Induction of Sp1 activity by prolactin and interleukin-2 in Nb2 T-cells: differential association of Sp1-DNA complexes with Stats.
The induction of the transcription factor Sp1 by prolactin (PRL) and interleukin-2 (IL-2) was investigated in the PRL- and IL-2 responsive rat Nb2 T-cell line. Western analysis showed a rapid increase in Sp1 synthesis in Nb2 cells in response to PRL or IL-2. Elevation of Sp1 protein levels occurred within 15 min following PRL or IL-2 stimulation, reached a maximum by 1 h and was inhibited by cycloheximide, indicating de novo protein synthesis. Interestingly, dilution of confluent, growth-arrested Nb2 cells to low density also caused a rapid elevation in Sp1 suggesting that growth arrest may down-regulate Sp1 synthesis. Electrophoretic mobility shift assays using an Sp1 consensus oligonucleotide as probe showed a rapid but transient formation of a single PRL-inducible complex at 30 min. In contrast, three IL-2-inducible complexes were formed at 30 min and persisted to at least 60 min. Mobility shift interference assays using specific Stat antibodies failed to detect Stat1alpha, Stat3 or Stat5 in the 30 min PRL-inducible complex. In contrast, the IL-2 induced complexes contained Stat3 alone at 30 min and both Stat3 and Stat5 at 60 min. The PRL- and IL-2-inducible complexes did not contain the tumor suppressor protein, p53. The time dependent association of the Stat proteins with the IL-2-inducible complexes, but not with the PRL-inducible complex, suggests that the two mitogens may selectively utilize specific promoter elements for transcriptional activation of PRL- and IL-2-responsive genes. Alternatively, the two mitogens may be activating different genes with Sp1-binding promoter elements for their mitogenic action in Nb2 cells.
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Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada. CTOO@IS.DAL.CA
D000818:Animals; D001483:Base Sequence; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007376:Interleukin-2 / Q000494:pharmacology*; D008934:Mitogens / Q000494:pharmacology; D015345:Oligonucleotide Probes / Q000235:genetics; D011388:Prolactin / Q000494:pharmacology*; D051381:Rats; D016329:Sp1 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D008934:Mitogens; D015345:Oligonucleotide Probes; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D011388:Prolactin; D004247:DNA