Abstract
Immediate-early genes, whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation, are postulated to play important regulatory roles in the growth response. The complement of immediate-early genes expressed must depend on the milieu of preexisting transcription factors in the quiescent cell as well as the type of mitogenic stimulation and, thus, may differ between cell types. We have begun characterizing the immediate-early response in regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells in comparison with previously published results from mitogen-stimulated Balb/c 3T3 fibroblasts. The proliferating H-35 and regenerating liver cells maintain their similarity to quiescent liver as demonstrated by their continued production of the liver-specific albumin, CCAAT/enhancer binding protein, and phosphoenolpyruvate carboxykinase messenger RNAs (mRNA). Surprisingly, the phosphoenolpyruvate carboxykinase gene, which undergoes down-regulation in insulin-treated H-35 cells, was cloned by differential screening of a subtraction-enriched regenerating liver cDNA library and is an immediate-early gene in regenerating liver. H-35 cells treated with either insulin or phorbol 12-myristate 13-acetate express elevated levels of the jun genes, and phorbol 12-myristate 13-acetate pretreatment fails to abolish the insulin response, indicating that it does not depend on protein kinase C. jun family gene expression in regenerating liver differs from that in mitogen-treated fibroblasts in that the time course of expression of c-jun and junB is prolonged, and junD mRNA levels distinctly increase. Additionally, although c-fos and egr-1 mRNAs are expressed at elevated levels in stimulated liver cells, fos-B, fra-1, and egr-2 are not, which suggests that factors in addition to the serum response factor participate in the regulation of immediate-early gene induction. Interestingly, gene 33, which was cloned from a regenerating liver cDNA library by differential screening and lacks a recognizable serum response element, functions as an immediate-early gene in regenerating liver and in mitogen-treated H-35 and Balb/c 3T3 cells. These results suggest that gene 33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. Overall, the results presented here suggest that the immediate-early response varies considerably between regenerating liver and mitogen-stimulated fibroblasts and could involve multiple, preexisting, tissue-specific, transcription-activating proteins.
Highlights
13-acetate pretreatment fails to abolish the insulin response, indicating that it does not depend on protein kinase C. jun family gene expression in regenerating liver differs from that in mitogen-treated fibroblasts in that the time course of expression of c-jun and junB is prolonged, and junD messenger RNAs (mRNA) levels distinctly increase
Gene 33, which was cloned from a regenerating liver cDNA library by differential screening and lacks a recognizable serum response element, func
The results presented here suggest that the immediateearly response varies considerably between regenerating liver and mitogen-stimulated fibroblasts and could involve multiple, preexisting, tissue-specific, transcription-activating proteins
Summary
CellCulture-H-35 cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 5% fetal bovine serum (GIBCO), 5% calf serum (GIBCO), 2 mM L-glutamine (Flow), and 100 units of penicillin and 50 units of streptomycin/ml (Flow) as reported previously [37]. Medium was changed to Dulbecco's modified Eagle's medium, plus 0.5% fetal bovine serum, glutamine, and penicillin/ streptomycin for 48 h to induce quiescence, and thereafter, cells were treated with various agents as described above and in the figure legends. CDNA Library Construction and Screening-The insulin-treated H-35 cell eDNA library was prepared for us by Invitrogen from RNA isolated as described above from serum-starved H-35 cells stimulated for 3 h with insulin (10-8 M) and cycloheximide (10 /lg/ml) and polyt A)" selected by passage over an oligotd'I') column (Collaborative Research). CDNA inserts from recombinant phage of interest were subcloned (H-35 library) into pGEM-4Z (Promega Biotec) or excised in vivo (regenerating liver libraries) to the pBluescript and nick-translated for initial analysis (hybridization to a panel of all recombinant phage clones of interest and to Northern blots containing mitogen-stimulated and quiescent RNA from H-35 cells, regenerating liver, and Balb/c 3T3 cells). Unique differentially expressed clones were preliminary sequenced (-300 base pairs at each end using the Sanger method and T -3, T -7 and SP6 primers (Promega Biotsc), and the sequence was compared with other reported sequences using the Intelligenetics software and data bases
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