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Impaired dermal wound healing in discoidin domain receptor 2-deficient mice associated with defective extracellular matrix remodeling.

Olaso E, Lin HC, Wang LH, Friedman SL - Fibrogenesis Tissue Repair (2011)

Bottom Line: DDR2-/- wounds showed decreased tensile strength during healing, which correlated with a significant reduction in collagen content and defective collagen crosslinking.Contraction of collagen gels was reduced in DDR2-/- fibroblasts, accompanied by significantly reduced phosphorylated SrcY418.Inhibition of either LOX activity by β-aminoproprionitrile or MMP activity by N-[(2R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001) reduced collagen gel contraction by skin fibroblasts after DDR2 induction with soluble collagen type I.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Histology, University of the Basque Country School of Medicine, Leioa, Spain. elvira.olaso@ehu.es.

ABSTRACT

Background: The wounding response relies on tightly regulated crosstalk between recruited fibroblasts and the collagenous extracellular matrix (ECM). Discoidin domain receptor 2 (DDR2) is a tyrosine kinase receptor for fibrillar collagen expressed during pathologic scarring, for example wound healing, arthritis and cancer. We have previously shown that DDR2 phosphorylation drives key wounding responses in skin fibroblasts including proliferation, chemotactic migration and secretion of both metalloproteinases and fibrillar collagen. In this study we compared healing of cutaneous wounds in DDR2+/+ and DDR2-/- mice and analyzed specific fibroblast responses.

Results: Cutaneous wound healing was significantly delayed in DDR2-/- mice compared with DDR2+/+ animals. Reduced α-smooth muscle actin (αSMA) expression and matrix metalloproteinase 2 (MMP2) activity in the DDR2-/- wound extracts indicated defective recruitment of skin fibroblasts. DDR2-/- wounds showed decreased tensile strength during healing, which correlated with a significant reduction in collagen content and defective collagen crosslinking. Non-wounded skin in DDR2-/- mice expressed less mRNA of the crosslinking enzymes lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and matricellular 'secreted protein, acidic and rich in cysteine' (SPARC; also known as osteonectin). Skin fibroblasts isolated from DDR2-/- mice displayed altered mRNA expression of a cluster of collagens, proteoglycans, integrins and MMPs that have been previously correlated with DDR2 expression, and reduced LOX, LH1 and SPARC mRNA levels and proteins. Stable reconstitution of wild-type DDR2 by retroviral infection restored LOX, LH1 and SPARC mRNA and protein levels in DDR2-/- fibroblasts. Contraction of collagen gels was reduced in DDR2-/- fibroblasts, accompanied by significantly reduced phosphorylated SrcY418. Inhibition of either LOX activity by β-aminoproprionitrile or MMP activity by N-[(2R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001) reduced collagen gel contraction by skin fibroblasts after DDR2 induction with soluble collagen type I.

Conclusions: DDR2 contributes to skin fibroblast responses during tissue injury. Defective synthesis of collagen type I, crosslinking molecules and MMP2 predispose DDR2-/- mice to defective dermal wounding.

No MeSH data available.


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Reconstitution of discoidin domain receptor 2 (DDR2) restores expression of crosslinking enzymes. (a) Quantitative reverse-transcription real-time (RT)-PCR analysis of lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and 'secreted protein, acidic and rich in cysteine' (SPARC) mRNA levels in cultured skin fibroblasts. Results are expressed as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. *P < 0.05, significantly different compared with DDR2-/- skin fibroblasts retrovirally infected with full-length DDR2 (-/-/WTR2). (b) Representative western blot analysis of LOX, LH1 and SPARC protein levels in cultured skin fibroblasts. Tubulin expression was used as control for protein loading. Bands were quantified by scanning densitometry analysis and relativized to protein expression in DDR2+/+ cells.
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Figure 4: Reconstitution of discoidin domain receptor 2 (DDR2) restores expression of crosslinking enzymes. (a) Quantitative reverse-transcription real-time (RT)-PCR analysis of lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and 'secreted protein, acidic and rich in cysteine' (SPARC) mRNA levels in cultured skin fibroblasts. Results are expressed as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. *P < 0.05, significantly different compared with DDR2-/- skin fibroblasts retrovirally infected with full-length DDR2 (-/-/WTR2). (b) Representative western blot analysis of LOX, LH1 and SPARC protein levels in cultured skin fibroblasts. Tubulin expression was used as control for protein loading. Bands were quantified by scanning densitometry analysis and relativized to protein expression in DDR2+/+ cells.

Mentions: Next, we investigated if defective collagen crosslinking in DDR2-/- skin correlates with reduced expression of collagen crosslinking molecules in DDR2-/- fibroblasts. As shown in Figure 4, DDR2-/- fibroblasts express significantly less LOX, LH1 and SPARC mRNAs (Figure 4a), and reduced LOX, LH1 and SPARC proteins (Figure 4b). We also analyzed DDR2-/- skin fibroblasts in which wild type DDR2 expression was reconstituted by retroviral infection (WTR2). We utilized WTR2 fibroblasts that expressed roughly similar levels of DDR2 wild-type cells (data not shown)[18]. LOX, LH1 and SPARC mRNA expression levels were restored in WTR2 cells and remained significantly higher than in the DDR2-/- cells (Figure 4a, dashed columns). As shown in Figure 4b, LOX, LH1 and SPARC protein expression in WTR2 cells was similar to that of DDR2+/+ cells. DDR2-/- skin fibroblasts retrovirally infected with control vector (internal ribosomal entry site and green fluorescent protein (GFP) cDNA) behaved as non-infected cells (data not shown).


Impaired dermal wound healing in discoidin domain receptor 2-deficient mice associated with defective extracellular matrix remodeling.

Olaso E, Lin HC, Wang LH, Friedman SL - Fibrogenesis Tissue Repair (2011)

Reconstitution of discoidin domain receptor 2 (DDR2) restores expression of crosslinking enzymes. (a) Quantitative reverse-transcription real-time (RT)-PCR analysis of lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and 'secreted protein, acidic and rich in cysteine' (SPARC) mRNA levels in cultured skin fibroblasts. Results are expressed as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. *P < 0.05, significantly different compared with DDR2-/- skin fibroblasts retrovirally infected with full-length DDR2 (-/-/WTR2). (b) Representative western blot analysis of LOX, LH1 and SPARC protein levels in cultured skin fibroblasts. Tubulin expression was used as control for protein loading. Bands were quantified by scanning densitometry analysis and relativized to protein expression in DDR2+/+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044108&req=5

Figure 4: Reconstitution of discoidin domain receptor 2 (DDR2) restores expression of crosslinking enzymes. (a) Quantitative reverse-transcription real-time (RT)-PCR analysis of lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and 'secreted protein, acidic and rich in cysteine' (SPARC) mRNA levels in cultured skin fibroblasts. Results are expressed as relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. *P < 0.05, significantly different compared with DDR2-/- skin fibroblasts retrovirally infected with full-length DDR2 (-/-/WTR2). (b) Representative western blot analysis of LOX, LH1 and SPARC protein levels in cultured skin fibroblasts. Tubulin expression was used as control for protein loading. Bands were quantified by scanning densitometry analysis and relativized to protein expression in DDR2+/+ cells.
Mentions: Next, we investigated if defective collagen crosslinking in DDR2-/- skin correlates with reduced expression of collagen crosslinking molecules in DDR2-/- fibroblasts. As shown in Figure 4, DDR2-/- fibroblasts express significantly less LOX, LH1 and SPARC mRNAs (Figure 4a), and reduced LOX, LH1 and SPARC proteins (Figure 4b). We also analyzed DDR2-/- skin fibroblasts in which wild type DDR2 expression was reconstituted by retroviral infection (WTR2). We utilized WTR2 fibroblasts that expressed roughly similar levels of DDR2 wild-type cells (data not shown)[18]. LOX, LH1 and SPARC mRNA expression levels were restored in WTR2 cells and remained significantly higher than in the DDR2-/- cells (Figure 4a, dashed columns). As shown in Figure 4b, LOX, LH1 and SPARC protein expression in WTR2 cells was similar to that of DDR2+/+ cells. DDR2-/- skin fibroblasts retrovirally infected with control vector (internal ribosomal entry site and green fluorescent protein (GFP) cDNA) behaved as non-infected cells (data not shown).

Bottom Line: DDR2-/- wounds showed decreased tensile strength during healing, which correlated with a significant reduction in collagen content and defective collagen crosslinking.Contraction of collagen gels was reduced in DDR2-/- fibroblasts, accompanied by significantly reduced phosphorylated SrcY418.Inhibition of either LOX activity by β-aminoproprionitrile or MMP activity by N-[(2R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001) reduced collagen gel contraction by skin fibroblasts after DDR2 induction with soluble collagen type I.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Histology, University of the Basque Country School of Medicine, Leioa, Spain. elvira.olaso@ehu.es.

ABSTRACT

Background: The wounding response relies on tightly regulated crosstalk between recruited fibroblasts and the collagenous extracellular matrix (ECM). Discoidin domain receptor 2 (DDR2) is a tyrosine kinase receptor for fibrillar collagen expressed during pathologic scarring, for example wound healing, arthritis and cancer. We have previously shown that DDR2 phosphorylation drives key wounding responses in skin fibroblasts including proliferation, chemotactic migration and secretion of both metalloproteinases and fibrillar collagen. In this study we compared healing of cutaneous wounds in DDR2+/+ and DDR2-/- mice and analyzed specific fibroblast responses.

Results: Cutaneous wound healing was significantly delayed in DDR2-/- mice compared with DDR2+/+ animals. Reduced α-smooth muscle actin (αSMA) expression and matrix metalloproteinase 2 (MMP2) activity in the DDR2-/- wound extracts indicated defective recruitment of skin fibroblasts. DDR2-/- wounds showed decreased tensile strength during healing, which correlated with a significant reduction in collagen content and defective collagen crosslinking. Non-wounded skin in DDR2-/- mice expressed less mRNA of the crosslinking enzymes lysyl oxidase (LOX), lysyl hydroxylase1 (LH1) and matricellular 'secreted protein, acidic and rich in cysteine' (SPARC; also known as osteonectin). Skin fibroblasts isolated from DDR2-/- mice displayed altered mRNA expression of a cluster of collagens, proteoglycans, integrins and MMPs that have been previously correlated with DDR2 expression, and reduced LOX, LH1 and SPARC mRNA levels and proteins. Stable reconstitution of wild-type DDR2 by retroviral infection restored LOX, LH1 and SPARC mRNA and protein levels in DDR2-/- fibroblasts. Contraction of collagen gels was reduced in DDR2-/- fibroblasts, accompanied by significantly reduced phosphorylated SrcY418. Inhibition of either LOX activity by β-aminoproprionitrile or MMP activity by N-[(2R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001) reduced collagen gel contraction by skin fibroblasts after DDR2 induction with soluble collagen type I.

Conclusions: DDR2 contributes to skin fibroblast responses during tissue injury. Defective synthesis of collagen type I, crosslinking molecules and MMP2 predispose DDR2-/- mice to defective dermal wounding.

No MeSH data available.


Related in: MedlinePlus