucleus by the action of PKC binding proteins and may not necessarily rely on the nuclear localization sequence.⁴⁸

Integrin binding induced a PKC isoform translocation to focal adhesions. That PKC isoforms can be associated with focal adhesions has been shown by our group and others.^{27 29 37 49 50} In contrast to the observations by Barry and Critchley,⁵¹ we could not detect an association of PKC with focal adhesions. However, these investigators used serum to induce the recruitment of the cytoskeletal proteins talin, vinculin, and paxillin, as well as the protein kinases pp125FAK and PKC, to newly formed focal adhesions. Our findings suggest that integrin occupation induces translocation of PKC and PKC, modulates the affinity of the fibronectin-binding integrins, and plays a role in integrin inside-out signaling.⁵² Several groups have shown that in different cell types direct activation of PKC via phorbol ester leads to an increase in integrin receptor affinity.⁵³ Such PKC activation has been demonstrated for ß₁, ß₂, and ß₃ integrins (for review see Reference 54⁵⁴ ). However, the mechanism whereby PKC activation alters integrin affinity remains elusive. The affinity modulation of the integrins may be mediated by both the ß- and -subunit integrins. Although reports of phosphorylation on - and ß-subunit cytoplasmic domains after PKC activation by phorbol ester treatment are numerous,^{38 54 55 56} there is no compelling evidence that direct phosphorylation of integrins by PKC serves as a physiological mechanism for cytoplasmic-induced affinity modulation.

The close association with FAK suggests that PKC may phosphorylate this kinase or other substrates within the focal adhesion complex. In fact, integrin-mediated pp125FAK tyrosine phosphorylation appears to be mediated by a PKC-dependent pathway. Vuori and Ruoslahti²² observed that PKC activation precedes the onset of cell spreading and suggested that PKC activation mediates or facilitates cell spreading via the phosphorylation of this tyrosine kinase. However, PKC did not directly act on pp125FAK, suggesting that other mechanisms are involved. In accordance with this hypothesis is the observation that the PKC-induced phosphorylation of pp125FAK depends on F-actin.⁵⁷ Another possible substrate of PKC within focal adhesions is paxillin. De Nichilo et al⁵⁸ have recently shown that paxillin localizes to focal contacts in the absence of FAK expression and is predominantly phosphorylated on serine residues in a PKC-dependent manner. That PKC may play an important role in the formation of focal adhesions has recently been suggested by Lewis et al.²³ They demonstrated that PKC activation by phorbol ester in _vß₅-expressing cells induced spreading, increased colocalization of -actinin, tensin, vinculin, and actin, and triggered tyrosine phosphorylation of FAK.

Our observations may have pathophysiological implications. Disturbances in PKC regulation may induce an alteration in mechanotransduction or cell migration. Berk et al⁵⁹ have proposed a model whereby PKC regulates the dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in the endothelial cell response to flow. Under conditions of increased PKC activity, such as diabetes,²⁶ PKC may influence cell adhesion. A preliminary report has shown increased tyrosine phosphorylation of pp125FAK and paxillin in glomeruli from patients with diabetes.⁶⁰

In summary, we showed that VSMC binding to fibronectin via integrins leads to an increase in DAG production and translocation of specific PKC isoforms. PKC is translocated to the nucleus and toward focal adhesions, whereas PKC is mainly associated with focal adhesions. Specific inhibition of these PKC isoforms by antisense ODN decreased cell spreading in an additive manner. These results suggest that PKC and PKC play a role in ligand-mediated outside-in integrin signaling as well as in cytoplasmic-initiated affinity modulation (inside-out). We conclude that PKC isoforms appear to play a major role in the promotion of integrin-induced cell spreading.

	Selected Abbreviations and Acronyms

 

DAG = diacylglycerol FAK = focal adhesion kinase ODN = oligodeoxyribonucleotide PKC = protein kinase C RGD = Gly-Arg-Gly-Asp-Asn-Pro VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by a grant in aid from the Deutsche Forschungsgemeinschaft to Hermann Haller.

Received August 14, 1997; accepted October 14, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Gumbiner BM. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 1996;84:345–357.[Medline] [Order article via Infotrieve]

2. Clark EA., Brugge JS. Integrins and signal transduction pathways: the road taken. Science. 1995;268:233–239.[Abstract/Free Full Text]

3. Damsky CH, Werb Z. Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curr Opin Cell Biol. 1992;4:772–781.[Medline] [Order article via Infotrieve]

4. Juliano R, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993;120:772–781.

5. Rosales C, O'Brien V, Kornberg L, Juliano R. Signal transduction by cell adhesion receptors. Biochim Biophys Acta. 1995;1242:77–98.[Medline] [Order article via Infotrieve]

6. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Biol. 1995;11:549–600.[Medline] [Order article via Infotrieve]

7. Yamada KM, Miyamoto S. Integrin transmembrane signalling and cytoskeletal control. Curr Opin Cell Biol. 1995;7:681–689.[Medline] [Order article via Infotrieve]

8. Krook A, Rapoport MJ, Anderson S, Pross H, Zhou YC, Denhardt DT, Delovitch TL, Haliotis T. p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts. Mol Cell Biol. 1993;13:1471–1479.[Abstract/Free Full Text]

9. Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature. 1996;380:538–540.[Medline] [Order article via Infotrieve]

10. McNamee HP, Ingber DE, Schwartz MA. Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown. J Cell Biol. 1993;121:673–678.[Abstract/Free Full Text]

11. Altieri DC, Stamnes SJ, Gahmber CG. Regulated Ca²⁺ signalling through leukocyte CD11b/CD18 integrin. Biochem J. 1992;288:465–473.

12. Chun JS, Jacobson BS. Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering. Mol Biol Cell. 1992;3:481–492.[Abstract]

13. Cybulsky AVS, Carbonetto S, Cyr MD, McTavish AJ, Huang Q. Extracellular matrix-stimulated phospholipase activation is mediated by beta 1-integrin. Am J Physiol. 1993;264:C323–C332.[Abstract/Free Full Text]

14. Hellberg C, Molony L, Zheng L, Andersson T. Ca²⁺ signalling mechanisms of the beta 2 integrin on neutrophils: involvement of phospholipase C gamma 2 and Ins(1,4,5)P3. Biochem J. 1996;317:403–409.

15. Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA. Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol. 1993;121:163–170.[Abstract/Free Full Text]

16. Menon RP, Pillai S, Sudhakaran PR. Binding of collagen causes intracellular mobilization of calcium in human mononuclear cells. Biochem Mol Biol Int. 1993;31:833–840.[Medline] [Order article via Infotrieve]

17. Schwartz MA. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Cell Biol. 1993;120:1003–1010.[Abstract/Free Full Text]

18. Schwartz MA, Brown EJ, Fazeli B. A 50-kDa integrin-associated protein is required for integrin-regulated calcium entry in endothelial cells. J Biol Chem. 1993;268:19931–19934.[Abstract/Free Full Text]

19. Schwartz MA, Denninghoff K. Alpha v integrins mediate the rise in intracellular calcium in endothelial cells on fibronectin even though they play a minor role in adhesion. J Biol Chem. 1994;269:11133–11137.[Abstract/Free Full Text]

20. Somogyi L, Lasic Z, Vukicevic S, Banfic H. Collagen type IV stimulates an increase in intracellular Ca²⁺ in pancreatic acinar cells via activation of phospholipase C. Biochem J. 1994;299:603–611.

21. Chun JS, Ha MJ, Jacobson BS. Differential translocation of protein kinase C epsilon during HeLa cell adhesion to a gelatin substratum. J Biol Chem. 1996;271:13008–13012.[Abstract/Free Full Text]

22. Vuori K, Ruoslahti E. Activation of protein kinase C precedes alpha 5 beta 1 integrin-mediated cell spreading on fibronectin. J Biol Chem. 1993;268:21459–21462.[Abstract/Free Full Text]

23. Lewis JM, Cheresh DA, Schwartz MA. Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J Cell Biol. 1996;134:1323–1332.[Abstract/Free Full Text]

24. Nishizuka Y. The Albert Lasker Medical Awards: the family of protein kinase C signal transduction. JAMA. 1989;262:1826–1833.[Abstract/Free Full Text]

25. Beckmann RC, Lindschau C, Haller H, Hucho F, Buchner K. Differential nuclear localization of protein kinase C isoforms in neuroblastomaxglioma hybrid cells. Eur J Biochem. 1994;222:335–343.[Medline] [Order article via Infotrieve]

26. Haller H, Baur E, Quass P, Behrend M, Lindschau C, Distler A, Luft FC. High glucose concentrations and protein kinase C isoforms in vascular smooth muscle cells. Kidney Int. 1995;47:1057–1067.[Medline] [Order article via Infotrieve]

27. Jaken S, Leach K, Klauck T. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol. 1989;109:697–704.[Abstract/Free Full Text]

28. Leach KL, Powers EA, Ruff VA, Jaken S, Kaufmann S. Type 3 protein kinase C localization to the nuclear envelope of phorbol ester treated NIH 3T3 cells. J Cell Biol. 1989;109:685–695.[Abstract/Free Full Text]

29. Kiley S, Parker PJ. Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation. J Cell Sci. 1995;108:1003–1016.[Abstract]

30. Neri LM, Billi AM, Manzoli L, Rubbini S, Gilmour RS, Cocco L, Martelli AM. Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF. FEBS Lett. 1994;347:63–68.[Medline] [Order article via Infotrieve]

31. Weinstein IB. The roles of specific isoforms of protein kinase C in growth control and human colon cancer. Princess Takamatsu Symp. 1991;22:277–283.[Medline] [Order article via Infotrieve]

32. Mochly-Rosen D, Khaner H, Lopez J. Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci U S A. 1991;88:3997–4000.[Abstract/Free Full Text]

33. Haller H, Ziegler W, Lindschau C, Luft FC. Endothelial cell tyrosine kinase receptor and G-protein coupled receptor activation involves distinct protein kinase C isoforms. Arterioscler Thromb Vasc Biol. 1996;16:678–686.[Abstract/Free Full Text]

34. Wrenn RW, Herman LE. Integrin-linked tyrosine phosphorylation increases membrane association of protein kinase C alpha in pancreatic acinar cells. Biochem Biophys Res Commun. 1995;208:978–984.[Medline] [Order article via Infotrieve]

35. Haller H, Lindschau C, Quass P, Distler A, Luft FC. Differentiation of vascular smooth muscle cells and the regulation of protein kinase C-. Circ Res. 1995;76:21–29.[Abstract/Free Full Text]

36. Kurscheid-Reich D, Throckmorton DC, Rasmussen H. Serotonin activates phospholipase D in rat mesangial cells. Am J Physiol. 1995;268:F997–F1003.[Abstract/Free Full Text]

37. Haller H, Lindschau C, Quass P, Distler A, Luft FC. Angiotensin II and PDGF induce differential distribution of PKC isoforms in vascular smooth muscle cells. Hypertension. 1994;23:848–852.[Abstract/Free Full Text]

38. Dean NM, McKay R. Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1994;91:11762–11766.[Abstract/Free Full Text]

39. Merrill JT, Winchester RJ, Buyon JP. Dynamic state of beta 2 integrin phosphorylation: regulation of neutrophil aggregation involves a phosphatase-dependent pathway. Clin Immunol Immunopathol. 1994;71:216–222.[Medline] [Order article via Infotrieve]

40. Merrill JT, Slade SG, Weissmann G, Winchester R, Buyon JP. Two pathways of CD11b/CD18-mediated neutrophil aggregation with different involvement of protein kinase C-dependent phosphorylation. J Immunol. 1990;145:2608–2615.[Abstract]

41. Buchner K. Protein kinase C in the transduction of signals toward and in the cell nucleus. Eur J Biochem. 1995;228:211–221.[Medline] [Order article via Infotrieve]

42. Irvine R, Divecha N. Nuclear functions of protein kinase C. Semin Cell Biol. 1992;3:225–235.[Medline] [Order article via Infotrieve]

43. Martelli AM, Gilmour RS, Falcieri E, Manzoli FA, Cocco L. Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement. Exp Cell Res. 1989;185:191–202.[Medline] [Order article via Infotrieve]

44. Martelli AM, Neri LM, Gilmour RS, Barker PJ, Huskisson NS, Manzoli FA, Cocco L. Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin-like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphospoinositides. Biochem Biophys Res Commun. 1991;177:480–487.[Medline] [Order article via Infotrieve]

45. Corbett AH, Fernald AW, Osheroff N. Protein kinase C modulates the catalytic activity of topoisomerase II by enhancing the rate of ATP hydrolysis: evidence for a common mechanism of regulation by phosphorylation. Biochemistry. 1993;32:2090–2097.[Medline] [Order article via Infotrieve]

46. Li L, Zhou J, James G, Heller-Harrison R, Czech MP, Olson EN. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domain. Cell. 1992;71:1181–1194.[Medline] [Order article via Infotrieve]

47. Malviya AN, Block C. A bipartite nuclear targeting motif in protein kinase C? Trend Biochem Sci. 1992;17:176–179.

48. James G, Olson E. Deletion of the regulatory domain of protein kinase C exposes regions in the hinge and catalytic domains that mediate nuclear targeting. J. Cell Biol. 1992;116:863–874.[Abstract/Free Full Text]

49. Jaken S. Measurement of phorbol ester receptors in intact cells and subcellular fractions. Methods Enzymol. 1989;141:275–289.

50. Woods A, Couchman JR. Protein kinase C involvement in focal adhesion formation. J Cell Sci. 1992;101:277–290.[Abstract/Free Full Text]

51. Barry ST, Critchley DR. The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions. J Cell Sci. 1994;107:2033–2045.[Abstract]

52. Ginsberg MH, Du X, Plow EF. Inside-out integrin signalling. Curr Opin Cell Biol. 1992;4:766–771.[Medline] [Order article via Infotrieve]

53. Shattil SJ, Haimovich B, Cunningham M, Lipfert L, Parsons JT, Ginsberg MH, Brugge JS. Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. J Biol Chem. 1994;269:14738–14745.[Abstract/Free Full Text]

54. Sastry SK, Horwitz AF. Integrin cytoplasmatic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr Opin Cell Biol. 1993;5:819–831.[Medline] [Order article via Infotrieve]

55. Freed E, Gailit J, van der Geer P, Ruoslahti E, Hunter T. A novel integrin ß subunit is associated with the vitronectin receptor a subunit (av) in a human osteosarcoma cell line and is a substrate for protein kinase C. Embo J. 1989;8:2955–2965.[Medline] [Order article via Infotrieve]

56. Pacifici R, Roman J, Kimble R, Civitelli R, Brownfield CM, Bizzarri C. Ligand binding to monocyte alpha 5 beta 1 integrin activates the alpha 2 beta 1 receptor via the alpha 5 subunit cytoplasmic domain and protein kinase C. J Immunol. 1994;153:2222–2233.[Abstract]

57. Zhang C, Qiu HE, Krafft GA, Klein WL. Protein kinase C and F-actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G-proteins and amyloid beta protein. FEBS Lett. 1996;386:185–188.[Medline] [Order article via Infotrieve]

58. De Nichilo MO, Yamada KM. Integrin alpha v beta 5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin. J Biol Chem. 1996;271:11016–11022.[Abstract/Free Full Text]

59. Berk BC, Corson MA, Peterson TE, Tseng H. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech. 1995 28;1439–1450.

60. Shikano T, Haneda M, Togawa M, Kikkawa R. Tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in glomeruli from diabetic rats [in Japanese]. Nippon Jinzo Gakkai Shi. 1996;38:57–64.[Medline] [Order article via Infotrieve]

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