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Journal of Zhejiang University. Science. B logoLink to Journal of Zhejiang University. Science. B
. 2023 Aug 15;24(8):682–697. doi: 10.1631/jzus.B2200506

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway

细胞因子受体样因子1(CRLF1)通过ERK1/2信号通路促进心脏纤维化

Shenjian LUO 1,*, Zhi YANG 2,*, Ruxin CHEN 3, Danming YOU 2, Fei TENG 1, Youwen YUAN 1, Wenhui LIU 2, Jin LI 4,✉, Huijie ZHANG 1,2,3,✉
PMCID: PMC10423965  PMID: 37551555

Abstract

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Keywords: Cytokine receptor-like factor 1 (CRLF1), TGF‍-‍β1/SMAD signaling pathway, ERK1/2 signaling pathway, Cardiac fibrosis, Myofibroblast transformation, Extracellular matrix (ECM)

1. Introduction

Cardiovascular disease is a serious disease that endangers people’s lives, and its incidence is increasing year by year. Cardiac fibrosis most commonly occurs in the wake of many kinds of cardiovascular diseases (Brown et al., 2005; Schuetze et al., 2014). A key characteristic of cardiac fibrosis is that extracellular matrix (ECM) components are deposited in myocardial stroma (Janicki and Brower, 2002; Berk et al., 2007; Bacmeister et al., 2019). During the process of cardiac fibrosis, multiple fibrosis-associated components in the ECM, especially type Ⅰ collagen (Col-1) and type Ⅲ collagen (Col-3), change significantly in stimulated hearts (Shirwany and Weber, 2006; Disertori et al., 2017; López et al., 2021). Collagen proteins are synthesized and secreted mainly by cardiac fibroblasts, and maintain a dynamic balance of synthesis and degradation under physiological conditions, which is broken under pathological conditions. This quantitative or proportional imbalance of collagen proteins in the ECM subsequently damages heart functions including cardiac systolic and diastolic functions (Fernández-Alfonso and Ruilope, 2014; Nguyen et al., 2014, 2017; Perestrelo et al., 2021). Although it is well known that increased collagen deposition and myofibroblast transformation occur during cardiac fibrosis, the underlying events and regulation mechanisms are still unclear and need to be explored urgently. In this study, we chose cytokine receptor-like factor 1 (CRLF1) as our target protein in the hope of discovering a new action molecule in cardiac fibrosis.

CRLF1 is a member of the cytokine family proteins. Mutation of CRLF1 is proposed as a clinical auxiliary diagnostic index of the cold-induced sweating syndrome (CISS) (Herholz et al., 2011; Busch et al., 2017). Subsequent research has shown that CRLF1 plays an important role in controlling facial muscles, heat production, and other autonomic nervous systems (Buers et al., 2020). Some studies have shown that CRLF1 can participate in the physiological and pathological processes of various tissues and organs (Yu ST et al., 2018, 2020; Li et al., 2021). In addition, the expression of CRLF1 is positively correlated with the level of liver fibrosis, and CRLF1 overexpression can up-regulate Col-3 expression and the α-smooth muscle actin (α‍-‍SMA) level in quiescent hepatic stellate cells (HSCs) (Stefanovic and Stefanovic, 2012). There has been very little research on the role of CRLF1 in the heart. Acute cocaine exposure can cause an increased expression of CRLF1 in both the ventricular tissue of the rabbit and H9C2 cell lines (Lattanzio et al., 2005). Here, we analyzed the expression of CRLF1 in two important kinds of heart cells (fibroblasts and cardiomyocytes) and found that CRLF1 was expressed in the heart, mostly in cardiac fibroblasts rather than cardiomyocytes (Tucker et al., 2020; Chaffin et al., 2022; Koenig et al., 2022). This finding suggested that CRLF1 might affect the regulation of cardiac fibrosis. Unfortunately, little was known about the functions of CRLF1 in cardiac fibrosis.

In this study, we carried out detailed and in-depth research about CRLF1 in cardiac fibrosis. Of note, we found that CRLF1 was predominantly expressed in cardiac fibroblasts, and its expression was induced during the fibrotic process both in vitro and in vivo. CRLF1 overexpression promoted the cardiac fibrotic process, including excessive cell proliferation, collagen protein synthesis and secretion, and myofibroblast transformation, while silencing of CRLF1 had the opposite effects. Furthermore, activation of the transforming growth factor-‍β1 (TGF‍-‍β1)/mothers against decapentaplegic homolog (SMAD) signaling pathway during the fibrotic process not only caused the production of collagen and myofibroblast transformation, but also increased CRLF1 expression. Up-regulation of CRLF1 in turn consolidated the outcome of the TGF-β1/SMAD signaling pathway in cardiac fibrosis by activating the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In summary, we elucidated the function of CRLF1 in the development of cardiac fibrosis and its relationship with the TGF‍-‍β1/SMAD signaling pathway. Our study provides a theoretical basis for CRLF1 to be applied as a novel promising marker for cardiac fibrosis diagnosis as well as a useful target for cardiac fibrosis remedies.

2. Results

2.1. CRLF1 expression during the cardiac fibrotic process

Firstly, the change in CRLF1 expression during cardiac fibrosis was investigated in a mouse cardiac fibrosis model achieved by myocardial infarction (MI) for four weeks. Masson’s trichrome staining suggested cardiac fibrosis had occurred in the mouse hearts after MI (Fig. 1a). The messenger RNA (mRNA) expression of Col-1 (consisting of the Col-1A1 and Col-1A2 subunits) and Col-3 (Col-3A1 subunit) was increased in the MI group, suggesting enhanced collagen production (Fig. 1b). In line with the pathogenesis of cardiac fibrosis, the MI group showed increased α‍-SMA expression, implying elevated myofibroblast transformation (Fig. 1b). Interestingly, compared to the sham group, the cardiac mRNA expression and protein levels of CRLF1 were also increased during the fibrotic process in vivo (Figs. 1b and 1c).

Fig. 1. Up-regulation of CRLF1 in the fibrotic process in vivo and in vitro. (a) Comparison of interstitial fibrosis detected by Masson's trichrome staining in the sham and MI groups. n=3. (b) mRNA expression of Collagen and α-SMA in mice hearts. n=8. (c) Protein level of CRLF1 in heart tissues. n=3. (d) mRNA expression of CRLF1 in NMCFs, AMCFs, NMCMs, and ECs. n=4. (e) Comparison of EdU staining of NMCFs in the Ctrl and TGF-β1 stimulation groups. n=4. (f) mRNA expression of Collagen and α-SMA in NMCFs under TGF-β1 stimulation. n=5. (g) mRNA expression of CRLF1 in NMCFs and HCFs under TGF-β1 stimulation. n=5. (h) Protein level of CRLF1 in NMCFs and HCFs under TGF-β1 stimulation. n=3. Data are shown as mean±SD. * P<0.05; ns: not significant. CRLF1: cytokine receptor-like factor 1; MI: myocardial infarction; mRNA: messenger RNA; α‍-SMA: α‍-smooth muscle actin; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; NMCFs: neonatal mice cardiac fibroblasts; AMCFs: adult mice cardiac fibroblasts; NMCMs: neonatal mice cardiomyocytes; ECs: endothelial cells; Ctrl: control; TGF‍-‍β1: transforming growth factor-‍β1; EdU: 5-ethynyl-2'-deoxyuridine; HCFs: human cardiac fibroblasts; SD: standard deviation.

Fig. 1

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Next, CRLF1 expression was compared among neonatal mice cardiac fibroblasts (NMCFs), adult mice cardiac fibroblasts (AMCFs), neonatal mice cardiomyocytes (NMCMs), and endothelial cells (ECs). Compared to NMCFs, CRLF1 expression was similar in AMCFs, while NMCMs and ECs expressed lower levels of CRLF1 (Fig. 1d). Staining with 5-ethynyl-2'-deoxyuridine (EdU) suggested that NMCFs were undergoing a fibrotic process of excessive proliferation under TGF‍-‍β1 stimulation (Fig. 1e). In addition, TGF-‍β1 stimulation in NMCFs induced collagen production and myofibroblast transformation as shown by increased expression of Col-1A1, Col-1A2, Col-3A1, and α-SMA (Fig. 1f). During this fibrotic process, CRLF1 expression was significantly increased in NMCFs and human cardiac fibroblasts (HCFs) under TGF-‍β1 stimulation (Fig. 1g). In addition, the CRLF1 protein level was detected by western blotting in NMCFs and HCFs under TGF-β1 stimulation. Compared to the control (Ctrl) group, CRLF1 had a higher level under TGF-‍β1 stimulation in both NMCFs and HCFs (Fig. 1h).

2.2. Effects of CRLF1 overexpression or silencing on fibrotic process in NMCFs under a physiological state

Gain- and loss-of-function strategies were used to explore the functions of CRLF1 in NMCFs by transfection of overexpression plasmids and small interfering RNAs (siRNAs). We screened the transfection concentrations of plasmids and finally selected 100 ng/mL as the concentration for subsequent study (Fig. 2a). Furthermore, transfection efficiency was verified at mRNA and protein levels accompanied by increased transcription and translation of CRLF1, collagen proteins (Col-1 and Col-3), and α-SMA (Figs. 2b and 2c). Cell counting kit-8 (CCK-8) assay results showed that CRLF1 overexpression increased the cell viability of NMCFs, which indirectly indicated that NMCFs had a higher proliferative capacity after CRLF1 overexpression (Fig. 2e). Similarly, CRLF1 overexpression increased collagen production of NMCFs (Fig. 2f).

Fig. 2. Effects of CRLF1 overexpression or silencing on fibrotic process in NMCFs. (a) mRNA expression of CRLF1 after transfection of its overexpression plasmids with different concentrations. n=3. (b) mRNA expression of Collagen and α ‍ -SMA in NMCFs after CRLF1 overexpression. n=3. (c) Protein levels in NMCFs after CRLF1 overexpression. n=3. (d) CRLF1 level detected by ELISA in cell supernatant of NMCFs after CRLF1 overexpression. n=4. (e) Cell viability of NMCFs after CRLF1 overexpression. n=4. (f) Collagen content of NMCFs after CRLF1 overexpression. n=4. (g) mRNA expression of CRLF1 after transfection of its different siRNAs. n=3. (h) mRNA expression of Collagen and α ‍ -SMA in NMCFs after CRLF1 silencing. n=3. (i) Protein levels in NMCFs after CRLF1 silencing. n=3. (j) CRLF1 level detected by ELISA in cell supernatant after CRLF1 silencing. n=4. (k) Cell viability of NMCFs after CRLF1 silencing. n=4. (l) Collagen content of NMCFs after CRLF1 silencing. n=4. Data are shown as mean±SD. * P<0.05; ns: not significant. CRLF1: cytokine receptor-like factor 1; NMCFs: neonatal mice cardiac fibroblasts; mRNA: messenger RNA; si-CRLF1: small interfering RNAs (siRNAs) for CRLF1; α-SMA: α-smooth muscle actin; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; ERK1/2: extracellular signal-regulated kinase 1/2; p-ERK1/2: phospho-ERK1/2; t-ERK1/2: total-ERK1/2; ELISA: enzyme-linked immunosorbent assay; NC: negative control; SD: standard deviation.

Fig. 2

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In the following loss-of-function experiments, we screened for siRNAs that could stably and efficiently silence CRLF1 expression (Fig. 2g). We ultimately chose siRNA-2 (si-CRLF1) for CRLF1 silencing, and its silencing efficiency was verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting experiments (Figs. 2h and 2i). Meanwhile, transcription levels of Col-1A1, Col-1A2, Col-3A1, and α‍-SMA were decreased (Fig. 2h). In addition, Col-1, Col-3, and α‍-SMA protein levels were also decreased after CRLF1 silencing in NMCFs (Fig. 2i). Moreover, cell viability (Fig. 2k) and collagen content (Fig. 2l) of the CRLF1 silencing group were significantly reduced. No change in CRLF1 was detected in the cell supernatant after CRLF1 overexpression or silencing (Figs. 2d and 2j). This suggested that CRLF1 might function in the cytoplasm rather than in the extracellular space.

2.3. Effect of CRLF1 overexpression on fibrotic process in NMCFs under a pathological state

Having detected the role of CRLF1 in a physiological state, we investigated whether CRLF1 also played a role in cells under a pathological state. We provoked NMCFs with TGF‍-‍β1 to proliferate excessively and synthesize collagen to imitate the fibrotic process in vitro. CRLF1 overexpression aggravated the increase of cell viability and collagen production activated by TGF-‍β1 (Figs. 3a and 3b). The EdU results directly showed that CRLF1 overexpression resulted in further proliferation induced by TGF‍-‍β1 (Fig. 3c). Furthermore, we tested the mRNA and protein levels of fibrosis-related molecules. The qRT-PCR results showed that the mRNA expression levels of Col-1 (Col-1A1 and Col-1A2), Col-3 (Col-3A1), and α-SMA were all up-regulated in response to TGF-β1 stimulation after CRLF1 overexpression (Fig. 3d). In addition, CRLF1 overexpression obviously raised the protein levels of Col-1, Col-3, and α‍-‍SMA compared with those of the TGF-β1 stimulation group (Fig. 3e).

Fig. 3. Effect of CRLF1 overexpression on fibrotic process in NMCFs under TGF-β1 stimulation. (a) Cell viability of NMCFs after CRLF1 overexpression under TGF-β1 stimulation. n=4. (b) Collagen content of NMCFs after CRLF1 overexpression under TGF-‍β1 stimulation. n=4. (c) EdU staining of NMCFs after CRLF1 overexpression under TGF‍-‍β1 stimulation. n=4. (d) mRNA expression of Collagen and α ‍ -SMA after CRLF1 overexpression in TGF-‍β1-treated NMCFs. n=4. (e) Protein levels of collagen and α‍-‍SMA after CRLF1 overexpression in TGF-‍β1‍-treated NMCFs. n=3. Data are shown as mean±SD. * P<0.05; # P<0.05. CRLF1: cytokine receptor-like factor 1; NMCFs: neonatal mice cardiac fibroblasts; TGF-β1: transforming growth factor-‍β1; EdU: 5-ethynyl-2'-deoxyuridine; mRNA: messenger RNA; α‍-SMA: α‍-smooth muscle actin; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; Ctrl: control; SD: standard deviation.

Fig. 3

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2.4. Effect of CRLF1 silencing on fibrotic process in NMCFs under a pathological state

In contrast, silencing of endogenous CRLF1 by si-CRLF1 inhibited the up-regulation of cell viability (Fig. 4a), collagen content (Fig. 4b), and cell proliferation (Fig. 4c) induced by TGF-β1. Moreover, CRLF1 silencing decreased the mRNA and protein levels of Col-1, Col-3, and α‍-SMA (Figs. 4d and 4e). These results suggested that CRLF1 overexpression promoted NMCFs to proliferate and synthesize collagen excessively, and transform myofibroblasts, while CRLF1 silencing had the opposite effects under physiological and pathological states.

Fig. 4. Effect of CRLF1 silencing on fibrotic process in NMCFs under TGF-‍β1 stimulation. (a) Cell viability of NMCFs after CRLF1 silencing under TGF-‍β1 stimulation. n=4. (b) Collagen content of NMCFs after CRLF1 silencing under TGF-‍β1 stimulation. n=4. (c) EdU staining of NMCFs after CRLF1 silencing under TGF-‍β1 stimulation. n=4. (d) mRNA expression of Collagen and α-SMA after CRLF1 silencing in TGF-‍β1-treated NMCFs. n=4. (e) Protein levels of collagen and α‍-SMA after CRLF1 silencing in TGF-β1-treated NMCFs. n=3. Data are shown as mean±SD. * P<0.05; # P<0.05. CRLF1: cytokine receptor-like factor 1; NMCFs: neonatal mice cardiac fibroblasts; TGF-β1: transforming growth factor-β1; si-CRLF1: small interfering RNAs for CRLF1; EdU: 5-ethynyl-2'-deoxyuridine; mRNA: messenger RNA; α‍-SMA: α-smooth muscle actin; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; Ctrl: control; NC: negative control; SD: standard deviation.

Fig. 4

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2.5. Activation of ERK1/2 signaling pathway after CRLF1 overexpression or silencing in NMCFs

The function of CRLF1 is regulated by the ERK1/2 signaling pathway in other tissues (Yu ST et al., 2018, 2020; Zheng et al., 2020), and the ERK1/2 signaling pathway also participates widely in the regulation of cardiac fibrosis (Yamaguchi et al., 2004; Westermann et al., 2007; Wang XW et al., 2021; Wen et al., 2022). Therefore, we examined whether the ERK1/2 signaling pathway changed when CRLF1 was overexpressed or silenced in NMCFs with or without TGF-β1 stimulation. Enzyme-linked immunosorbent assay (ELISA) results showed that CRLF1 overexpression increased the phospho-ERK1/2 (p-ERK1/2) protein level in NMCFs without TGF‍-‍β1 stimulation (Fig. 5a). Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) results also showed that the ratio of p-ERK1/2 to total-ERK1/2 (t-ERK1/2) was increased by CRLF1 overexpression in NMCFs without TGF-β1 stimulation (Fig. 5b). Then, NMCFs were co-treated with TGF-‍β1 and CRLF1 overexpression plasmids. The ELISA and AlphaLISA results showed that CRLF1 overexpression increased p-ERK1/2 protein activation in TGF-‍β1-treated NMCFs (Figs. 5c and 5d). Furthermore, western blotting results indicated that CRLF1 overexpression increased the p-/t-ERK1/2 protein level after TGF-‍β1 stimulation (Fig. 5e).

Fig. 5. ERK1/2 protein activation after CRLF1 overexpression or silencing in NMCFs with or without TGF-‍β1 stimulation. (a) p-ERK1/2 level detected by ELISA in NMCFs after CRLF1 overexpression without TGF-‍β1 stimulation. n=3. (b) Relative AlphaLISA signal (p-/t-ERK1/2) in NMCFs after CRLF1 overexpression without TGF-‍β1 stimulation. n=3. (c) p-ERK1/2 level detected by ELISA in NMCFs after CRLF1 overexpression with TGF-β1 stimulation. n=3. (d) Relative AlphaLISA signal (p-/t-ERK1/2) in NMCFs after CRLF1 overexpression with TGF-β1 stimulation. n=3. (e) p-/t-ERK1/2 level detected by western blotting in NMCFs after CRLF1 overexpression under TGF-β1 stimulation. n=3. (f) p-ERK1/2 level detected by ELISA in NMCFs after CRLF1 silencing without TGF-‍β1 stimulation. n=3. (g) Relative AlphaLISA signal (p-/t-ERK1/2) in NMCFs after CRLF1 silencing without TGF-‍β1 stimulation. n=3. (h) p-ERK1/2 level detected by ELISA in NMCFs after CRLF1 silencing with TGF-‍β1 stimulation. n=3. (i) Relative AlphaLISA signal (p-/t-ERK1/2) in NMCFs after CRLF1 silencing with TGF-‍β1 stimulation. n=3. (j) p-/t-ERK1/2 level detected by western blotting in NMCFs after CRLF1 silencing under TGF-‍β1 stimulation. n=3. Data are shown as mean±SD. * P<0.05; # P<0.05. ERK1/2: extracellular signal-regulated kinase 1/2; p-ERK1/2: phospho-ERK1/2; t-ERK1/2: total-ERK1/2; CRLF1: cytokine receptor-like factor 1; NMCFs: neonatal mice cardiac fibroblasts; TGF-‍β1: transforming growth factor-‍β1; si-CRLF1: small interfering RNAs for CRLF1; ELISA: enzyme-linked immunosorbent assay; AlphaLISA: amplified luminescent proximity homogeneous assay-linked immunosorbent assay; Ctrl: control; NC: negative control; SD: standard deviation.

Fig. 5

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In contrast, CRLF1 silencing showed the opposite effects of CRLF1 overexpression (Figs. 5f‍‒‍5j). We also detected the changes of the p-ERK1/2 protein level after CRLF1 overexpression or silencing in NMCFs without TGF-‍β1 stimulation. The ratio of p-ERK1/2 to t-ERK1/2 was also increased after CRLF1 overexpression, but decreased after CRLF1 silencing in non-activated NMCFs (Figs. 2c and 2i). These results suggested that CRLF1 could influence the ERK1/2 signaling pathway in NMCFs under pathological and physiological states.

2.6. Functions of CRLF1 via the ERK1/2 signaling pathway in NMCFs

We wondered if the ERK1/2 signaling pathway could mediate the functions of CRLF1 in NMCFs. Firstly, by consulting the medical literature (Wang ZY et al., 2019; Wang JC et al., 2021), we tested 1 and 2 µmol/L doses of the ERK1/2 inhibitor Temuterkib (LY3214996) to select an appropriate inhibition concentration. Both 1 and 2 µmol/L of Temuterkib significantly reduced the basal p-ERK1/2 and t-ERK1/2 protein levels (Fig. 6a). Both doses of Temuterkib clearly reduced activation of the ERK1/2 signaling pathway. We finally chose 1 µmol/L because of its low dose and favorable potency. After treating NMCFs with CRLF1 overexpression plasmids, qRT-PCR showed that CRLF1 overexpression no longer increased the expression of Col-1 (Col-1A1 and Col-1A2), Col-3 (Col-3A1), or α‍- SMA mRNAs after inhibition of the ERK1/2 signaling pathway (Figs. 6b‒6e). Col-1, Col-3, and α‍-SMA protein levels showed the same trend (Fig. 6f). These results suggested that CRLF1 worked in NMCFs by activating the ERK1/2 signaling pathway.

Fig. 6. Functions of CRLF1 via the ERK1/2 signaling pathway in NMCFs. (a) Protein levels of p-ERK1/2 and t-ERK1/2 under different stimulation concentrations of the ERK1/2 inhibitor Temuterkib. n=4. (b‒e) mRNA expression of Col-1A1 (b), Col-1A2 (c), Col-3A1 (d), and α-SMA (e) in NMCFs. n=4. (f) Protein levels of collagen and α‍-‍SMA in NMCFs. n=3. Data are shown as mean±SD. * P<0.05; # P<0.05. CRLF1: cytokine receptor-like factor 1; ERK1/2: extracellular signal-regulated kinase 1/2; p-ERK1/2: phospho-ERK1/2; t-ERK1/2: total-ERK1/2; NMCFs: neonatal mice cardiac fibroblasts; DMSO: dimethyl sulfoxide; mRNA: messenger RNA; α-SMA: α‍-smooth muscle actin; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; SD: standard deviation.

Fig. 6

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2.7 CRLF1up‍-‍regulation via TGF‍-‍β1/SMAD signaling pathway in cardiac fibrosis

Through the above research, we had identified p-ERK1/2 as the downstream target in the regulation of CRLF1 function, but it was unclear why CRLF1 was up-regulated in NMCFs under TGF-‍β1 stimulation. TGF‍-‍β1 is the best characterized fibrogenic growth factor (Frangogiannis, 2020; Lodyga and Hinz, 2020). TGF-‍β1 signaling cascades are divided into SMAD-dependent and SMAD-independent pathways (Derynck and Zhang, 2003; Schmierer and Hill, 2007; Hu et al., 2018). In SMAD-dependent pathways (SMAD signaling pathways), the phosphorylated SMAD2 and SMAD3 complicate with SMAD4 and translocate to the nucleus to induce transcription of profibrotic molecules (Meng et al., 2016; Hanna and Frangogiannis, 2019). We speculated that the SMAD signaling pathway could regulate the expression of CRLF1 because it is widely involved in cardiac fibrosis (Li et al., 1997; Dewald et al., 2004; Xia et al., 2011; Biernacka et al., 2015; Nagaraju et al., 2019). Following a comprehensive review of the medical literature (Tu et al., 2011; Sundararaj et al., 2016; Meng et al., 2020; Shen et al., 2020), we first screened 1 and 2 µmol/L doses of the SMAD signaling pathway inhibitor, specific inhibitor of SMAD3 (SIS3), to test its effect on the mRNA expression of Col-1 (Col-1A1 and Col-1A2), Col-3 (Col-3A1), and α-SMA (Fig. 7a). Both doses of SIS3 had good potency. Then, NMCFs were treated with TGF-β1, followed by treatment with DMSO (a solvent for SIS3) or SIS3 (1 µmol/L). Up-regulated expression of CRLF1 induced by TGF-β1 was blocked by SIS3 in mRNA and protein levels (Figs. 7b and 7c).

Fig. 7. CRLF1 up-regulation via TGF-‍β1/SMAD signaling pathway. (a) mRNA expression in NMCFs under different concentrations of SIS3. n=5. (b) mRNA expression of CRLF1 under SIS3 treatment in TGF-β1-treated NMCFs. n=4. (c) Protein level of CRLF1 under SIS3 treatment in TGF-‍β1‍-treated NMCFs. n=3. (d) mRNA expression of CRLF1 under treatment of different inhibitors of SMAD-independent pathways in TGF‍-‍β1‍-treated NMCFs. n=4. (e) Protein level of CRLF1 under treatment with different inhibitors of SMAD-independent pathways in TGF-‍β1-treated NMCFs. n=3. Data are shown as mean±SD. * P<0.05. CRLF1: cytokine receptor-like factor 1; TGF-β1: transforming growth factor-β1; SMAD: mothers against decapentaplegic homolog; mRNA: messenger RNA; NMCFs: neonatal mice cardiac fibroblasts; SIS3: specific inhibitor of SMAD3; DMSO: dimethyl sulfoxide; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; α‍-SMA: α-smooth muscle actin; SD: standard deviation.

Fig. 7

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In addition, in SMAD-independent pathways, TGF‍-‍β1 can regulate the activation of three known mitogen-activated protein kinase (MAPK) pathways, namely ERK1/2, c-Jun N-terminal kinase (JNK), and protein 38 (p38) MAPK, as well as the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Ras homolog gene family, member A (RhoA)/Rho-associated kinase (ROCK) pathways (Horbelt et al., 2012; Scalise et al., 2021). Therefore, we applied the ERK1/2 inhibitor Temuterkib (1 µmol/L) and other inhibitors within the same category, at a concentration based on the literature: SP600125 (25 µmol/L), SB203580 (10 µmol/L), Y-27632 (10 µmol/L), and LY294002 (10 µmol/L). These inhibitors were used to inhibit the following pathways in NMCFs under TGF-β1 stimulation: SP600125 for JNK (Singh et al., 2021), SB203580 for p38 MAPK (Stratton et al., 2019), Y-27632 for ROCK (Childers et al., 2019), and LY294002 for PI3K (Lei et al., 2015; Yu P et al., 2020). We then applied qRT-PCR and western blotting to detect CRLF1 levels after these inhibitors were used under TGF-‍β1 stimulation. The results of qRT-PCR (Fig. 7d) and western blotting (Fig. 7e) showed that CRLF1 mRNA and protein levels did not change after these inhibitors were used. These results suggested that up-regulation of CRLF1 in NMCFs under TGF-β1 stimulation depended on the activation of the SMAD-dependent pathway rather than SMAD-independent pathways.

3. Discussion

In this study, we identified the function and mechanism of action of CRLF1 in cardiac fibrosis and provided a theoretical basis for the development of CRLF1 as a potential pharmaceutical target of cardiac fibrosis. Firstly, we clarified the expression pattern of CRLF1 in different in vivo and in vitro models of cardiac fibrosis in humans and mice. CRLF1, which was enriched in cardiac fibroblasts, was up-regulated not only in a mouse heart fibrotic model induced by MI, but also in NMCFs and HCFs provoked by TGF‍-‍β1. We then explored the roles of CRLF1 in NMCFs in detail. CRLF1 increased collagen production and aggravated the cardiac fibrotic process under physiologic-al and pathological conditions. Mechanistically, up-regulation of CRLF1 was attributed to the activation of the TGF-β1/SMAD signaling pathway. CRLF1 increased fibrosis-related proteins and up-regulated the activity of the ERK1/2 signaling pathway by increasing the p-ERK1/2 level in the cytoplasm of NMCFs. Finally, we summarized our research and drew a schematic diagram (Fig. 8).

Fig. 8. Role of CRLF1 in cardiac fibrosis. CRLF1: cytokine receptor-like factor 1; TGF-β1: transforming growth factor-β1; SMAD: mothers against decapentaplegic homolog; SIS3: specific inhibitor of SMAD3; P: phosphorylation; Col-1: type Ⅰ collagen; Col-3: type Ⅲ collagen; α-SMA: α-smooth muscle actin; ERK1/2: extracellular signal-regulated kinase 1/2. Created with BioRender.com.

Fig. 8

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Cardiac fibrosis often accompanies various cardiac pathological conditions and may initially be an adaptive activation of cardiac maladaptive processes (Berk et al., 2007; Frangogiannis, 2019; Umbarkar et al., 2021). However, when this adaptive activation becomes inappropriate, excessive, or unrestrained, cardiac fibrosis can contribute to cardiac dysfunction, eventually leading to heart failure (Wynn, 2008; Gupta et al., 2021; Umbarkar et al., 2021). TGF-β1 is a key factor of collagen production and cell proliferation in the heart, and induces fibroblasts to differentiate into myofibroblasts, thereby promoting the transcription and translation of α-SMA (Scalise et al., 2021). We applied TGF-β1 in NMCFs to induce cardiac fibrosis in vitro. Under TGF-β1 stimulation, collagen production and cell proliferation were increased, and CRLF1 was up-regulated. These results indicated that NMCFs were undergoing fibrotic process, and this process was associated with an increase in the expression of CRLF1. The fibrotic process was further up-regulated by CRLF1 overexpression, and down-regulated by CRLF1 silencing. These results suggested that CRLF1 obviously participated in the regulation of cardiac fibrosis induced by TGF‍-‍‍‍β1. In addition, a change of CRLF1 expression alone in NMCFs could also influence the fibrotic process. In particular, the inhibition of the fibrotic process by CRLF1 silencing indicated that CRLF1 silencing could be used not only as a therapeutic strategy but also as a prevention method for cardiac fibrosis. Anti-fibrosis therapy can be a useful treatment for heart diseases (Roubille et al., 2014; Schuetze et al., 2014; Tuleta and Frangogiannis, 2021). Our findings provide strong support for CRLF1 becoming a novel prevention and remedy target for heart diseases.

Clarifying the regulation of collagen synthesis and degradation is the key to clarify the basis of the regulatory mechanism of cardiac fibrosis. In addition, Col-1 and Col-3 are the major structural components of the collagenous matrix in the heart. In particular, Col-1 accounts for about 85% to 90% of the matrix (Weber, 1989; Bashey et al., 1992; Jugdutt, 2003; Frangogiannis, 2017). In our research, the mRNA and protein levels of Col-1 and Col-3 were further increased by CRLF1 overexpression compared with the levels in the TGF-‍β1 stimulation group. Conversely, CRLF1 silencing inhibited the up-regulation of Col-1 and Col-3 mRNA expression and protein levels induced by TGF-β1. Activated fibroblasts and myofibroblasts are the main sources of matrix proteins (Umbarkar et al., 2021). In our study, α‍-SMA, a myofibroblast transformation indicator, showed the same trend as Col-1 and Col-3 when CRLF1 was overexpressed or silenced under TGF‍-‍β1 stimulation. Moreover, CRLF1 overexpression increased Col-1, Col-3, and α‍-SMA mRNA and protein levels in cardiac fibroblasts without TGF-β1 stimulation, while CRLF1 silencing showed the opposite effects. These results suggest that CRLF1 not only increases collagen production and myofibroblast transformation, but also functions in cardiac fibroblasts under pathological and physiological states. To confirm and extend our findings, further research in vivo needs to be carried out in the future. Although we have found that CRLF1 is increased in HCFs treated with TGF-β1, the function of CRLF1 in HCFs with or without TGF-‍β1 stimulation is yet to be elucidated. The wider use of human specimens in future studies would help to confirm the clinical potential of CRLF1 as a remedy for cardiac fibrosis.

We attempted to find a target for CRLF1 in cardiac fibroblasts. Previous studies in other organs or cells have shown that CRLF1 can activate the Janus-activated kinase (JAK)/signal transducer and activator of transcription (STAT), PI3K/Akt, and MAPK signal pathways via its membrane receptor (Looyenga et al., 2013; Yu et al., 2018). However, CRLF1 overexpression or silencing in NMCFs did not change the CRLF1 protein level in the cell supernatant, which suggested that CRLF1 might function in the cytoplasm via a new regulation mode. Using western blotting, ELISA, and the highly sensitive AlphaLISA methods, we found that CRLF1 could change the p-ERK1/2 level in NMCFs with or without TGF-β1 stimulation. Therefore, we chose to study the ERK1/2 signaling pathway as a downstream target of CRLF1, although many other signaling pathways could also regulate cardiac fibrosis (Chou et al., 2018; Magaye et al., 2020, 2021a, 2021b). To confirm the uniqueness of the ERK1/2 signaling pathway in the process of CRLF1 regulation, inhibitors of other pathways should be applied in the future to assess whether CRLF1 exerts its function also via these pathways. Fortunately, we found that CRLF1 overexpression no longer had a stimulatory effect in NMCFs when the ERK1/2 signaling pathway was inhibited. Therefore, we concluded that the ERK1/2 signaling pathway was the most likely downstream target of CRLF1 regulating cardiac fibrosis.

CRLF1 was up-regulated in NMCFs provoked by TGF-‍β1. However, the downstream signaling cascades of TGF-‍β1 are divided into SMAD-dependent and SMAD-independent pathways (Derynck and Zhang, 2003; Schmierer and Hill, 2007; Hu et al., 2018). TGF-β1 binds to its type I and type II cell surface receptors (TGFβRI and TGFβRII) to form heteromeric complexes to activate SMAD-dependent or SMAD-independent pathways (Horbelt et al., 2012; Meng et al., 2016; Hanna and Frangogiannis, 2019; Scalise et al., 2021). Therefore, we investigated which kind of molecule or signaling pathway was the most immediate upstream of CRLF1 in cardiac fibrosis. Firstly, we checked whether SMAD-dependent pathways were the upstream pathways or not by using SIS3, because SMAD3 could directly regulate transcription in the nucleus. After treating NMCFs with TGF-β1 and SIS3, we found that CRLF1 expression decreased. We also applied inhibitors of SMAD-independent pathways to clarify the upstream pathways of CRLF1. We separately treated NMCFs with different inhibitors under TGF-β1 stimulation. The CRLF1 mRNA and protein levels did not change after these inhibitors were used. Combining the above results, we concluded that CRLF1 expression was up-regulated in NMCFs under TGF-β1 stimulation mainly through the TGF-‍β1/SMAD signaling pathway. Interestingly, CRLF1 was up-regulated under TGF-‍β1 stimulation via a SMAD-dependent pathway, namely the TGF-‍β1/SMAD signaling pathway, but in turn, CRLF1 strengthened the effects of TGF‍-‍β1 by influencing the SMAD-independent ERK1/2 signaling pathway in NMCFs.

Based on the above studies, we have shed light on the role and mechanism of action of CRLF1 in cardiac fibrosis. However, there were some limitations in our study. Firstly, we observed that CRLF1 was up-regulated in a mouse heart fibrotic model induced by MI. Once we have ascertained the relationship between the level of CRLF1 and the degree of cardiac fibrosis, it would be useful to determine the most effective intervention time node. In addition, apart from TGF-‍β1, some inflammatory factors, such as interleukin (IL)‍-4, IL-6, and IL-13, could also activate the cardiac fibrosis pathway (Mantovani et al., 2005; Barron and Wynn, 2011; Kong et al., 2014; Zhao et al., 2021). In this study, we focused only on the effect of TGF-‍β1 on CRLF1 expression in vitro and ignored the effect of inflammatory factors. Whether inflammatory factors are upstream or downstream targets of CRLF1 needs to be clarified in the future. This will contribute to a comprehensive elucidation of the mechanism of CRLF1 in cardiac fibrosis. Secondly, by examining published single cell sequences (Tucker et al., 2020; Chaffin et al., 2022) and searching a human database (The Human Protein Atlas, https://www.‍proteinatlas.‍org) and mice database (https://tabula-muris.ds.czbiohub.‍org) (The Tabula Muris Consortium et al., 2018), we found that CRLF1 expressed mostly in cardiac fibroblasts, especially activated fibroblasts. Furthermore, we measured the expression of CRLF1 in the heart and found that CRLF1 was more highly expressed in NMCFs than in NMCMs and ECs. However, there are many cell types other than fibroblasts, cardiomyocytes, and ECs in heart. It may be useful to test CRLF1 expression patterns in more types of cells, such as immune cells, in future studies. This will be helpful to identify the target cells of CRLF1. Thirdly, we have verified that the ERK1/2 signaling pathway is the downstream target of CRLF1. RNA sequencing (RNA-seq) could be used as a reliable and accurate method to find out whether there are other downstream pathways affected by CRLF1. Fourthly, we did not investigate how CRLF1 interacted with the p-ERK1/2 protein. Protein co-immunoprecipitation experiments are needed to determine whether CRLF1 directly interacts with p-ERK1/2. Finally, we speculated that CRLF1 worked in the cell cytosol because we did not detect a change of CRLF1 after its overexpression or silencing in the cell supernatant using ELISA. Other more sensitive measurements may be needed to detect CRLF1 in the cell supernatant. Taken together, these intriguing questions merit further research.

4. Conclusions

CRLF1 is up-regulated in cardiac fibrosis and aggravates cardiac fibrosis by increasing the p-ERK1/2 protein level. The activation of the TGF-β1/SMAD signaling pathway in cardiac fibrosis can increase CRLF1 expression, and the up-regulation of CRLF1 in turn can strengthen collagen production and myofibroblast transformation by increasing the p-ERK1/2 level in the cytosol. These findings imply that CRLF1 has the potential to become a novel prevention and remedy target for cardiac fibrosis.

Materials and methods

Detailed methods are provided in the electronic supplementary materials of this paper.

Supplementary information

Materials and methods

Acknowledgments

This work was supported by the National Key Research and Development Project of China (No. 2018YFA0800404), the National Natural Science Foundation of China (Nos. 82100255 and 81970736), and the China Postdoctoral Science Foundation (Nos. 2021M691459 and 2022T150299).

Author contributions

Huijie ZHANG, Shenjian LUO, and Jin LI contributed to conception and designed this study. Zhi YANG and Ruxin CHEN carried out the experiments. Shenjian LUO performed the statistical analysis and revised the manuscript. Danming YOU, Fei TENG, and Wenhui LIU wrote the first manuscript version. Shenjian LUO, Zhi YANG, and Ruxin CHEN discussed and modified all sections of the manuscript. Youwen YUAN supervised the experiments. All authors have reviewed and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.

Compliance with ethics guidelines

Shenjian LUO, Zhi YANG, Ruxin CHEN, Danming YOU, Fei TENG, Youwen YUAN, Wenhui LIU, Jin LI, and Huijie ZHANG declare that they have no conflict of interest.

The animal experiments were carried out under the approval of the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (No. NFYY-2021-1047).

References

  1. Bacmeister L, Schwarzl M, Warnke S, et al. , 2019. Inflammation and fibrosis in murine models of heart failure. Basic Res Cardiol, 114(3): 19. 10.1007/s00395-019-0722-5 [DOI] [PubMed] [Google Scholar]
  2. Barron L, Wynn TA, 2011. Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages. Am J Physiol Gastrointest Liver Physiol, 300(5): G723-728. 10.1152/ajpgi.00414.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bashey RI, Martinez-Hernandez A, Jimenez SA, 1992. Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components. Circ Res, 70(5): 1006-1017. 10.1161/01.res.70.5.1006 [DOI] [PubMed] [Google Scholar]
  4. Berk BC, Fujiwara K, Lehoux S, 2007. ECM remodeling in hypertensive heart disease. J Clin Invest, 117(3): 568-575. 10.1172/JCI31044 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biernacka A, Cavalera M, Wang JH, et al. , 2015. Smad3 signaling promotes fibrosis while preserving cardiac and aortic geometry in obese diabetic mice. Circ Heart Fail, 8(4): 788-798. 10.1161/CIRCHEARTFAILURE.114.001963 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown RD, Ambler SK, Mitchell MD, et al. , 2005. The CARDIAC FIBROBLAST: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol, 45: 657-687. 10.1146/annurev.pharmtox.45.120403.095802 [DOI] [PubMed] [Google Scholar]
  7. Buers I, Persico I, Schöning L, et al. , 2020. Crisponi/cold-induced sweating syndrome: differential diagnosis, pathogenesis and treatment concepts. Clin Genet, 97(1): 209-221. 10.1111/cge.13639 [DOI] [PubMed] [Google Scholar]
  8. Busch A, Žarković M, Lowe C, et al. , 2017. Mutations in CRLF1 cause familial achalasia. Clin Genet, 92(1): ‍104-108. 10.1111/cge.12953 [DOI] [PubMed] [Google Scholar]
  9. Chaffin M, Papangeli I, Simonson B, et al. , 2022. Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy. Nature, 608(7921): 174-180. 10.1038/s41586-022-04817-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Childers RC, Sunyecz I, West TA, et al. , 2019. Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure. Am J Physiol Heart Circ Physiol, 316(3): H596-H608. 10.1152/ajpheart.00095.2018 [DOI] [PubMed] [Google Scholar]
  11. Chou CH, Hung CS, Liao CW, et al. , 2018. IL-6 trans-signalling contributes to aldosterone-induced cardiac fibrosis. Cardiovasc Res, 114(5): 690-702. 10.1093/cvr/cvy013 [DOI] [PubMed] [Google Scholar]
  12. Derynck R, Zhang YE, 2003. Smad-dependent and Smad-independent pathways in TGF-‍β family signalling. Nature, 425(6958): 577-584. 10.1038/nature02006 [DOI] [PubMed] [Google Scholar]
  13. Dewald O, Ren GF, Duerr GD, et al. , 2004. Of mice and dogs: species-specific differences in the inflammatory response following myocardial infarction. Am J Pathol, 164(2): 665-677. 10.1016/S0002-9440(10)63154-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Disertori M, Masè M, Ravelli F, 2017. Myocardial fibrosis predicts ventricular tachyarrhythmias. Trends Cardiovasc Med, 27(5): 363-372. 10.1016/j.tcm.2017.01.011 [DOI] [PubMed] [Google Scholar]
  15. Fernández-Alfonso MS, Ruilope LM, 2014. One step forward for serelaxin as a promising therapy in cardiac fibrosis. Hypertension, 64(2): 229-230. 10.1161/HYPERTENSIONAHA.114.03642 [DOI] [PubMed] [Google Scholar]
  16. Frangogiannis NG, 2017. The extracellular matrix in myocardial injury, repair, and remodeling. J Clin Invest, 127(5): 1600-1612. 10.1172/JCI87491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frangogiannis NG, 2019. Cardiac fibrosis: cell biological mechanisms, molecular pathways and therapeutic opportunities. Mol Aspects Med, 65: 70-99. 10.1016/j.mam.2018.07.001 [DOI] [PubMed] [Google Scholar]
  18. Frangogiannis NG, 2020. Transforming growth factor-‍β in tissue fibrosis. J Exp Med, 217(3): e20190103. 10.1084/jem.20190103 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gupta S, Ge Y, Singh A, et al. , 2021. Multimodality imaging assessment of myocardial fibrosis. JACC Cardiovasc Imaging, 14(12): 2457-2469. 10.1016/j.jcmg.2021.01.027 [DOI] [PubMed] [Google Scholar]
  20. Hanna A, Frangogiannis NG, 2019. The role of the TGF‍-‍β superfamily in myocardial infarction. Front Cardiovasc Med, 6: 140. 10.3389/fcvm.2019.00140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Herholz J, Meloni A, Marongiu M, et al. , 2011. Differential secretion of the mutated protein is a major component affecting phenotypic severity in CRLF1-associated disorders. Eur J Hum Genet, 19(5): 525-533. 10.1038/ejhg.2010.253 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horbelt D, Denkis A, Knaus P, 2012. A portrait of transforming growth factor β superfamily signalling: background matters. Int J Biochem Cell Biol, 44(3): 469-474. 10.1016/j.biocel.2011.12.013 [DOI] [PubMed] [Google Scholar]
  23. Hu HH, Chen DQ, Wang YN, et al. , 2018. New insights into TGF‍-‍β/Smad signaling in tissue fibrosis. Chem Biol Interact, 292: 76-83. 10.1016/j.cbi.2018.07.008 [DOI] [PubMed] [Google Scholar]
  24. Janicki JS, Brower GL, 2002. The role of myocardial fibrillar collagen in ventricular remodeling and function. J Card Fail, 8(6): S319-S325. 10.1054/jcaf.2002.129260 [DOI] [PubMed] [Google Scholar]
  25. Jugdutt BI, 2003. Ventricular remodeling after infarction and the extracellular collagen matrix: when is enough enough? Circulation, 108(11): 1395-1403. 10.1161/01.CIR.0000085658.98621.49 [DOI] [PubMed] [Google Scholar]
  26. Koenig AL, Shchukina I, Amrute J, et al. , 2022. Single-cell transcriptomics reveals cell-type-specific diversification in human heart failure. Nat Cardiovasc Res, 1(3): 263-280. 10.1038/s44161-022-00028-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kong P, Christia P, Frangogiannis NG, 2014. The pathogenesis of cardiac fibrosis. Cell Mol Life Sci, 71(4): 549-574. 10.1007/s00018-013-1349-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lattanzio FA Jr, Tiangco D, Osgood C, et al. , 2005. Cocaine increases intracellular calcium and reactive oxygen species, depolarizes mitochondria, and activates genes associated with heart failure and remodeling. Cardiovasc Toxicol, 5(4): 377-389. 10.1385/ct:5:4:377 [DOI] [PubMed] [Google Scholar]
  29. Lei H, Wu D, Wang JY, et al. , 2015. C1q/tumor necrosis factor-related protein-6 attenuates post-infarct cardiac fibrosis by targeting RhoA/MRTF-A pathway and inhibiting myofibroblast differentiation. Basic Res Cardiol, 110(4): 35. 10.1007/s00395-015-0492-7 [DOI] [PubMed] [Google Scholar]
  30. Li RK, Li GM, Mickle DAG, et al. , 1997. Overexpression of transforming growth factor-‍β1 and insulin-like growth factor-I in patients with idiopathic hypertrophic cardiomyopathy. Circulation, 96(3): ‍874-881. 10.1161/01.cir.96.3.874 [DOI] [PubMed] [Google Scholar]
  31. Li YF, Xun J, Wang BT, et al. , 2021. miR-3065-3p promotes stemness and metastasis by targeting CRLF1 in colorectal cancer. J Transl Med, 19: 429. 10.1186/s12967-021-03102-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lodyga M, Hinz B, 2020. TGF‍-‍β1—a truly transforming growth factor in fibrosis and immunity. Semin Cell Dev Biol, 101: 123-139. 10.1016/j.semcdb.2019.12.010 [DOI] [PubMed] [Google Scholar]
  33. Looyenga BD, Resau J, MacKeigan JP, 2013. Cytokine receptor-like factor 1 (CRLF1) protects against 6-hydroxydopamine toxicity independent of the gp130/JAK signaling pathway. PLoS ONE, 8(6): e66548. 10.1371/journal.pone.0066548 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. López B, Ravassa S, Moreno MU, et al. , 2021. Diffuse myocardial fibrosis: mechanisms, diagnosis and therapeutic approaches. Nat Rev Cardiol, 18(7): 479-498. 10.1038/s41569-020-00504-1 [DOI] [PubMed] [Google Scholar]
  35. Magaye RR, Savira F, Hua Y, et al. , 2020. Exogenous dihydrosphingosine 1 phosphate mediates collagen synthesis in cardiac fibroblasts through JAK/STAT signalling and regulation of TIMP1. Cell Signal, 72: 109629. 10.1016/j.cellsig.2020.109629 [DOI] [PubMed] [Google Scholar]
  36. Magaye RR, Savira F, Hua Y, et al. , 2021a. Attenuating PI3K/Akt-mTOR pathway reduces dihydrosphingosine 1 phosphate mediated collagen synthesis and hypertrophy in primary cardiac cells. Int J Biochem Cell Biol, 134: 105952. 10.1016/j.biocel.2021.105952 [DOI] [PubMed] [Google Scholar]
  37. Magaye RR, Savira F, Xiong X, et al. , 2021b. Dihydrosphingosine driven enrichment of sphingolipids attenuates TGFβ induced collagen synthesis in cardiac fibroblasts. IJC Heart Vasc, 35: 100837. 10.1016/j.ijcha.2021.100837 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mantovani A, Sica A, Locati M, 2005. Macrophage polarization comes of age. Immunity, 23(4): 344-346. 10.1016/j.immuni.2005.10.001 [DOI] [PubMed] [Google Scholar]
  39. Meng JX, Qin YY, Chen JZ, et al. , 2020. Treatment of hypertensive heart disease by targeting Smad3 signaling in mice. Mol Ther Methods Clin Dev, 18: 791-802. 10.1016/j.omtm.2020.08.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Meng XM, Nikolic-Paterson DJ, Lan HY, 2016. TGF‍-‍β: the master regulator of fibrosis. Nat Rev Nephrol, 12(6): 325-338. 10.1038/nrneph.2016.48 [DOI] [PubMed] [Google Scholar]
  41. Nagaraju CK, Robinson EL, Abdesselem M, et al. , 2019. Myofibroblast phenotype and reversibility of fibrosis in patients with end-stage heart failure. J Am Coll Cardiol, 73(18): 2267-2282. 10.1016/j.jacc.2019.02.049 [DOI] [PubMed] [Google Scholar]
  42. Nguyen MN, Kiriazis H, Gao XM, et al. , 2017. Cardiac fibrosis and arrhythmogenesis. Compr Physiol, 7(3): 1009-1049. 10.1002/cphy.c160046 [DOI] [PubMed] [Google Scholar]
  43. Nguyen TP, Qu ZL, Weiss JN, 2014. Cardiac fibrosis and arrhythmogenesis: the road to repair is paved with perils. J Mol Cell Cardiol, 70: 83-91. 10.1016/j.yjmcc.2013.10.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perestrelo AR, Silva AC, Oliver-De La Cruz J, et al. , 2021. Multiscale analysis of extracellular matrix remodeling in the failing heart. Circ Res, 128(1): 24-38. 10.1161/CIRCRESAHA.120.317685 [DOI] [PubMed] [Google Scholar]
  45. Roubille F, Busseuil D, Merlet N, et al. , 2014. Investigational drugs targeting cardiac fibrosis. Expert Rev Cardiovasc Ther, 12(1): 111-125. 10.1586/14779072.2013.839942 [DOI] [PubMed] [Google Scholar]
  46. Scalise RFM, de Sarro R, Caracciolo A, et al. , 2021. Fibrosis after myocardial infarction: an overview on cellular processes, molecular pathways, clinical evaluation and prognostic value. Med Sci (Basel), 9(1): 16. 10.3390/medsci9010016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmierer B, Hill CS, 2007. TGFβ‍-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol, 8(12): 970-982. 10.1038/nrm2297 [DOI] [PubMed] [Google Scholar]
  48. Schuetze KB, McKinsey TA, Long CS, 2014. Targeting cardiac fibroblasts to treat fibrosis of the heart: focus on HDACs. J Mol Cell Cardiol, 70: 100-107. 10.1016/j.yjmcc.2014.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shen Y, Teng YS, Lv YP, et al. , 2020. PD-1 does not mark tumor-infiltrating CD8+ T cell dysfunction in human gastric cancer. J Immunother Cancer, 8(2): e000422. 10.1136/jitc-2019-000422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shirwany A, Weber KT, 2006. Extracellular matrix remodeling in hypertensive heart disease. J Am Coll Cardiol, 48(1): 97-98. 10.1016/j.jacc.2006.04.004 [DOI] [PubMed] [Google Scholar]
  51. Singh R, Kaundal RK, Zhao BY, et al. , 2021. Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling. Pharmacol Res, 167: 105414. 10.1016/j.phrs.2020.105414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stefanovic L, Stefanovic B, 2012. Role of cytokine receptor-like factor 1 in hepatic stellate cells and fibrosis. World J Hepatol, 4(12): 356-364. 10.4254/wjh.v4.i12.356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stratton MS, Bagchi RA, Felisbino MB, et al. , 2019. Dynamic chromatin targeting of BRD4 stimulates cardiac fibroblast activation. Circ Res, 125(7): 662-677. 10.1161/CIRCRESAHA.119.315125 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sundararaj K, Pleasant DL, Moschella PC, et al. , 2016. mTOR complexes repress hypertrophic agonist-stimulated expression of connective tissue growth factor in adult cardiac muscle cells. J Cardiovasc Pharmacol, 67(2): 110-120. 10.1097/FJC.0000000000000322 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. The Tabula Muris Consortium, Coordination Overall, Coordination Logistical, et al. , 2018. Single-cell transcriptomics of 20 mouse organs creates a Tabula muris . Nature, 562(7727): 367-372. 10.1038/s41586-018-0590-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tu YG, Wu TQ, Dai AZ, et al. , 2011. Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-‍β in diabetic nephropathy. Kidney Int, 79(2): 199-209. 10.1038/ki.2010.374 [DOI] [PubMed] [Google Scholar]
  57. Tucker NR, Chaffin M, Fleming SJ, et al. , 2020. Transcriptional and cellular diversity of the human heart. Circulation, 142(5): 466-482. 10.1161/CIRCULATIONAHA.119.045401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tuleta I, Frangogiannis NG, 2021. Fibrosis of the diabetic heart: clinical significance, molecular mechanisms, and therapeutic opportunities. Adv Drug Deliv Rev, 176: 113904. 10.1016/j.addr.2021.113904 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Umbarkar P, Ejantkar S, Tousif S, et al. , 2021. Mechanisms of fibroblast activation and myocardial fibrosis: lessons learned from FB-specific conditional mouse models. Cells, 10(9): 2412. 10.3390/cells10092412 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang JC, Zhao HK, Zheng L, et al. , 2021. FGF19/SOCE/NFATc2 signaling circuit facilitates the self-renewal of liver cancer stem cells. Theranostics, 11(10): 5045-5060. 10.7150/thno.56369 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang XW, Ma JP, Zhang SS, et al. , 2021. G protein-coupled estrogen receptor 30 reduces transverse aortic constriction-induced myocardial fibrosis in aged female mice by inhibiting the ERK1/2-MMP-9 signaling pathway. Front Pharmacol, 12: 731609. 10.3389/fphar.2021.731609 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wang ZY, Shen JK, Sun W, et al. , 2019. Antitumor activity of raddeanin A is mediated by jun amino-terminal kinase activation and signal transducer and activator of transcription 3 inhibition in human osteosarcoma. Cancer Sci, 110(5): 1746-1759. 10.1111/cas.14008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weber KT, 1989. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol, 13(7): 1637-1652. 10.1016/0735-1097(89)90360-4 [DOI] [PubMed] [Google Scholar]
  64. Wen JX, Li MJ, Zhang WW, et al. , 2022. Role of higenamine in heart diseases: a mini-review. Front Pharmacol, 12: 798495. 10.3389/fphar.2021.798495 [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Westermann D, van Linthout S, Dhayat S, et al. , 2007. Tumor necrosis factor-alpha antagonism protects from myocardial inflammation and fibrosis in experimental diabetic cardiomyopathy. Basic Res Cardiol, 102(6): 500-507. 10.1007/s00395-007-0673-0 [DOI] [PubMed] [Google Scholar]
  66. Wynn TA, 2008. Cellular and molecular mechanisms of fibrosis. J Pathol, 214(2): 199-210. 10.1002/path.2277 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Xia Y, Dobaczewski M, Gonzalez-Quesada C, et al. , 2011. Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism. Hypertension, 58(5): ‍902-911. 10.1161/HYPERTENSIONAHA.111.175323 [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yamaguchi O, Watanabe T, Nishida K, et al. , 2004. Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. J Clin Invest, 114(7): 937-943. 10.1172/JCI20317 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Yu P, Ma SC, Dai XC, et al. , 2020. Elabela alleviates myocardial ischemia reperfusion-induced apoptosis, fibrosis and mitochondrial dysfunction through PI3K/AKT signaling. Am J Transl Res, 12(8): 4467-4477. [PMC free article] [PubMed] [Google Scholar]
  70. Yu ST, Zhong Q, Chen RH, et al. , 2018. CRLF1 promotes malignant phenotypes of papillary thyroid carcinoma by activating the MAPK/ERK and PI3K/AKT pathways. Cell Death Dis, 9(3): 371. 10.1038/s41419-018-0352-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Yu ST, Sun BH, Ge JN, et al. , 2020. CRLF1-MYH9 interaction regulates proliferation and metastasis of papillary thyroid carcinoma through the ERK/ETV4 axis. Front Endocrinol (Lausanne), 11: 535. 10.3389/fendo.2020.00535 [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zhao K, Zhang J, Xu TH, et al. , 2021. Low‐intensity pulsed ultrasound ameliorates angiotensin II-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(10): 818-838. 10.1631/jzus.B2100130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zheng ZY, Ao X, Li P, et al. , 2020. CRLF1 is a key regulator in the ligamentum flavum hypertrophy. Front Cell Dev Biol, 8: 858. 10.3389/fcell.2020.00858 [DOI] [PMC free article] [PubMed] [Google Scholar]

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