DNA-BINDING BY C EBP PROTEINS CORRELATES WITH HEPATOCYTE PROLIFERATION/

The leucine zipper transcription factors C/EBP alpha and C/EBP beta ex hibit growth-related variations of expression and DNA binding during l iver regeneration. We examined the expression of C/EBP proteins in rel ation to hepatocyte proliferation by studying their DNA-binding activi ty in primary mouse hepatocytes in vitro. Mouse hepatocytes were disso ciated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell prote in extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consens us sequence oligomer (bZIP). C/EBP alpha is the major bZIP-binding pro tein present in the dissociated cells prior to plating. With the cultu re conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBP bet a binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBP alpha binding continued to be gr eatly reduced and no C/EBP delta binding was observed. C/EBP beta bind ing remained elevated for the duration of the experiment. Additional g rowth factor treatment (EGF, FGF, TGF alpha, and HGF) of the hepatocyt es did not appreciably alter the pattern of C/EBP binding. However, TG F beta treatment, known to decrease hepatocyte proliferation, increase d C/EBP beta binding activity earlier and more actively than in contro l cells. This study confirms a negative correlation between DNA bindin g by the C/EBP transactivator proteins and the proliferation of primar y mouse hepatocytes in vitro.