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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.02-0089fje. |
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Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
2Correspondence: Department of Pathology, University of Pittsburgh, 707B Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261, USA. E-mail: carywu{at}imap.pitt.edu
SPECIFIC AIMS
Integrin-linked kinase (ILK) is a cytoplasmic component of the cell-extracellular matrix contact sites. In this study, we have investigated the role of ILK and its binding proteins PINCH and CH-ILKBP (calponin homology domain-containing ILK binding protein) in renal glomerular mesangial cell proliferation and fibronectin matrix deposition, processes critically involved in the pathogenesis of glomerulosclerosis.
PRINCIPAL FINDINGS
1. ILK, PINCH, and CH-ILKBP form a ternary complex in primary glomerular mesangial cells and colocalize in fibrillar fibronectin matrix contacts
To test whether ILK, PINCH, and CH-ILKBP form a complex in primary glomerular mesangial cells, we immunoprecipitated CH-ILKBP from lysates of mesangial cells with a monoclonal anti-CH-ILKBP antibody. Analyses of the anti-CH-ILKBP immunoprecipitates with anti-CH-ILKBP, anti-ILK, anti-PINCH, and anti-paxillin antibodies showed that ILK and PINCH, but not paxillin, were coimmunoprecipitated with CH-ILKBP, indicating that ILK, PINCH, and CH-ILKBP, but not paxillin, form a complex in mesangial cells. To test whether PINCH and CH-ILKBP colocalize with ILK in mesangial cell-matrix contacts (focal adhesions and fibrillar adhesions), we transfected mesangial cells with expression vectors encoding GFP-tagged PINCH and CH-ILKBP, respectively. Immunofluorescent staining of the cells with anti-ILK and anti-fibronectin antibodies showed that GFP-PINCH and GFP-CH-ILKBP colocalized with ILK in fibronectin fibrillar adhesions as well as in focal adhesions.
2. Overexpression of the ILK amino-terminal ANK fragment inhibits the PINCH-ILK interaction in mesangial cells
To assess the functional significance of the ILK-PINCH-CH-ILKBP complex formation in mesangial cells, we sought to disrupt formation of the ILK-PINCH-CH-ILKBP complex by overexpression of a FLAG-tagged PINCH binding amino-terminal ANK fragment of ILK (FLAG-ANK) in these cells. To do this, we infected primary rat mesangial cells with an adenoviral vector encoding FLAG-ANK. Analyses of the ILK-PINCH-CH-ILKBP complex in mesangial cells overexpressing FLAG-ANK showed that although the amount of ILK associated with CH-ILKBP was not significantly altered, the amount of PINCH associated with the ILK-CH-ILKBP complex was markedly reduced in mesangial cells overexpressing FLAG-ANK, indicating that overexpression of FLAG-ANK effectively inhibits the ILK-PINCH-CH-ILKBP complex formation in mesangial cells by disrupting the ILK-PINCH interaction.
3. Inhibition of PINCH-ILK interaction reduces fibronectin matrix deposition
We next analyzed the effect of inhibition of the PINCH-ILK interaction on fibronectin matrix deposition. We isolated fibronectin matrix from mesangial cells infected with the adenoviral FLAG-ANK expression vector and from those infected with the ß-galactosidase control adenoviral vector. Western blotting analyses showed that mesangial cells overexpressing FLAG-ANK deposited much less fibronectin matrix than the control cells. The level of total fibronectin was not significantly altered in mesangial cells overexpressing FLAG-ANK. These results suggest that the PINCH-ILK interaction is critically involved in the mesangial fibronectin matrix deposition.
4. Inhibition of the PINCH-ILK interaction decreases cell proliferation
To test whether PINCH-ILK interaction is involved in the regulation of mesangial cell proliferation, we analyzed the effect of FLAG-ANK overexpression on mesangial cell proliferation by 1) directly counting the cell number and 2) measuring the percentage of nuclear BrdU incorporation, an index for proliferating cells. The results showed that overexpression of FLAG-ANK significantly reduced the increase in cell number. Consistent with the results of direct cell counting, the percentage of BrdU incorporation in nuclei was also significantly reduced in mesangial cells overexpressing FLAG-ANK.
5. Overexpression of the ILK carboxyl-terminal fragment inhibits ILK-CH-ILKBP interaction in mesangial cells
To further analyze the functions of the PINCH-ILK-CH-ILKBP complex, we generated an adenoviral expression vector encoding a FLAG-tagged CH-ILKBP binding carboxyl-terminal fragment of ILK, which, we reasoned, could potentially inhibit the PINCH-ILK-CH-ILKBP complex formation by disrupting the ILK-CH-ILKBP interaction. The expression of FLAG-ILKC in mesangial cells infected with the FLAG-ILKC adenovirus but not in those infected with the control adenovirus was confirmed by Western blotting with anti-FLAG antibodies. Overexpression of FLAG-ILKC significantly reduced the amounts of ILK and PINCH associated with CH-ILKBP, indicating that FLAG-ILKC indeed functions as an effective inhibitor of the PINCH-ILK-CH-ILKBP complex formation.
6. Inhibition of ILK-CH-ILKBP interaction reduces mesangial fibronectin matrix deposition and proliferation
We next tested the effects of inhibition of ILK-CH-ILKBP interaction on mesangial fibronectin matrix deposition and cell proliferation. We isolated fibronectin matrix from the FLAG-ILKC overexpressing mesangial cells and the control cells, respectively, and analyzed fibronectin deposited into the extracellular matrix and that in the total cellular extracts by Western blotting. The results showed that mesangial cells overexpressing FLAG-ILKC, like mesangial cells overexpressing FLAG-ANK, deposited much less fibronectin into extracellular matrix than the control cells (Fig. 1
A). Abundant fibronectin was detected in the total cellular extracts of all cells, albeit the level of the total fibronectin in the FLAG-ILKC overexpressing cells appeared to be slightly reduced (Fig. 1B
).
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To evaluate the effect of inhibition of the ILK-CH-ILKBP interaction on mesangial cell proliferation, we measured the rate of cell proliferation in mesangial cells overexpressing FLAG-ILKC by directly counting the cell number and measuring the percentage of nuclear BrdU incorporation. The results showed that overexpression of FLAG-ILKC significantly reduced the rate of mesangial cell proliferation (Fig. 2
). These results, together with those obtained with FLAG-ANK, suggest that inhibition of the PINCH-ILK-CH-ILKBP complex, by either disrupting the ILK-CH-ILKBP interaction or disrupting the PINCH-ILK interaction, reduces mesangial cell proliferation.
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CONCLUSIONS AND SIGNIFICANCE
We have shown in this report that ILK forms a complex with PINCH and CH-ILKBP in primary mesangial cells and they are coclustered at fibrillar fibronectin matrix contacts, sites involved in the fibronectin matrix deposition. We have analyzed the functional significance of the PINCH-ILK-CH-ILKBP complex formation by overexpression of two different mutants of ILK that disrupt the PINCH-ILK and ILK-CH-ILKBP interactions, respectively, in mesangial cells. Disruption of either the PINCH-ILK interaction or the ILK-CH-ILKBP interaction significantly reduced mesangial fibronectin matrix deposition and proliferation. These results suggest that the PINCH-ILK-CH-ILKBP complex plays a crucial role in the regulation of glomerular mesangial cell proliferation and fibronectin matrix deposition (Fig. 3
).
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How does the PINCH-ILK-CH-ILKBP complex regulate fibronectin matrix deposition? Because 1) fibronectin matrix deposition is an integrin-mediated process that requires integrin cytoplasmic interactions with the actin cytoskeleton, 2) components of the PINCH-ILK-CH-ILKBP complex interact with integrin cytoplasmic domains as well as the actin filaments, and 3) the three components of the PINCH-ILK-CH-ILKBP complex are coclustered at fibrillar fibronectin matrix contacts, the PINCH-ILK-CH-ILKBP complex most likely functions in the fibronectin matrix deposition by providing an important physical connection between integrins and the actin cytoskeleton at the cell-fibronectin matrix contact sites. Thus, disruption of the PINCH-ILK-CH-ILKBP complex could weaken the connection between integrins and the actin cytoskeleton at fibronectin matrix contacts and thereby reduce fibronectin matrix deposition (Fig. 3)
.
Previous studies have shown that ILK signaling is crucially involved in cell proliferation. The findings described in this report suggest that the ILK-mediated protein-protein interactions, in addition to its kinase activity, are required for ILK signaling in the regulation of cell proliferation.
We have recently found that mesangial ILK expression is increased in human patients with diabetic nephropathy. Kretzler et al. recently identified ILK as a candidate effector in proteinuria in patients with congenital nephritic syndrome of the Finnish type. Thus, ILK, and the PINCH-ILK-CH-ILKBP complex as shown in this paper likely represent a key regulator of the glomerular cell behavior. Elucidation of the molecular mechanism by which cells control the assembly and functions of the PINCH-ILK-CH-ILKBP complex will be of considerable value to understanding the molecular basis underlying the pathogenesis of renal failure and could potentially lead to novel approaches in the therapeutic control of progressive renal failure and other pathological processes involving abnormal cell proliferation and fibronectin matrix deposition.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0089fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.02-0089fje 
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