FIG. 1.

CD40 stimulation induces the upregulation of IRF-1 expression. (A) CD40 ligation upregulates IRF-1 protein expression in EJ bladder carcinoma cells. EJ cells were stimulated with recombinant soluble CD40L at 0.5 μg/ml for the indicated times, and lysates were analyzed for IRF-1, IRF-3, or β-tubulin levels by immunoblotting. (B) Generality of the effect of CD40 stimulation on IRF-1 upregulation. AGE60 and VM-CUB-1 cells were treated as described in the legend to panel A, and IRF-1 levels were determined by immunoblotting. (C) CD40 stimulation induces the rapid induction of IRF-1 RNA expression. EJ cells were stimulated with recombinant soluble CD40L at 0.5 μg/ml for the indicated times before RNA was isolated. cDNA was synthesized and used as a template for semiquantitative PCR with primers specific for IRF-1 or the GAPDH housekeeping gene, which served as a loading and amplification control. The results shown are representative of results from three independent experiments. 15′, 15 min. (D) Graphical representation of CD40 and its TRAF binding domains. TRAF6 interacts with a membrane-proximal region, whereas TRAF2 and TRAF3 associate with a membrane-distal domain at the cytoplasmic C terminus of CD40. A double Q²³⁴E²³⁵→AA mutation, yielding CD40mT6 (mT6), selectively abolishes the interaction of TRAF6 with CD40, whereas a T²⁵⁴→A mutation, yielding CD40mT2/T3 (mT2/T3), inhibits TRAF2 and TRAF3 binding but leaves TRAF6-CD40 interactions intact. The CD40mT2/T3/T6 construct (mT2/T3/T6) contains a triple Q²³⁴E²³⁵ T²⁵⁴→AAA mutation which perturbs the binding of all TRAFs. These constructs are described in detail in reference 8. WT, wild type. (E) CD40L-induced IRF-1 synthesis depends on TRAF binding to the cytoplasmic C terminus of CD40. HeLa cervical carcinoma cells, which do not express CD40, were transfected with the CD40 constructs depicted in panel D. Selected stable clones were stimulated with CD40L for 3 h, and lysates were analyzed for CD40 or IRF-1 levels by immunoblotting. The results shown are representative of results from three independent experiments. +, present; −, absent.