Bradykinin modulates focal adhesions and induces stress fiber remodeling in renal papillary collecting duct cells

Abstract

Márquez MG, del Carmen Fernández-Tome M, Favale NO, Pescio LG, Sterin-Speziale NB. Bradykinin modulates focal adhesions and induces stress fiber remodeling in renal papillary collecting duct cells. Am J Physiol Renal Physiol 294: F603-F613, 2008. First published December 26, 2007 doi:10.1152/ajprenal.00234.2007 are specialized regions of cell attachment to the extracellular matrix. Previous works have suggested that bradykinin (BK) can modulate cell-matrix interaction. In the present study, we used a physiological cellular model to evaluate the potential role of BK in modulating FAs and stress fibers. We performed a quantitative morphometric analysis of FAs in primary cultured rat renal papillary collecting duct cells, which included size, axial ratio (shape), and average length. After 1, 5, or 10 min of incubation with BK, cultured cells were immunostained and analyzed by confocal microscopy. Although the shape of FAs was not altered, BK induced a decrease in the number of vinculin-stained FAs per cell, and a decrease in both their size and their average length, but not in talin-containing FAs, thus suggesting that BK could be inducing a restructuring of FAs. BK also induced a remodeling of the actin filament assemblies rather than their dissipation. Since we have previously demonstrated that BK stimulates activation of PLC␤ in rat renal papillae, we attempted to determine whether BK can modulate FA restructuring by this mechanism, by pretreating cultured cells with the PLC␤ inhibitor U73122. The present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLC␤. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function. stress fibers; cell-matrix adhesion; vinculin; talin; actin filaments FOCAL ADHESIONS (FAs) are specialized regions of the cell involved in the attachment to the extracellular matrix, where actin stress fibers and the extracellular matrix are connected (4, 35). FA assembly occurs through the binding of the integrinextracellular domain to extracellular matrix proteins, followed by the interaction of the ␤-integrin cytoplasmic domain with talin. Talin can then recruit vinculin, which, in turn, binds other FA proteins (15,21,33). In typical adherent cultured cells, a large number of distinct FAs, ranging in size from less than a square micrometer to several square micrometers, can be detected. The number of FAs, their size, and distribution can vary from one cell to another or even within a single cell, and their morphological diversity may be affected by multiple factors (36). In migrating cells, integrin-mediated adhesions are molecularly heterogeneous and appear in different forms such as "classic" FAs, fibrillar adhesions, and focal complexes. Each of these has a typical morphology and molecular composition (34). Besides h...

Mechanisms and Functions of Vinculin Interactions with Phospholipids at Cell Adhesion Sites

“…The endogenous intrarenal hormone bradykinin restructures FAs transiently in renal papillary collecting duct cells, causing a reversible dissociation of vinculin but not talin from FAs and inducing the reorganization of the actin cytoskeleton while preserving cell attachment to the ECM and neighboring cells (58). Subsequent studies provided evidence that some of the vinculin released upon bradykinin stimulation was located in cytoplasmic vesicles containing PIP 2 (59).…”

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2in renal papillary collecting duct cells

“…Using this experimental strategy, we demonstrated that BK treatment induces the dissipation of most of the vinculin-stained FAs and that the pretreatment of cultured cells with the PLC␤ inhibitor U73122 abolishes the loss of vinculinstained FAs resulting from the BK treatment. By contrast, BK does not induce dissipation of talin-stained FAs, as occurs with vinculin-stained FAs (27).…”

“…In a previous study (27), we took advantage of the fact that primary cultured papillary collecting duct cells preserve their tendency to attach with their self-formed extracellular matrix through FAs by mimicking their behavior in intact tissue. Using this experimental strategy, we demonstrated that BK treatment induces the dissipation of most of the vinculin-stained FAs and that the pretreatment of cultured cells with the PLC␤ inhibitor U73122 abolishes the loss of vinculinstained FAs resulting from the BK treatment.…”

“…Since we have previously demonstrated that BK stimulates the activation of PLC␤ in rat renal papillae (39) and modulates FAs through a mechanism that involves the activation of PLC␤ in rat renal papilla collecting duct cells (27), we attempted to determine whether BK-induced vinculin-stained FA dissipation depends on BK-B2R activation. To this end, we incubated nonconfluent primary cultures of rat renal papillary collecting duct cells with 100 nM HOE-140, a selective BK B 2 R antagonist, for 30 min before BK stimulation and subsequently performed an analysis by confocal microscopy.…”

“…We recently demonstrated (27) that BK modulates cytoskeleton organization through a PLC␤-dependent mechanism. Indeed, BK dissipates vinculin-but not talin-stained FAs, thus inducing a remodeling of the actin cytoskeleton in a reversible fashion and preserving cell attachment to the extracellular matrix, thus maintaining the cell-to-cell connection (27). Since FAs are the most stable points of cell tethering to the extracellular matrix, their dissipation is expected to cause cell detachment.…”