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.


CONCLUSIONS
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.
McGaha
Tracy L
TL
Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Le
Maithao
M
Kodera
Takao
T
Stoica
Cristina
C
Zhu
Jinfang
J
Paul
William E
WE
Bona
Constantin A
CA
D000818:Animals; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D005355:Fibrosis; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D015847:Interleukin-4 / Q000494:pharmacology*; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins; D019948:Receptors, Interleukin-4 / Q000235:genetics; D050800:STAT6 Transcription Factor; D012595:Scleroderma, Systemic / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D015534:Trans-Activators / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D024042:Collagen Type I; D011518:Proto-Oncogene Proteins; D019948:Receptors, Interleukin-4; D050800:STAT6 Transcription Factor; C494100:Stat6 protein, mouse; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D015847:Interleukin-4; D011505:Protein-Tyrosine Kinases; C507905:Jak1 protein, mouse; C507926:Jak2 protein, mouse; D053613:Janus Kinase 1; D053614:Janus Kinase 2; C080813:emt protein-tyrosine kinase