DETECTION AND CHARACTERIZATION OF SP1 BINDING-ACTIVITY IN HUMAN CHONDROCYTES AND ITS ALTERATIONS DURING CHONDROCYTE DEDIFFERENTIATION

We have detected DNA binding activity for a synthetic oligonucleotide containing an Sp1 consensus sequence in nuclear extracts from human ch ondrocytes. Changes in the levels of Sp1 oligonucleotide binding activ ity were examined in nuclear extracts from freshly isolated human chon drocytes, from chondrocytes that had beets cultured under conditions t hat allowed the maintenance of a chondrocyte-specific phenotype on pla stic dishes coated with the hydrogel poly(2-hydroxyethyl methacrylate) , and from chondrocytes induced to dedifferentiate into fibroblast-lik e cells by passage in monolayer culture on plastic substrate, It was o bserved that Spl binding was 2-3-fold greater in nuclear extracts from dedifferentiated chondrocytes than in nuclear extracts from either fr eshly isolated chondrocytes or from cells cultured in suspension. The Sp1 binding activity was specific, since it was competed by unlabeled Sp1 but not by AP1 or AP2, The addition of a polyclonal antibody again st Sp1 to nuclear extracts from freshly isolated chondrocytes or to ex tracts isolate from chondrocytes cultured in monolayer decreased the b inding of Sp1 by similar to 85%, However, when the same experiment was carried out with nuclear extracts prepared from cells cultured on pol y(2-hydroxyethyl methacrylate)-coated plates, only a very slight inhib ition of Sp1 binding was observed, When fragments of the COL2A1 promot er containing putative Spl binding sites amplified by polymerase chain reaction were examined, it was found that the amounts of DNA-protein complex formed with nuclear extracts from dedifferentiated chondrocyte s were 2-3-fold greater than the amounts formed with nuclear extracts from freshly isolated chondrocytes or from cells cultured in suspensio n, Quantitation of DNA binding activity by titration experiments demon strated that nuclear extracts from fibroblast-like cells contained app roximately 2-fold greater Sp-1 specific binding activity than nuclear extracts from chondrocytes. The direct role of Sp1 in type II collagen gene transcription was demonstrated by co-transfection experiments of COL2A1 promoter-CAT constructs in Drosophila Schneider line L2 cells that lack Sp1 homologs. This is the first demonstration of Sp1 binding activity in human chondrocytes and of differences in Sp1 DNA binding activity between differentiated and dedifferentiated chondrocytes.