Molecular mechanisms of interleukin-4-induced up-regulation of type I collagen gene expression in murine fibroblasts

Tracy L. McGaha, Maithao Le, Takao Kodera, Cristina Stoica, Jinfang Zhu, William E. Paul, Constantin A. Bona

Research output: Contribution to journalArticle

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Abstract

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 α2(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 α 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 α1(1) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine α2(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 α1(1) 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.

Original languageEnglish (US)
Pages (from-to)2275-2284
Number of pages10
JournalArthritis and Rheumatism
Volume48
Issue number8
DOIs
StatePublished - Aug 1 2003

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Collagen Type I
Interleukin-4
Up-Regulation
Fibroblasts
Gene Expression
Collagen
STAT6 Transcription Factor
Interleukin-4 Receptors
Transcription Factors
Western Blotting
Luciferases
Flow Cytometry
Janus Kinase 1
Messenger RNA
Extracellular Matrix Proteins
Transcription Factor AP-1
Electrophoretic Mobility Shift Assay
Hyperplasia
Real-Time Polymerase Chain Reaction
Signal Transduction

ASJC Scopus subject areas

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

Cite this

Molecular mechanisms of interleukin-4-induced up-regulation of type I collagen gene expression in murine fibroblasts. / McGaha, Tracy L.; Le, Maithao; Kodera, Takao; Stoica, Cristina; Zhu, Jinfang; Paul, William E.; Bona, Constantin A.

In: Arthritis and Rheumatism, Vol. 48, No. 8, 01.08.2003, p. 2275-2284.

Research output: Contribution to journalArticle

McGaha, Tracy L. ; Le, Maithao ; Kodera, Takao ; Stoica, Cristina ; Zhu, Jinfang ; Paul, William E. ; Bona, Constantin A. / Molecular mechanisms of interleukin-4-induced up-regulation of type I collagen gene expression in murine fibroblasts. In: Arthritis and Rheumatism. 2003 ; Vol. 48, No. 8. pp. 2275-2284.
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AU - McGaha, Tracy L.

AU - Le, Maithao

AU - Kodera, Takao

AU - Stoica, Cristina

AU - Zhu, Jinfang

AU - Paul, William E.

AU - Bona, Constantin A.

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N2 - 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 α2(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 α 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 α1(1) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine α2(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 α1(1) 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.

AB - 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 α2(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 α 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 α1(1) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine α2(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 α1(1) 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.

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