Figure 5.

FOSL2 transcriptionally activates Wnt5a. (A) The predicted target genes regulated by FOSL2 from the Cistrome database and a set of genes related to angiogenesis and the reported proteins in the extracellular matrix downloaded from AmiGO Gene Ontology were used in our analysis to acquire the FOSL2-regulated angiogenesis genes. Sixty-eight putative FOSL2 targets are shown. (B) The significantly altered cytokine genes (fold changes>1.5 times, CAFs vs NFs) were identified by microarray analysis for primary CAFs and NFs. (C-D) Expression of Wnt5a was determined by qRT-PCR (C) or western blotting (D) in the indicated stromal fibroblasts (**p<0.01). (E) Schematic diagram of canonical FOSL2-binding motif (JASPAR Database) and potential FOSL2 responsive elements (E1, E2, E3) in the Wnt5a promoter. Full-length and truncated Wnt5a promoter or E2 mutated promoter are shown. (F) Transcriptional activities of FOSL2 on Wnt5a were determined by luciferase reporter assay as CAFs were transfected with full-length or truncated Wnt5a promoter (upper panel) and E2 wild-type or mutated reporter plasmids (down panel). (G) ChIP assays were performed to analyze FOSL2 binding to the Wnt5a promoter using an anti-FOSL2 antibody. The E2 site was significantly enriched in comparison with the E1 or E3 site. (H) western blotting to determine Wnt5a collected from CM derived from the indicated cells.