3-DIMENSIONAL MATRIX PRIMES MESANGIAL CELLS TO DOWN-REGULATION OF ALPHA-SMOOTH MUSCLE ACTIN VIA DEACTIVATION OF CARG BOX ELEMENTS

Prolonged culture of mesangial cells forms multifocal nodular structur es, termed ''hillocks,'' composed of cells and extracellular matrix (E CM), which may mimic the situation in the glomerular mesangium. Mesang ial cells incorporated in hillocks show repressed expression of alpha- smooth muscle actin, a marker of mesangial cell activation/dedifferent iation. The aim of this study is to elucidate molecular mechanisms inv olved in this phenomenon, focusing on the activity of CArG box element s located in 5'-flanking region of the alpha-smooth muscle actin gene. Reporter mesangial cells were created to monitor the activity of CArG elements. These clones expressed beta-galactosidase gene (lacZ) under the control of CArG boxes. Within the hillocks, reporter cells showed repressed expression of lacZ as well as a-smooth muscle actin compare d to the cells in two-dimensional cultures. Consistent with this resul t, the reporter cells embedded in collagen gel exhibited down-regulati on of lacZ and alpha-smooth muscle actin transcripts. Deactivation of CArG box elements by transfection with either a dominant negative muta nt of serum response factor or a dominant negative form of ternary com plex factor Elk-l led to depressed expression of a-smooth muscle actin gene. These data suggested that three-dimensional ECM primes mesangia l cells to down-regulation of alpha-smooth muscle actin via deactivati on of CArG box elements.