Transforming growth factor (TGF)‐β1 is well established as a critical IgA isotype switching factor and Smad molecules have been reported to act as transducers and transcriptional factors in the expression of TGF‐β1‐targeted genes. We examined the involvement of Smad proteins in TGF‐β1‐induced IgA expression. First, we found that TGF‐β1 significantly increases endogenous germ‐line (GL) α transcripts by LPS‐stimulated CH12.LX.4933 (μ+) B lymphoma cells. To investigate its signaling mechanisms, the lymphoma cell line was transfected with pFL3 that contains the TGF‐β‐responsive element of the GLα promoter, and stimulated with TGF‐β1. Similar to endogenous GLα transcripts, TGF‐β1 induces GLα promoter activity and overexpression of Smad3 markedly enhances the promoter activity. This activity is further augmented by cotransfected Smad4. On the other hand, Smad7 substantially abrogates the synergistic effect of Smad3/4 onGLα promoter activity. In addition, overexpression of Smad3/4 enhances TGF‐β1‐induced endogenous GLα transcripts in normal spleen B cells. Finally, in the presence of TGF‐β1, overexpression of Smad3/4 selectively increases both surface IgA expression and IgA production. The results from the present study indicate that Smad3, Smad4, and Smad7, at least in part, serve as mediators linking TGF‐β1 to transcriptional regulation of IgA switching related gene and regulation of IgA class switching.

VanR is a negative transcriptional regulator of bacteria that belongs to the PadR family and modulates the expression of vanillate transport and degradation proteins in response to vanillate. Although VanR plays a key role in the utilization of vanillate as a carbon source, it is barely understood how VanR recognizes its effector. Thus, our knowledge concerning the gene regulatory mechanism of VanR is limited. Here, we reveal the vanillate-binding mode of VanR through structural, biophysical, and mutational studies. Similar to other PadR family members, VanR forms a functional dimer, and each VanR subunit consists of an N-terminal DNA-binding domain (NTD) and a C-terminal dimerization domain (CTD). One VanR dimer simultaneously binds two vanillate molecules using two interdomain cavities, as observed in PadR. In contrast to these common features, VanR contains an additional α-helix, αi, that has not been found in other PadR family members. The αi helix functions as an interdomain crosslinker that mediates interactions between the NTD and the CTD. In addition, the VanR-specific αi helix plays a key role in the formation of a unique effector-binding site. As a result, the effector-binding mode of VanR is distinguishable from that of PadR in the location and accessibility of the effector-binding site as well as the orientation of its bound effector. Furthermore, we propose the DNA-binding mode and vanillate-mediated transcriptional regulation mechanism of VanR based on comparative structural and mutational analyses. DATABASES: The atomic coordinates and the structure factors for VanR (PDB ID 5Z7B) have been deposited in the Protein Data Bank, www.pdb.org.

We recently reported that lactoferrin (LF) induces Foxp3 + Treg differentiation through binding to TGFβ receptor III (TβRIII), and this activity was further enhanced by TGFβ1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3 + Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, and lactoferrampin [aa 265-284]), along with TGFβ1 on Foxp3 + Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFβ1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFβ1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFβ1 receptor III (sTβRIII). Consistent with this, the activity of LFch on TGFβ1-induced Foxp3 expression was also abrogated by treatment with sTβRIII. Finally, the TGFβ1/LFchinduced T cell population substantially suppressed the proliferation of responder CD4 + T cells. These results indicate that LFch robustly enhances TGFβ1-induced Foxp3 + Treg differentiation by diminishing TCR/CD28 signal intensity.