β1-Integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+signaling

Abstract

Because β1-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 β1-integrin to be an important part of the primary ciliary mechanosensory apparatus in MDCK cells. We observed that β1-integrin, α3-integrin, and perhaps α5-integrin were localized to the primary cilium of MDCK cells by combining lectin and immunofluorescence confocal microscopy. β1-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 β1-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 β1-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 β1-integrin with RGD peptide prevented the Ca2+ signal. Cross-linking of β1-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 β1-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 β1-integrin.