Fig. 5. Cholesterol-mediated Ctsk induction requires the transcription factor MITF.

A. CD-chol induces the expression of Mitf and Ctsk, as well as p38 MAP kinase activation. ConA-elicited macrophages were treated with 5 mM CD-cho (2.5:1, M:M) for indicated time. mRNA levels were measured by Taqman real-time PCR. Phospho- and total p38 protein were detected with specific antibodies. B. Inducation of Ctsk by CD-chol was abolished by Mitf dominant negative mutation. Splenic macrophages from wt or Mitf^mi/mi mice were treated with 5 mM CD-chol (2.5:1, M:M) for indicated time. Ctsk mRNA level in untreated condition was set as 1. C. Disruption of three upstream E boxes on human CTSK promoter blunted the induction by CD-chol loading. RAW cells were transfected with indicated promoter-luciferase constructs and TK-Renilla luciferase. Transfection media was changed to DMEM/10%FBS (control) or 5 mM CD-chol (2.5:1, M:M) in DMEM/10%FBS (CD-chol) 6 hours after transfection. Cells were collected for luciferase assay 28 hours later. A schematic representation of human Ctsk promoter was shown at the bottom. The sequences of wild type and mutated E boxes used were shown in the boxes. D. CD-chol loading enriched MITF and phospho-p38 on Ctsk promoter. Splenic macrophages from wt mice were loaded with 5 mM CD-chol (2.5:1, M:M) for 18 hours. ChIP assay was performed in duplicates to determine the MITF, PU.1 and phospho-p38 on Ctsk promoter. N=2.