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₁-Integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca²⁺ signaling

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 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 ₁-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that ₁-integrin, ₃-integrin, and perhaps ₅-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. 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 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 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 ₁-integrin with RGD peptide prevented the Ca²⁺ signal. Cross-linking of 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 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 ₁-integrin.

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

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