Abstract
Purpose :
To determine if integrin activation by talin-1 is needed for TGFβ2-induced increases in fibronectin fibril formation.
Methods :
Inactivation of αvβ3 integrin was done by transducing the human trabecular meshwork (TM) TM-1 cell line with pLKO1 lentiviral vector expressing human TLN1 shRNA to knockout talin-1 expression. An empty pLKO1 vector was used as a control. Stable clones of talin-1 deficient (TLNKO) or control (pLKO1) cells were established using puromycin selection. Lack of talin-1 expression and αvβ3 integrin inactivation was verified by immunofluorescence microscopy (IF) using a mouse anti-talin1 antibody or αvβ3 integrin antibody LM609 followed by Alexa fluorophore conjugated secondary antibody. Flow cytometry analysis was also performed to determine changes in αvβ3 integrin expression levels on the cell surface. The effect of integrin inactivation on TGFβ2-induced increase in fibronectin protein expression was determined by treating TLNKO or pLKO1 cells for 96hrs in the absence or presence of 5ng/ml TGFβ2 in media containing 1% FBS. Fibronectin fibril formation was determined by IF using a rabbit anti-fibronectin antibody and an Alexa fluorophore conjugated secondary antibody. A pEGFP-C1 vector expressing full length talin-1 was used to rescue talin-1 expression in TLNKO and pLKO1 cells prior to TGFβ2 treatment.
Results :
TLNKO cells showed decreased talin-1 and αvβ3 integrin expression and fewer focal adhesions compared to pLKO1 cells. Flow cytometry showed less αvβ3 integrin on the cell surface of TLNKO versus pLKO1 cells. TGFβ2 treatment increased fibronectin protein levels and incorporation into fibrils in pLKO1 cells whereas TLNKO cells showed very little increase in fibronectin fibril levels. Transfection of full length talin-1 into TLNKO cells rescued the increased fibronectin protein expression induced by TGFβ2 treatment and talin-1 localization in focal adhesions.
Conclusions :
These studies suggest that the increase in fibronectin fibrils induced by TGFβ2 in TM cells may be dependent on talin-1 mediated activation of αvβ3 integrin.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.