Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells

Abstract

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages. All types of cultured cells expressed PARP-1 in Western blot when grown to sub-confluence. However, a dramatic reduction was observed at post-confluence. Similarly, high levels of Sp1/Sp3 were observed by both Western blot and EMSAs (electrophoretic mobility-shift assays) in sub-confluent, but not post-confluent, cells. Consistent with these results, the promoter of the rPARP-1 (rat PARP-1) gene directed high levels of activity in sub-confluent, but not confluent, cells upon transfection of various CAT (chloramphenicol acetyltransferase)–rPARP-1 promoter constructs into cultured cells. The positive regulatory influence of Sp1 was not solely exerted on the rPARP-1 promoter constructs, as inhibition of endogenous Sp1 expression in HDKs (human dermal keratinocytes) through the transfection of Sp1 RNAi (RNA interference) considerably reduced endogenous hPARP-1 (human PARP-1) expression as well. The reduction in PARP-1 protein expression as cells reached confluence also translated into a corresponding reduction in PARP-1 activity. In addition, expression of both Sp1/Sp3, as well as that of PARP-1, was dramatically reduced as cells were passaged in culture and progressed towards irreversible terminal differentiation. PARP-1 gene expression therefore appears to be co-ordinated with that of Sp1 and Sp3 in primary cultured cells, suggesting that PARP-1 may play some important functions during the proliferative burst that characterizes wound healing.