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

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 rPARP1 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-I 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.