Characterisation of the expression of adenosine and estrogen receptors in myofibroblast differentiation in Peyronie's disease

Introduction: Peyronie’s disease (PD) is a fibroproliferative disease of the penis in which myofibroblast differentiation has a central role. Adenosine and estrogen signalling mediate their effect via interaction with their respective receptors which have been associated with the pathophysiology of a variety of fibroproliferative diseases, but this has yet to be studied in PD. Hypothesis: Adenosine and/or estrogen receptor expression is involved in myofibroblast differentiation in PD and may, therefore, be novel potential targets for anti-fibrotic therapies in this disease. Aim: The aim of this project was to characterise the myofibroblast differentiation process in human tunica albuginea-derived fibroblasts and to investigate the role of adenosine and estrogen receptors in this process. Methods: Tissue samples from non-PD TA tissue and PD plaque tissue were obtained and fibroblast cell lines were established from each sample. Cells were exposed to transforming growth factor (TGF)-β1. The mRNA levels and the protein levels of several targets of interest, alpha-smooth muscle actin (α-SMA), four adenosine receptors and two estrogen receptors (ERs), were evaluated using real-time RT-PCR (RT-qPCR), immunocytochemistry (ICC), immunohistochemistry (IHC), Western blot and In-Cell Western (ICW). The effect of compound modulators of two of the receptors on TGF-β1-induced myofibroblast transformation was assessed. Results: Both RT-qPCR and ICW methods were successfully developed and/or validated. The α-SMA mRNA and protein levels increased in cells isolated from non-PD TA tissue and PD plaque tissue when exposed to TGF-β1. The two cell groups expressed ADORA1 and ADORA2B and there was a differential effect to TGF-β1 in these two receptors. An ADORA2B agonist (BAY 60-6583) significantly inhibited TGF-β1-induced myofibroblast differentiation in the two cell types investigated. The two cell groups expressed ERβ but did not express ERα. Two selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, significantly inhibited TGF-β1-induced myofibroblast differentiation, suggesting that these SERMs interact with ERβ. Conclusions: Cells isolated from non-PD TA tissue and PD plaque tissue expressed ADORA1, ADORA2B and ERβ. TGF-β1 had differential effects on the receptors investigated depending on the cell type. An adenosine receptor agonist and two SERMs significantly inhibited TGF-β1-induced myofibroblast differentiation in a concentration-dependent manner suggesting that these receptors and consequently the adenosine and estrogen pathways may be involved in the differentiation of myofibroblasts and may be potential novel therapeutic targets in PD.