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

被引:68
作者
Praetorius, HA
Praetorius, J
Nielsen, S
Frokiaer, J
Spring, KR
机构
[1] Univ Aarhus, Water & Salt Res Ctr, DK-8200 Aarhus, Denmark
[2] Univ Aarhus, Inst Clin, DK-8200 Aarhus, Denmark
[3] Univ Aarhus, Inst Anat, DK-8200 Aarhus, Denmark
[4] NHLBI, Kidney & Electrolyte Metab Lab, NIH, Bethesda, MD 20892 USA
关键词
intracellular calcium ion; antibody; Madin-Darby canine kidney cells; fibronectin; fluorescence imaging;
D O I
10.1152/ajprenal.00096.2004
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
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. Crosslinking 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.
引用
收藏
页码:F969 / F978
页数:10
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