TGFbeta2 influences alpha5-beta1 integrin distribution in human lens cells

TGFbeta plays a central role in posterior capsule opacification, in which cell proliferation and matrix deposition, accompanied by capsular wrinkling, are largely responsible for the increased light scatter involved. Human FHL124 cells were plated onto uncoated glass coverslips to form circular patches so that the central cells reached confluency while the peripheral cells grew outwards. Cell patches were exposed to serum free (SF) EMEM (control) or TGFbeta supplemented (10 ng ml(-1)) EMEM. Fibronectin (Fn), alpha5beta1 integrin and F-actin were localized by immunofluorescence techniques and analysed by confocal microscopy. In the confluent, central cells in SF medium alpha5beta1 showed a punctate distribution while Fn was present in strongly staining fibres. TGFbeta had no effect on integrin or Fn distribution in confluent cells. In the peripheral, motile cells of the patches in SF conditions alpha5beta1 was localized in well-defined focal adhesion plaques at the ends of actin stress fibres, while Fn was distributed in a punctate perinuclear pattern. TGFbeta had a profound dispersing effect on the integrin causing a widespread distribution of alpha5beta1 in the membrane with no apparent association with the actin filaments. The cells had a more fibroblastic morphology with increased deposition of Fn near the nucleus. All the TGFbeta-induced changes were inhibited by the TGFbeta antibody CAT152 (Cambridge Antibody Technology). Culture with a function-blocking alpha5 antibody or Fn antibody resulted in detachment of the peripheral cells from the patches, but the central cells remained intact. The patch culture method therefore provides a convenient means of investigating the differences between confluent and growing lens cells both in terms of the patterns of alpha5beta1 integrin and Fn and also in the response of the molecular arrangements of both to TGFbeta2.