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 chondrocytes. Changes in the levels of Sp1 oligonucleotide binding activity were examined in nuclear extracts from freshly isolated human chondrocytes, from chondrocytes that had been cultured under conditions that allowed the maintenance of a chondrocyte-specific phenotype on plastic dishes coated with the hydrogel poly(2-hydroxyethyl methacrylate), and from chondrocytes induced to dedifferentiate into fibroblast-like cells by passage in monolayer culture on plastic substrata. It was observed that Sp1 binding was 2-3-fold greater in nuclear extracts from dedifferentiated chondrocytes than in nuclear extracts from either freshly 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 against Sp1 to nuclear extracts from freshly isolated chondrocytes or to extracts isolated from chondrocytes cultured in monolayer decreased the binding of Sp1 by approximately 85%. However, when the same experiment was carried out with nuclear extracts prepared from cells cultured on poly(2-hydroxyethyl methacrylate)-coated plates, only a very slight inhibition of Sp1 binding was observed. When fragments of the COL2A1 promoter containing putative Sp1 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 chondrocytes were 2-3-fold greater than the amounts formed with nuclear extracts from freshly isolated chondrocytes or from cells cultured in suspension. Quantitation of DNA binding activity by titration experiments demonstrated that nuclear extracts from fibroblast-like cells contained approximately 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.
Dharmavaram
R M
RM
Division of Rheumatology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Liu
G
G
Mowers
S D
SD
Jimenez
S A
SA
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics*; D004331:Drosophila melanogaster; D005333:Fetus; D005347:Fibroblasts; D006132:Growth Plate / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D011102:Polyhydroxyethyl Methacrylate; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000302:isolation & purification / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D011102:Polyhydroxyethyl Methacrylate; D003094:Collagen