Molecular Mechanisms of Interleukin-4-induced Up-regulation of Type I Collagen Gene Expression in Murine Fibroblasts

Objective: There is evidence that interleukin-4 (IL-4) plays a major role in the induction of extracellular matrix protein synthesis in fibrotic disease. We therefore examined the effect of IL-4 on collagen synthesis in primary fibroblasts isolated from normal and TSK/+ mice, which spontaneously develop a scleroderma-like syndrome characterized by diffuse cutaneous hyperplasia.

Methods: Expression of the IL-4 receptor was determined by flow cytometry and Western blotting. The IL-4 signal transduction cascade was analyzed by Western blotting. We assessed the role of signal transducer and activator of transcription 6 (STAT-6) in IL-4 induction of alpha2(I) collagen promoter activity and message levels via luciferase reporter assay and real-time polymerase chain reaction. The activation status of the transcription factors activator protein 1 (AP-1) and Sp-1 upon stimulation with IL-4 in normal and TSK/+ fibroblasts was examined by electrophoretic mobility shift assay.

Results: Flow cytometry and Western blotting showed that IL-4 receptor alpha expression was elevated in TSK/+ fibroblasts compared with normal fibroblasts. After IL-4 stimulation, janus-activated kinase 1 (JAK-1) and JAK-2 were phosphorylated to a greater degree in TSK/+ fibroblasts than in C57BL/6 fibroblasts. TSK/+ fibroblasts appeared to be hyperresponsive to IL-4, displaying increased synthesis of alpha1(I) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine alpha2(I) collagen promoter. Overexpression of STAT-6 enhanced this effect, whereas expression of a dominant-negative STAT-6 abrogated the ability of IL-4 to induce alpha1(I) collagen mRNA in TSK/+ fibroblasts. Moreover, IL-4 induced increased DNA binding activity of transcription factors that are important for collagen synthesis.

Conclusion: Our observations indicate that IL-4 has a profound effect on several factors that have been identified as playing major roles in the regulation of collagen synthesis and suggest that IL-4 increases the expression of type I collagen through a mechanism involving the activation of transcription factors that bind to and activate collagen promoter.