TGF-beta induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-beta1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta1, whereas cotransfection of HIF-1alpha/beta and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-beta1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1alpha/beta and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-beta1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF-beta1 and IFN-gamma induce BAFF expression to see if TGF-beta1 and IFN-gamma regulate B cell differentiation via macrophages. We found that TGF-beta1 stimulated mouse macrophages to express BAFF and that a typical TGF-beta signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad-binding elements. Importantly, TGF-beta1 enhanced the expression of membrane-bound and soluble forms of BAFF. IFN-gamma further augmented TGF-beta1-induced BAFF expression. IFN-gamma caused phosphorylation of CREB, and overexpression of CREB increased IFN-gamma-induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1alpha (a well-known transducing molecule of IFN-gamma) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN-gamma. Taken together, these results demonstrate that TGF-beta1 and IFN-gamma up-regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF-β1 or IFN-γ express membrane-bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN-γ or TGF-β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF-receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP-1 have similar activities. Our findings suggest that macrophage-derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP-1 pathways.

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