Analyses of TGF-beta1-inducible Ig germ-line gamma2b promoter activity: involvement of Smads and NF-kappaB.

ABSTRACT

TGF-beta1 directs class switch recombination to IgG2b as well as IgA. We have shown that Smad3/4, Runx3, and p300 mediate TGF-beta1-induced germ-line (GL) gamma2b transcription and that there is a potential Smad-binding element (SBE, CAGAC, -38/-34) and Runx-binding element (TGTGGGT, +41/+47) in the promoter region. Here, we have characterized more putative transcription factor-binding elements in the promoter. Site-directed mutagenesis revealed that two more putative SBE (GTCTG, -67/-63 and +38/+42) are relevant to TGF-beta1-induced GLgamma2b promoter activity, a finding that was confirmed by EMSA. However, neither overexpression of Ets (i.e. Elf-1, Fli-1, or Pu.1) nor a mutation deleting a putative Ets-binding element (CAGGAA, -4/+2) affected basal or TGF-beta1-induced promoter activity. On the other hand, NF-kappaB repressed promoter activity without direct binding to two putative NF-kappaB-binding elements (GGACTCCCC, -63/-55; GGGCCTTTCC,+237/+246). Instead, NF-kappaB overexpression increased the expression of Smad7 transcripts. Moreover, p300 overexpression failed to rescue the inhibitory effect of NF-kappaB on GLgamma2b promoter activity. These results indicate that there are multiple SBE relevant to GLgamma2b promoter activity and that NF-kappaB acts in cooperation with p300 to downregulate promoter activity through increasing the gene expression of inhibitory Smad7.