Identification Of A Novel Interaction Between Integrin-Linked Kinase And Beta-Arrestin 1

Integrin-linked kinase (ILK) is a ubiquitous scaffold protein essential for the development of front-rear polarity and directional migration of epidermal keratinocytes. β-arrestin 1 is another adaptor protein which has recently emerged as a key factor in modulating proliferation and migration of various cell types. Previous studies have demonstrated an association between β-arrestin 2 and ILK in cerebellar granule precursor cells. I have now identified a novel interaction between ILK and β-arrestin 1 in primary keratinocytes that occurs directly and without post-translational modifications. The N-terminal 67 residues of ILK and multiple regions in β-arrestin 1 are important for this interaction. This association occurs regardless of the conformational state of β-arrestin 1. Furthermore, ILK and β-arrestin 1 colocalize at cell extensions, suggesting a possible role for this complex in the development of front-rear cell polarity and migration. My analysis of this novel interaction provides new insight into mechanisms of keratinocyte regulation

Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.

<p>Genes involved with a hyperproliferative response are shown.</p><p>Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.</p

(5Z)-7-oxozeaenol inhibits TGFβ1-induced mRNA expression in human gingival fibroblasts.

<p>(A) Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml^-1 (90 pM)) ligand or left untreated. Total RNA was harvested six hours later and subjected to gene expression profiling using GeneChip Human Gene 1.0 ST arrays (N = 2) as described in Methods. 147 genes were up-regulated in response to TGFβ1 (1.7 fold induction compared to DMSO control group) and 139 genes of the latter group were found to be (5<i>Z</i>)-7-Oxozeaenol sensitive. (B) Human gingival fibroblasts were treated as in (A) and subject to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown. * = p<0.05; ** = p<0.01; *** = p<0.001, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 protein expression in human gingival fibroblasts.

<p>(A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-CCN2 and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 4 separate occasions and relative CCN2 expression in response to TGFβ1 was calculated using densitometry (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) Indirect immunofluorescence analysis. Human gingival fibroblasts cultured on glass coverslips as treated as in (A). Cells were fixed and stained with an anti-CCN2 antibody and DyLight 594 conjugated secondary antibody. Cells were counterstained with DAPI to detect nuclei. Representative photographs are shown. Experiments were conducted four times, and relative fluoresce intensity ratio was calculated as described in methods (N = 4, averages+/-SEM are shown. * = p<0.05, One-Way ANOVA). (C) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-phospho-TAK1 and anti-beta actin antibodies, as indicated.</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 mRNA expression in human gingival fibroblasts.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. Values are expressed relative to untreated control. (N = 3; averages+/-SEM are shown; **** = p<0.0001, * = p<0.05 One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1 induced gingival fibroblast proliferation.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol ((5Z)-7-oxo; 400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml-1 (90 pM)) ligand or left untreated. Cultures were grown in the presence of BrdU for up to 72 hours as described in methods. One of three representative experiments is shown; (N = 4; averages+/-SEM are shown * p<0.05 for: DMSO vs TGFβ1, (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1; ** p<0.05 for: DMSO vs TGFβ1; (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1. Two-Way ANOVA followed by Tukey's Post Hoc analysis).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced collagen expression.

<p>A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-collagen type I and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 3 separate occasions. (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) mRNA analysis Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1 (90 pM)). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown; * = p<0.05, One-Way ANOVA).</p

Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts.

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents

TGFβ1-induced CCN2 and αSMA protein expression in human dermal and gingival fibroblasts is reduced by N-acetylcysteine and diphenyleneiodonium.

<p>Human dermal fibroblasts (A) and human gingival fibroblasts (B) were serum-starved overnight. Cells were incubated with either N-acetylcysteine (10 mM) or diphenyleneiodonium (10 μM) for 45 min followed by treatment with or without TGFβ1 (4ng/ml) for 24 hours. Protein lysates were prepared and subjected to western blot analysis with the indicated antibodies. β-actin was used to normalize for protein loading. Representative western blots are shown (n = 3).</p

Inhibition of NOX4 reduces TGFβ1-induced CCN2 and αSMA protein expression in human dermal and gingival fibroblasts and the overexpression of CCN2 and α-SMA, as well as the contractility of lesional SSc dermal fibroblasts.

<p>Human dermal fibroblasts (A) and human gingival fibroblasts (B) were serum-starved overnight. Cells were incubated with GKT-137831 (30 μM) for 45 min followed by treatment with or without TGFβ1 (4ng/ml) for 24 hours. Protein lysates were prepared and subjected to western blot analysis with the indicated antibodies. β-actin was used to normalize for protein loading. Representative western blots are shown (n = 3). <b>(C)</b> Dermal fibroblasts cultured from healthy individuals (NF, normal fibroblasts) and those with scleroderma (systemic sclerosis, SSc) were serum-starved overnight. Cells were incubated with GKT-137831 (30μM) for 24 hours. Protein lysates were prepared and subjected to western blot analysis with the indicated antibodies. GAPDH was used to normalize for protein loading. Representative western blots containing lysates from three different patients are shown (n = 3). (D) NF and SSc fibroblasts were cultured within 3D-collagen lattices in the presence and absence of GKT-137831 (30 μM). After polymerization, the gels were mechanically detached from the wells, and the contraction of the gel was quantified. Results are expressed as a mean +/- SD (n = 3). One-Way ANOVA with post-hoc Tukey test was conducted. * = p<0.05 relative to SSc (-) GKT.</p