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. 2013;15(3):R58.
doi: 10.1186/ar4229.

Adenosine A(2A) receptors promote collagen production by a Fli1- and CTGF-mediated mechanism

Edwin S L Chan, Hailing Liu, Patricia Fernandez, Alex Luna, Miguel Perez-Aso, Andreea M Bujor, Maria Trojanowska, Bruce N Cronstein

Adenosine A(2A) receptors promote collagen production by a Fli1- and CTGF-mediated mechanism

Edwin S L Chan et al. Arthritis Res Ther. 2013.

Abstract

Introduction: Adenosine, acting through the A(2A) receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A(2A) receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A(2A) receptor produces its fibrogenic effects.

Methods: The effects of A(2A) receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A(2A) receptor stimulation on CTGF and TGF-β1 expression are also examined.

Results: A(2A) receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A(2A) receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A(2A) receptor-mediated enhancement of collagen type I production. A(2A)R activation, however, resulted in a decrease in TGF-β1 protein release.

Conclusions: Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A(2A) receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.

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Figures

Figure 1
Figure 1
Adenosine A2A receptor occupancy suppresses Fli1 mRNA expression in human dermal fibroblasts. NHDF cells were incubated with CGS-26180 at A) 10 µM at the indicated time-points or B) 1 µM or 10 µM for four or eight hours. C) Incubation with the A2A receptor-selective antagonist ZM241385 (1 µM) prior to CGS-26180 (10 µM) for four hours prevents the decrease on Fli1 expression. Data represent means ± S.E.M. of four to eight independent experiments. *P <0.05 vs. non-stimulated control (One-way ANOVA, post-hoc analysis by Newman-Keuls test); ###P <0.001, CGS-26180 + ZM241385 vs. CGS-26180 (unpaired t-test).
Figure 2
Figure 2
Adenosine A2A receptor occupancy inhibits nuclear Fli1 protein expression in human dermal fibroblasts. NHDF cells were incubated with CGS-26180 at 1 to 10 µM over 24 hours and cellular fractionation was performed as described under "Methods". A) Representative Western-blot images showing Fli1 and the nuclear marker p84. B) Bands were quantified and data represent means ± S.E.M. from three independent experiments. Statistics were performed by one-way ANOVA with post-hoc analysis by Newman-Keuls test, *P <0.05 vs. non-stimulated control.
Figure 3
Figure 3
Adenosine A2A receptor occupancy increases CTGF mRNA and protein expression and secretion. NHDF cells were incubated with CGS-26180 at 1 µM A) for 4 hours for CTGF mRNA analysis or B) for 24 hours prior to cellular fractionation and protein concentration from supernates as described under "Methods". Bands were quantified and data represent means ± S.E.M. from three to eight independent experiments. Statistics were performed by one-way ANOVA with post-hoc analysis by Newman-Keuls test, *P <0.05 vs. non-stimulated control.
Figure 4
Figure 4
CTGF is involved in the A2A receptor-mediated collagen production. NHDF cells were incubated with CGS-26180 at 1 µM over A) 4 h for COL1A1 mRNA analysis or B) during 24 h for Western-blotting after 16 h-starvation. Where indicated, the neutralizing antibody to CTGF (Ab; 1:250) or the A2AR antagonist ZM241385 (10 µM) was added prior to incubation with CGS-21680. Bands were quantified and data represent means ± S.E.M. from three to six independent experiments. Statistics were performed by one-way ANOVA with post-hoc analysis by Newman-Keuls test, ***P <0.001 CGS-26180 vs. non-stimulated control; ###P <0.001 CGS-26180 vs. CGS-26180 + CTGF Ab; and NS = non-significant, CGS-26180 vs. CGS-26180 + Normal Serum.
Figure 5
Figure 5
TGF-β1 secretion is decreased by A2AR activation at earlier time points and restored after 24 hours. NHDF cells were serum-starved and incubated with CGS-26180 1 µM at the indicated time periods and released TGF-β1 levels were analyzed by ELISA. Data represent means ± S.E.M. from four independent experiments. Statistics were performed by unpaired-t test, ***P <0.001, **P <0.01, vs. non-stimulated control.
Figure 6
Figure 6
Schema depicting the contribution of the Fli1 and CTGF pathways on the A2A receptor-mediated increase in collagen production.
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