Beta1-integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling.
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 Ca(2+) 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 IC(50) of 0.02 mg/l. In immature, nonciliated cells or in deciliated mature cells, the IC(50) was 0.40 mg/l. Blocking the fibronectin-binding sites of beta(1)-integrin with RGD peptide prevented the Ca(2+) signal. Cross-linking of beta(1)-integrins by Sambucus nigra agglutinin produced a Ca(2+) response similar to the addition of fibronectin. Furthermore, the fibronectin-induced response was not dependent on flow or a flow-induced Ca(2+) response. Finally, the flow-induced Ca(2+) response was not prevented by the fibronectin-induced signal. Although beta(1)-integrin on the primary cilium greatly potentiates the fibronectin-induced Ca(2+) signaling in MDCK cells, the flow-dependent Ca(2+) signal is not mediated through activation of beta(1)-integrin.
Praetorius
H A
HA
The Water and Salt Research Center, Clinical Institute, University of Aarhus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. Helle.Praetorius@ki.au.dk
Praetorius
J
J
Nielsen
S
S
Frokiaer
J
J
Spring
K R
KR
D000814:Aniline Compounds; D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002697:Chloral Hydrate / Q000494:pharmacology; D002923:Cilia / Q000378:metabolism; D004285:Dogs; D005353:Fibronectins / Q000494:pharmacology*; D006993:Hypnotics and Sedatives / Q000494:pharmacology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D019012:Integrin beta1 / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D037102:Lectins; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014966:Xanthenes
D000814:Aniline Compounds; D005353:Fibronectins; C409648:Fluo 4; D006993:Hypnotics and Sedatives; D019012:Integrin beta1; D037102:Lectins; D014966:Xanthenes; D002697:Chloral Hydrate