Abstract
We have shown previously that the vasoactive peptide bradykinin (BK) stimulates proliferation of a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) via transactivation of epidermal growth factor receptor (EGFR) by a mechanism that involves matrix metalloproteinases (collagenase-2 and -3). Because collagenases lack an integral membrane domain, we hypothesized that receptors for extracellular matrix proteins, integrins, may play a role in BK-induced signaling by targeting collagenases to the membrane, thus forming a functional signaling complex. BK-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) in mIMCD-3 cells was reduced by ∼65% by synthetic peptides containing an Arg-Gly-Asp sequence, supporting roles for integrins in BK-induced signaling. Neutralizing antibody against α5β1 integrin partially (∼60%) blocked BK-induced ERK activation but did not affect EGF-induced ERK activation. Silencing of α5 and β1 expression by transfecting cells with small interfering RNAs (siRNA) significantly decreased BK-induced ERK activation (∼80%) and EGFR phosphorylation (∼50%). This effect was even more pronounced in cells that were cotransfected with siRNAs directed against both collagenases and α5β1 integrin. On the basis of our results, we suggested that integrin α5β1 is involved in BK-induced signaling in mIMCD-3 cells. Using immunoprecipitation/Western blotting, we demonstrated association of BK B2 receptor with α5β1 integrin upon BK treatment. Furthermore, BK induced association of α5β1 integrin with EGFR. These data provide the first evidence that specific integrins are involved in BK B2 receptor-induced signaling in kidney cells, and ultimately might lead to development of new strategies for treatment of renal tubulointerstitial fibrosis.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK52448], the National Institutes of Health National Institute of General Medical Sciences [Grant GM3909], the Department of Veterans Affairs Merit and Research Enhancement Award Program (to J.R.R. and M.N.G.), the American Heart Association [Grant-in-Aid 0655445U], and a laboratory endowment jointly supported by the Medical University of South Carolina Division of Nephrology and Dialysis Clinics, Incorporated.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064840.
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ABBREVIATIONS:
- BK
- bradykinin
- ERK
- extracellular signal-regulated protein kinase
- EGFR
- epidermal growth factor receptor
- MMP
- matrix metalloproteinase
- GPCR
- G protein-coupled receptor
- FAK
- focal adhesion kinase
- EGF
- epidermal growth factor
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- PCR
- polymerase chain reaction
- RIPA
- radioimmunoprecipitation assay
- ANOVA
- analysis of variance
- siRNA
- small interfering RNA.
- Received March 18, 2010.
- Accepted April 12, 2010.
- U.S. Government work not protected by U.S. copyright
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