Extracellular generation of adenosine by the ectonucleotidases CD39 and CD73 promotes dermal fibrosis

Abstract

Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-β1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.

Figure 1

Deficiency of CD39 and/or CD73 limits adenosine levels and dermal fibrosis after bleomycin treatment. A: Skin adenosine levels were measured by high-performance liquid chromatography in supernates after 2 hours of skin culture. ^∗P < 0.05 (n = 6 to 12 skin samples per group). Skinfold thickness (B) and breaking tension (C) measurements were performed on freshly excised skin and 6-mm skin punch biopsy specimens. D: Dermal hydroxyproline content was assessed on 6-mm skin biopsy specimens. Data represent means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, comparisons versus WT + bleomycin (BLM); analysis of variance, followed by Dunnett's post-test analyses. E: Skin histological sections were stained with H&E (top row) and picrosirius red, viewed under polarized microscopy (middle and bottom rows). Original magnifications: ×20 (E, top row); ×10 (E, middle row); ×40 (E, bottom row).

Figure 2

Deficiency of CD39 and/or CD73 prevents synthesis of profibrotic mediators after bleomycin (BLM) treatment. mRNA levels of connective tissue growth factor (CTGF) (A) and TGF-β1 (B) were measured by quantitative RT-PCR in skin lysates. mRNA levels of CTGF and TGF-β are increased by bleomycin only in the WT mice. ^††P < 0.01, bleomycin- versus PBS-treated WT mice. Protein levels of α-SMA and TGF-β1 were assessed by using Western blot analysis, and representative blots are shown (C). Band intensities were quantified and normalized to β-actin as loading control (D and E). Data represent means ± SEM (A, B, D, and E). ^∗P < 0.05, ^∗∗P < 0.01 for comparisons made versus WT-bleomycin (BLM) by analysis of variance followed by Dunnett's post-test analyses.

Figure 3

Deficiency of CD39 and/or CD73 prevents myofibroblast accumulation after bleomycin treatment. Anti–α-SMA immunohistochemistry was performed on skin histological sections on CD39/CD73DKO mice (A) and on CD39- or CD73-deficient mice (B). Fast Red or DAB substrates were used to develop immunostaining, respectively. Representative photomicrographs are shown for each group. C: Number of α-SMA⁺ cells were counted in skin cross sections, and data represent means ± SEM. Original magnification, ×20. ^∗∗P < 0.01. All comparisons were made versus WT-bleomycin (BLM) by analysis of variance, followed by Dunnett's post-test analyses.