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Am J Physiol Renal Physiol 287: F969-F978, 2004. First published June 29, 2004; doi:10.1152/ajprenal.00096.2004
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{beta}1-Integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling

H. A. Praetorius,1,2 J. Praetorius,1,3 S. Nielsen,1,3 J. Frokiaer,1,2 and K. R. Spring4

1The Water and Salt Research Center, 2Clinical Institute and 3Institute of Anatomy, University of Aarhus, DK-8200 Aarhus, Denmark; and 4Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603

Submitted 23 March 2004 ; accepted in final form 23 June 2004

Because {beta}1-integrin is involved in sensing of fluid flow rate in endothelial cells, a function that in Madin-Darby canine kidney (MDCK) cells is confined to the primary cilium, we hypothesized {beta}1-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that {beta}1-integrin, {alpha}3-integrin, and perhaps {alpha}5-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. {beta}1-Integrin was also colocalized with tubulin to the primary cilia of the rat renal collecting ducts, as well as to the cilia of proximal tubules and thick ascending limbs. Immunogold-electron microscopy confirmed the presence of {beta}1-integrin on primary cilia of MDCK cells and rat collecting ducts. Intracellular Ca2+ levels, monitored by fluorescence microscopy on fluo 4-loaded MDCK cells, significantly increased on addition of fibronectin, a {beta}1-integrin ligand, to mature MDCK cells with an IC50 of 0.02 mg/l. In immature, nonciliated cells or in deciliated mature cells, the IC50 was 0.40 mg/l. Blocking the fibronectin-binding sites of {beta}1-integrin with RGD peptide prevented the Ca2+ signal. Cross-linking of {beta}1-integrins by Sambucus nigra agglutinin produced a Ca2+ response similar to the addition of fibronectin. Furthermore, the fibronectin-induced response was not dependent on flow or a flow-induced Ca2+ response. Finally, the flow-induced Ca2+ response was not prevented by the fibronectin-induced signal. Although {beta}1-integrin on the primary cilium greatly potentiates the fibronectin-induced Ca2+ signaling in MDCK cells, the flow-dependent Ca2+ signal is not mediated through activation of {beta}1-integrin.

intracellular calcium ion; antibody; Madin-Darby canine kidney cells; fibronectin; fluorescence imaging


Address for reprint requests and other correspondence: H. A. Praetorius, The Water and Salt Research Center, Clinical Institute, Univ. of Aarhus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark (E-mail: Helle.Praetorius{at}ki.au.dk)




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