TGFβ is one of the most widely studied pro-fibrotic factors in the context of fibrosis. The TGFβ superfamily includes TGFβ, bone morphogenetic proteins (BMPs), growth/differentiation factors (GDFs), activins, and inhibins [18]. TGFβ plays a crucial role in transitioning fibroblasts into activated myofibroblasts, which are responsible for the excessive production of ECM in fibrosis [19]. TGFβ signals via interaction with two receptor serine/threonine kinases, known as type I and type II receptors, which form a heterotetrameric complex upon ligand binding [20]. Upon complexing, the autophosphorylation of type I and II receptors mediates the docking and phosphorylation of Smad 2/3, which in turn interact with Smad 4 to create a transcriptional complex that translocates to the nucleus and activates or represses multiple target genes [21]. While TGFβ works mainly via activation of the Smad pathway, it can also activate other non-canonical pathways [18]. In fibroblasts, TGFβ signaling shifts the gene expression profile to a profibrotic phenotype, inducing the expression of profibrotic and ECM genes, while suppressing the antifibrotic and matrix-degrading genes, leading to tissue fibrosis [19,22]. TGFβ can also exert similar effects on other cell types, such as epithelial and endothelial cells, and can induce their transition into alpha smooth muscle actin-expressing myofibroblasts [19,23].

In SSc, TGFβ-regulated genes are differentially expressed in the fibrotic lungs of patients, which is correlated with the severity of the disease [24]. This is consistent with the notion that TGFβ plays a central role in SSc pathology [25]. In fact, we previously demonstrated that fibroblasts derived from the lungs of SSc patients express higher TGFβ1 and TGFβ2 levels than fibroblasts from healthy lungs [26]. This supports previous findings obtained by Christmann et al. indicating that lung tissues from SSc patients show amplified expression of genes regulated by TGFβ [11]. Recent evidence has shown that macrophages polarized to the alternatively activated phenotype (M2) are also a major source of TGFβ [27]. A follow-up study showed that the expression of TGFβ by M2 macrophages is amplified by methyl-CpG-binding domain 2 (MBD2) protein, which suppresses the expression of an inhibitor upstream of TGFβ, and MBD2 was found to be overexpressed in SSc-ILD lung tissues [28]. Zehender et al. recently demonstrated a novel mechanism for TGFβ-induced fibrosis in SSc, which involves a loss of epigenetic control over autophagy via a Smad3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, mediating the activation of fibroblasts [29]. Core regulators of autophagy, BECLIN1 and ATG7, were consequently found to be upregulated in SSc dermal fibroblasts, as well as fibrotic skin and lungs of mice overexpressing TGFBRI, while their knockdown alleviated fibrosis [29].

Efforts to target TGFβ as a therapeutic strategy to reduce SSc lung fibrosis have not been effective, and concerns about potential adverse complications due to the pleiotropic roles of TGFβ in lung physiology have led to efforts focusing on targeting other pro-fibrotic factors and molecular pathways as a therapeutic strategy to treat SSc [1,30,31].

It is worth noting that several members of the TGFβ family have been largely overlooked in SSc lung research to date, although they are likely to play important roles in disease pathogenesis. Unlike TGFβ, whose active form is generated when and where it is needed, activin A and BMP4 are generally readily active and thus possess distinct signaling dynamics from TGFβ-induced fibrosis [32].

PDGF has also been shown to play a central role in organ fibrosis, since stromal mesenchymal cells, including fibroblasts, express PDGF receptor isoforms that are activated and drive processes implicated in fibrosis, such as proliferation, migration, and ECM deposition [33]. In fact, lung fibrosis of various etiologies, whether environmental exposure, transplant rejection, autoimmune, or idiopathic, have been associated with increased PDGF levels in bronchoalveolar lavage fluid (BALF) or lung tissues [34]. There are two PDGF receptor isoforms, PDGFRα and PDGFRβ, which are tyrosine kinase receptors recognized by four ligand isoforms, PDGF-A, PDGF-B, PDGF-C, and PDGF-D [35]. Upon ligand binding, the homo- or hetero-dimerization of the receptors leads to autophosphorylation events of their cytoplasmic domain, which activates downstream signaling pathways, including phosphatidylinositol 3 kinase (PI3K), Ras-MAPK, Src, and phospholipase Cγ (PLCγ) pathways [35]. More recently, we showed that PDGF can also signal via melanin-concentrating hormone receptor 1 (MCHR1), modulating intracellular cyclic adenosine monophosphate (cAMP) production and inducing TGFβ1 and connective tissue growth factor (CTGF) expression in fibroblasts, thus promoting a fibrotic response [36].

PDGF-A and PDGF-B levels are elevated in the BALF of SSc patients [37]. Interestingly, SSc-derived fibroblasts exhibit unique, positive cross-talk between PDGF and TGFβ signaling, which does not occur in healthy fibroblasts [38]. In addition, microRNA miR-30b, which suppresses the expression of PDGFRβ, is downregulated in the serum of SSc patients [39]. Reducing the expression of PDGFRβ in SSc dermal fibroblasts with miR-30b inhibited collagen synthesis and myofibroblast activation [39]. Since miR-30b levels are decreased in the circulation of SSc patients, it is reasonable to extrapolate these findings to lung fibroblasts. Recently, Svegliati et al. demonstrated that PDGF and anti-PDGFR autoantibodies, which are elevated in SSc patient serum [40,41], stimulated higher growth rate, migration, and expression of collagen in human pulmonary artery smooth muscle cells, which was attributed to the generation of reactive oxygen species, and elevated NOX4 and mammalian target of rapamycin (mTOR) [42]. All these findings have made PDGF an attractive molecular target for therapeutic treatment of SSc lung fibrosis [43]. In fact, nintedanib, a drug that blocks the ATP-binding pocket of PDGFR and other tyrosine kinase receptors, such as fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR), was approved by the FDA for the treatment of SSc-ILD [6,44]. At the cellular level, nintedanib blocks the PDGF-induced differentiation of lung fibroblasts into myofibroblasts, reduces their proliferation and migration, and suppresses the expression of collagen and fibronectin, supporting the antifibrotic outcome of blocking PDGF signaling.

FGFs are a family of signaling proteins that can act in an endocrine, paracrine, or even intracrine manner [45]. Under paracrine or endocrine conditions, target cells interact with FGF ligands via four receptor tyrosine kinases, FGFR1, FGFR2, FGFR3, and FGFR4 [45]. Intracrine FGFs are nonsignaling, in that they act independently of FGFRs and mainly serve as cofactors for voltage-gated sodium channels [45,46]. Upon ligand binding, FGFRs activate multiple pathways, including PI3K, Ras-MAPK, PLCγ, and STAT signaling pathways [45]. The roles of the different FGF ligands in fibrosis have been variable in the experimental models, with some promoting lung fibrosis and others protecting against it [47]. For example, members of the FGF family of proteins can activate fibroblasts and induce their proliferation and ECM deposition [48]. In contrast, one member of the family, FGF19, was found to be protective against lung fibrosis in mice, and its levels were decreased in the plasma of IPF patients [49]. However, studies about the specific role of FGFs in SSc-related lung fibrosis are scarce. Recently, Chakraborty et al. demonstrated a mechanistic involvement of FGF9 and its receptor FGFR3 in SSc, both of which are upregulated in SSc fibrotic skin [50]. FGF9 was shown to bind FGFR3 and activate dermal fibroblasts from SSc skin, leading to the downstream stimulation of AKT, p38, extracellular signal-regulated kinase (ERK), and calcium/calmodulin-dependent protein kinase 2 (CAMK2) and promoting cyclic adenosine 3′,5′-monophosphate response element binding protein (CREB) activation, which induced the expression of profibrotic mediators [50]. These findings from SSc skin have not yet been validated in tissues or primary cells derived from lungs of SSc patients.

Widely known for its role in organ and tissue development, the Wnt/β-catenin signaling pathway has more recently been implicated in fibrotic disorders in different organs [51,52,53,54]. The binding of Wnt ligands to their Frizzled (Fz) receptors triggers downstream effects inhibiting the degradation of β-catenin, stabilizing it in the cytoplasm, and promoting its translocation to the nucleus, where it results in the transcription of Wnt target genes [55]. Earlier studies have confirmed the involvement of the Wnt/β-catenin pathway in the pathogenesis of lung fibrosis and, specifically, in SSc [56,57,58]. In addition, SSc skin fibroblasts express high levels of Wnt proteins such as Wnt1 and Wnt10b, coupled with decreased expression of the Wnt antagonists SFRP1, DKK1, and WIF1 [59]. Our group recently reported similar results in SSc lung fibroblasts, showing increased Wnt5a and decreased SFRP1 expression in SSc lung fibroblasts compared with normal lung fibroblasts [14]. More recently, increased levels of a novel isoform of CD146 that activates myofibroblasts were noted in the serum of SSc patients with pulmonary fibrosis, a process driven by Wnt5a [60]. This is consistent with previous studies showing that the Wnt/β-catenin pathway is activated in SSc lung fibrosis, allowing its downstream pro-fibrotic effects to be promoted [57,61].

Interleukins (ILs) are a group of cytokines with immunoregulatory functions known to be secreted by white blood cells, but also several other cell types, such as epithelial and stromal cells [62,63]. There are more than 40 distinct ILs, each eliciting different functions across multiple different cell types via binding to high-affinity receptors [64]. Several of these ILs have been shown to directly interact with fibroblasts to promote lung fibrosis [65].

IGFs and IGFBPs have been implicated in the pathogenesis of SSc lung fibrosis. IGF-I levels are increased in the BALF of SSc patients, and the protein itself promotes the proliferation of fibroblasts [83]. IGF-II expression is also increased in fibrotic SSc lung tissues and fibroblasts and induces ECM deposition via the activation of PI3K and Jun N-terminal kinase pathways [84]. A follow-up study demonstrated that IGF-II signaled via type 1 IGF receptor (IGF1R), insulin receptor (IR), and a hybrid IGF1R/IR complex receptor, promoting fibrosis through multiple mechanisms: by directly activating myofibroblasts, by increasing ECM production while reducing its degradation, and by stimulating the expression of TGFβ2 and TGFβ3 [26]. We showed that IGFBP-3 and IGFBP-5 are profibrotic proteins that stimulate the production of ECM by lung fibroblasts [85,86,87]. IGFBP-5 is significantly overexpressed in SSc lung tissues and fibroblasts and induces the expression of collagen type I, fibronectin, CTGF, LOX, and DOK5 [88,89]. Mice expressing human IGFBP-5 showed sustained increased expression of ECM genes [90]. Recently, IGFBP-2 serum levels were found to have prognostic value for assessing the development of ILD in SSc patients, but further studies are needed to understand its potential role in lung disease [91].

Yes-associated protein (YAP) and transcription coactivator with PDZ-binding motif (TAZ) are key components of the Hippo pathway [92]. Mammalian STE20-like (MST) and large tumor suppressor kinase (LATS) are the core proteins upstream of YAP/TAZ in the Hippo pathway [93]. When the Hippo pathway is activated, the sequential phosphorylation of MST, LATS, and then YAP/TAZ occurs, causing the retention of YAP/TAZ in the cytoplasm [94]. YAP/TAZ signaling mediates its effect via cross-talk with multiple other pathways, including the TGFβ and Wnt/β-catenin pathways [94]. YAP/TAZ was shown to promote myofibroblast proliferation, contraction, and ECM synthesis [95]. Myofibroblast-specific YAP/TAZ deficiency ameliorates fibrosis across multiple organs, including lungs, kidneys, and liver, supporting previous findings on the critical role that YAP/TAZ signaling plays in fibrogenesis [96]. Further delineation of the role of YAP/TAZ signaling in SSc-related fibrosis is warranted. Toyoma et al. demonstrated that targeting YAP/TAZ with dimethyl fumarate is a viable therapeutic strategy for dermal fibrosis in SSc [97]. More recently, Wu et al. showed that skin fibroblasts and serum from SSc patients had increased levels of YAP and TAZ compared with healthy controls, and they demonstrated that knockdown of YAP/TAZ in mice alleviated bleomycin-induced lung fibrosis [98].

Chemokines are a family of small proteins that play a critical role in maintaining homeostasis and regulating inflammation and tissue-specific leukocyte migration [99]. Upon tissue damage, chemokines initiate and maintain the inflammatory process, and the fine-tuning of their expression is imperative for the resolution of inflammation, culminating in tissue repair and wound healing [100]. Chemokines can activate fibroblasts and perpetuate their ECM production and deposition [100]. This has led to research on the roles of certain chemokines in SSc lung disease. Specifically, C-C motif ligand-2 (CCL2) levels were elevated in the BALF of SSc patients compared with healthy controls [101]. Furthermore, CCL2 protein was overexpressed in fibroblasts from fibrotic lungs of SSc patients [102]. Wu et al. demonstrated that CCL2 plasma levels can serve as a biomarker and a potential therapeutic target for ILD progression in SSc patients, as higher CCL2 levels predicted a faster decline in forced vital capacity (FVC%) over time [103]. Another chemokine, CXCL4, was reported by van Bon et al. to be elevated in SSc patients and correlated with the severity of lung fibrosis [104]. A follow-up study by Affandi et al. demonstrated that CXCL4 is required for bleomycin-induced lung fibrosis in mice, where it promoted myofibroblast transformation, leading to excessive ECM deposition [105]. Multiple other chemokines have been implicated in SSc lung fibrosis, such as CCL5, CCL7, CCL18, CXCL3, and CXCL8, mostly as potential biomarkers of ILD [100,106,107]. Further studies are still needed to elucidate which of these chemokines are central to the pathogenesis of SSc lung fibrosis.