Dermatopontin Expression is Decreased in Hypertrophic Scar and Systemic Sclerosis Skin Fibroblasts and is Regulated by Transforming Growth Factor-β1,

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Dermatopontin Expression is Decreased in Hypertrophic Scar and Systemic Sclerosis Skin Fibroblasts and is Regulated by Transforming Growth Factor-β1, Interleukin-4, and Matrix Collagen Kei Kuroda, Hiroshi Shinkai Journal of Investigative Dermatology Volume 112, Issue 5, Pages 706-710 (May 1999) DOI: 10.1046/j.1523-1747.1999.00563.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reduced expression of dermatopontin mRNA in HS fibroblast cultures. (a) Confluent fibroblasts from scar lesions (S) and normal skin (N) of five patients (HS1–HS5) were incubated with serum-free DMEM for 24 h. After extraction of total RNA, northern blot analysis were performed. (b) Levels of dermatopontin mRNA were quantitated by densitometric scanning and corrected for GAPDH mRNA. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reduced dermatopontin protein levels in HS fibroblast cultures. (a) Confluent fibroblasts from scar lesions (S) and normal skin (N) of five patients (HS1–HS5) were incubated with serum-free DMEM for 24 h. After the precipitation of cell culture media, the expression of dermatopontin was studied by immunoblotting analysis. (b) Dermatopontin protein levels were quantitated by densitometric scanning and corrected for total radiolabeled protein. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Significant reduction of dermatopontin mRNA levels in SSc fibroblasts. (a) Confluent fibroblasts from normal healthy controls and SSc patients were incubated with serum-free DMEM for 24 h. After extraction of total RNA, northern blot analysis was performed. (b) Levels of dermatopontin mRNA were quantitated by densitometric scanning and corrected for GAPDH mRNA. The results from control and SSc fibroblast groups were compared using the Mann–Whitney U test. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Reduction of dermatopontin protein levels in SSc fibroblasts. (a) Confluent fibroblasts of normal healthy controls and SSc patients were incubated with serum-free DMEM for 24 h. After precipitation of cell culture media, the expression of dermatopontin were studied by immunoblotting analysis. (b) Dermatopontin protein levels were quantitated by densitometric scanning and corrected for total radiolabeled protein. The results from control and SSc fibroblast groups were compared using the Mann–Whitney U test. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Concentration-dependent and time-dependent regulation of dermatopontin mRNA levels by TGF-β1 and IL-4 in normal fibroblasts. Confluent normal fibroblasts were rinsed with PBS followed by synchronization with serum-free DMEM for 6 h, then incubated in serum-free DMEM for 24 h with various cytokine concentrations or for various intervals with 2.5 ng per ml TGF-β1 or 5 units per ml IL-4. After extraction of total RNA, the expression of dermatopontin mRNAs was studied by northern blot analysis. (a) TGF-β1; (b) IL-4. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of TGF-β1 and IL-4 on dermatopontin protein expression in normal fibroblasts. (a) Confluent normal fibroblasts were rinsed with PBS followed by synchronization with serum-free DMEM for 6 h, then incubated in serum-free DMEM for 24 h with or without 5 ng per ml TGF-β1 or 5 units per ml IL-4. After precipitation of cell culture media, the expression of dermatopontin were studied by immunoblotting analysis. (b) Dermatopontin protein levels were quantitated by densitometric scanning and corrected for total radiolabeled protein. Data are mean ± SD of triplicate cultures. TGF-β1; p < 0.001; IL-4, p < 0.001. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Matrix collagen downregulates dermatopontin gene expression. (a) Normal fibroblasts from subconfluent cultures were seeded on to plastic culture dishes coated with various concentrations of type I collagen, and incubated in serum-free DMEM for 24 h. After extraction of total RNA, northern blot analysis were performed. (b) Normal fibroblasts from subconfluent cultures were grown on untreated plastic culture dishes as monolayer cultures (ML) or in three-dimensional collagen gel (CG) for 24 h with 1% FBS/DMEM. Northern blot analysis were performed and levels of dermatopontin mRNA were quantitated by densitometric scanning and corrected for respective 28S rRNA levels. Journal of Investigative Dermatology 1999 112, 706-710DOI: (10.1046/j.1523-1747.1999.00563.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions