Background
Transforming growth factor-beta 1
(TGF-β1) is widely implicated in fibrotic diseases including Dupuytren’s disease (DD; also known as palmar fascia fibrosis). DD is the most common heritable fibroproliferative disorder of the palmar fascia of the hand. It affects 5–25% of people of European descent, and there is evidence that the prevalence of DD is increasing [
1,
2]. There is a strong genetic predisposition to DD, and there is a strong indication that DD has a substantial hereditary component [
3]. Similarly, there is evidence that multiple non-genetic factors, such as smoking, alcohol intake, diabetes, and hyperlipidemia, also play a role in disease development [
4]. In DD pathogenesis, subcutaneous fat is progressively replaced by a relatively avascular fibrous tissue, which develops as nodules in the palm and extends distally into the digits as thickened cords [
5], which draw the fingers into a flexed posture. The options for treating DD include the percutaneous release of the offending cords [
6], injection of collagenase followed 1–2 days later with manipulation to rupture the cords [
7] and surgical resection of the cords [
8]. All of these treatment strategies have a high risk of recurrence [
9].
While the molecular and cellular triggers remain difficult to elucidate during DD development, the progressive increase in extracellular matrix protein and subsequent contraction clearly suggests a role for myofibroblasts. Myofibroblasts have the ability to synthesize collagen and other extracellular matrix components in the palmar fascia leading to digital contractures [
10,
11]. A number of studies indicate that growth factors control the growth and proliferation of myofibroblasts. A role for several cytokines including FGF, IL-1, TGF-β1, and PDGF, and all have been shown to influence DD pathogenesis [
12,
13]. TGF-β1, a well-known pro-fibrotic growth factor, is up-regulated in DD- tissue [
14,
15] and is a major factor in the transformation of fibroblasts to myofibroblasts in DD [
16‐
18]. Previous studies have described the activation of fibroblasts by TGF- β and other cytokines as a major mechanism driving the fibrotic processes in DD and other fibroses [
19]. TGF-β
1 stimulation leads to increased contractile force in DD-cells [
20‐
22] and also leads to up-regulation of key ECM components, such as fibronectin and type I collagen [
16].
TGF-β1 signaling can occur through both canonical and non-canonical pathways. In canonical TGF- β1 signaling, binding of TGF-β1 to its receptors activates SMAD 2/3-dependent signaling; activated SMAD 2/3 complexes with SMAD 4 and translocates into the nucleus and regulates the expression of TGF-β-responsive genes [
23]. The TGF-β-induced non-canonical, SMAD-independent pathways include various branches of MAP kinase (MAPK) pathways, Rho-like GTPase signaling pathways, and phosphatidylinositol-3-kinase (PI3K)/AKT pathways [
24]. TGF-β1’s pleiotropic effects on DD cells, especially fibroblasts, make it an ideal candidate to target in the fight against Dupuytren’s fibrosis.
Pirfenidone (PFD; 5-methyl-1-phenyl-2 (1H)-pyridone) is currently used to treat patients with Idiopathic Pulmonary Fibrosis, a condition with similarities to Dupuytren’s Disease [
25]. We have shown that PFD inhibits TGF-β1-induced transformation of fibroblasts to myofibroblasts and inhibits ECM production, mainly type I/type III-collagens and fibronectin in DD-derived fibroblasts. PFD also inhibits TGF-β1-induced cell migration, proliferation and contractile ability in DD-derived fibroblasts. We further showed that TGF-β1-induced phosphorylation of SMAD 2/SMAD 3, a key factor in the TGF-β1 signaling pathway, was attenuated by the addition of PFD [
21]. In the present study, we investigated the action of PFD on TGF-β
1-induced non-SMAD signaling pathways in DD-derived fibroblasts as both SMAD-dependent and- independent signaling pathways elicited by TGF-β1 have a significant role in mediating fibrosis. Our focus was to examine the changes that PFD could elicit in the basal- and TGF-β1-induced phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2); Akt, a serine/threonine-specific protein kinase; p38 mitogen-activated protein kinases and myosin light chain (MLC), a subunit of myosin.
Discussion
DD is a complex disease that is characterized by the involvement of various growth factors, signaling molecules, and pathways that drive the process of fibrosis in the palm of the hands [
26]. TGF-β1 has been identified as a central growth factor in activating fibroblasts to transform to myofibroblasts, resulting in excessive accumulation of ECM proteins, especially collagen. Previous biochemical and immunochemical studies have reported a decrease in type III/I collagen ratio with DD progression [
27,
28]. In contrast, a study by Lam et al. (2010) quantified the different collagen types by computer analysis using stained DD tissue and documented a decrease in the amount of type III collagen as a percentage of the total collagen with the progression of the disease [
29]. There is strong evidence that the blockade of TGF-β1 alone is sufficient to block experimental fibrosis in liver, kidney, lung, heart, and skin [
30]. Targeting TGF- β1 may alleviate some of the adverse effects leading to fibrosis, and it may prevent the progression of DD. The small molecule PFD is an approved oral anti-fibrotic agent for patients affected with idiopathic pulmonary fibrosis [
25]. PFD mainly targets TGF-β1- induced fibrosis [
31]. Our previous in-vitro studies demonstrated that PFD could prevent the TGF- β
1-induced fibroblast to myofibroblast transformation and that it inhibits ECM protein production, especially type I and type III collagen and fibronectin in DD-derived fibroblasts [
21].
TGF- β1, a known pro-fibrotic growth factor elicits its effects by inducing both canonical and non-canonical signaling pathways. We have previously shown that PFD can inhibit TGF-β1- induced phosphorylation of SMAD 2/SMAD 3 in DD-derived fibroblasts, a key factor in the TGF-β1-induced canonical signaling pathway. We also reported in our previous studies that PFD inhibits TGF-β
1-induced cell migration, cell contraction and cell proliferation in both carpal tunnel (CT) - and DD-derived fibroblasts, but that the effect was more pronounced in DD-derived fibroblasts [
21]. Several studies have reported the effects of PFD on fibroblasts derived from other tissues namely, ocular, intestine, dermis and lung. In primary lung fibroblasts derived from idiopathic pulmonary fibrosis (IPF) patients authors show that combination of pirfenidone and rapamycin widen the inhibition range of ECM fibrogenic markers and prevents fibroblast migration [
32] showing possibility for using combination of small molecules for anti-fibrotic treatment. Similarly, Stahnke et al. (2017) [
33] show in ocular fibroblasts PFD inhibition of ECM protein deposition again suggesting PFD as a promising candidate for treating fibrosis following glucoma surgery. Sun et al. (2018) [
34] present findings using intestinal fibroblasts that PFD could inhibit TGF-β1-induced SMAD and PI3K/AKT signaling pathways and suggested the application of PFD as a potential therapeutic agent for intestinal fibrosis. Another interesting finding originates from Hall et al. 2018 [
35] showing evidence for PFD inhibiting TGF-β1-induced type I- and type III- collagens and targeting the p38 MAPK signaling pathway in human dermal fibroblasts which shows the capability of PFD to target dermal fibrosis.
In continuation of these findings, our interest was to determine if treatment of CT- and DD- fibroblasts with PFD could attenuate non-canonical signaling pathways induced by TGF-β1, particularly those involving pERK, pAKT, p38, and pMLC as each of these molecules have been found to play a role in cell migration, proliferation, differentiation, and contraction. Interestingly, our results indicate that TGF-β1-stimulated phosphorylation of ERK 1/2, AKT, MLC and p38 were all significantly inhibited by PFD in both CT-and DD-derived fibroblasts. PFD treatment alone also significantly inhibited the basal phosphorylation levels of AKT in both CT- and DD-derived fibroblasts. An inhibition in the basal phosphorylation levels in the other molecules, ERK1/2, MLC, and p38 was noted although a statistical significance was not achieved.
ERK1/2 belongs to the mitogen-activated protein kinase superfamily and mediates cell proliferation, migration, and apoptosis [
36]. p38 MAPKs normally respond to environmental stress and inflammatory cytokines and mediate cell differentiation and apoptosis [
37]. Krause et al. (2011) have previously reported the increased basal expression of phosphorylated ERK1/2 MAP kinase levels in Dupuytren’s fibroblasts [
14]. Further authors also reported that co-treatment with MAPKK1 inhibitor PD98059 and TGF-β type I receptor kinase inhibitor SB-431542 abrogated proliferation and contractile efficiency of Dupuytren’s fibroblasts. We observe here that PFD is able to accomplish a similar pharmacologic inhibition of ERK 1/2 inhibitors.
Akt is a downstream protein kinase of PI3K, and it is activated by phosphorylation of IP3K. The activated Akt kinase phosphorylates a number of key substrates involved in the stimulation of intermediary metabolism and promotion of cell survival, proliferation, and growth [
38]. Similar to Akt kinase activated p38 has numerous possible downstream targets driving cell proliferation and ECM deposition [
39,
40]. Earlier studies by Ratkaj et al. (2012) report higher levels of phosphorylated p38 and Akt kinases along with typical myofibroblast markers, α-SMA, and palladin in DD fibroblasts. Decreasing p-p38 and Akt levels using the inhibitor SB203580 reduced DD fibroblasts’ proliferative and differentiation potential to myofibroblasts [
41]. In lieu of the findings of Ratkaj et al. (2012) [
41] we show that basal phosphorylation levels of Akt was inhibited by PFD (trending towards statistical significance) in DD-derived fibroblasts. However, a significant decrease in basal phosphorylation levels of p38 by addition of PFD was noted in DD-derived fibroblasts. Our results further show that TGF- β1-induced phosphorylation of Akt and p38 was significantly inhibited by PFD in DD-cells again reiterating the potential of PFD in targeting molecules that mediate cell survival, proliferation, and growth.
Phosphorylation of myosin light chain (MLC) is associated with actin-myosin interaction to form stress fibers and contractile rings, facilitating cell contraction and motility [
42]. We observed increased basal phosphorylation levels of MLC in DD fibroblasts significantly more than that was seen in the fibroblasts derived from CT (Fig.
4). In our earlier studies, we observed increased cell contractile property at the basal level in DD- derived fibroblasts versus CT-derived fibroblasts [
21], which may be partly due to the elevated phosphorylation of MLC. Addition of PFD did not inhibit the basal phosphorylation levels of MLC in both CT-and DD-derived fibroblasts but inhibited the basal contractile property of both CT- and DD-derived fibroblasts [
21] indicating that other factors may also play a role in the contractile machinery of cells.
Overall, results from our previous and present studies demonstrate that PFD may serve as a potential candidate to fight against Dupuytren’s fibrosis as it could attenuate both canonical and non-canonical profibrotic signaling molecules elicited by TGF-β1. Whereas other pharmacological inhibitors may only be of benefit in targeting a single specific signaling molecule in DD, using PFD may negate a requirement for multiple pharmacological inhibitors by targeting several signaling molecules simultaneously. Future studies will determine PFD’s actions on other growth factors, namely PDGF, VEGF, and FGF as each of these has been implicated in the progression and development of DD and collectively the pro-fibrotic effects elicited by all of these growth factors need to be targeted.