The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB.

We previously showed that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, we set out to elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. We first observed that the apoptotic protein p53 and the anti-apoptotic protein NFκB are simultaneously induced in MyoFBs. When the expression level of p53 was suppressed using a siRNA, CCN5 did not induce apoptosis in MyoFBs. By contrast, when NFκB signaling was inhibited using IKK VII, an IκB inhibitor, MyoFBs underwent apoptosis even in the absence of CCN5. SMAD7 is one of the downstream targets of CCN5 and it was previously shown to potentiate apoptosis in epithelial cells through inhibition of NFκB. In accordance with these reports, when the expression of SMAD7 was suppressed using a siRNA, NFκB signaling was enhanced, and CCN5 did not induce apoptosis. Lastly, we used a luciferase reporter construct to show that CCN5 positively regulated SMAD7 expression at the transcriptional level. Collectively, our data suggest that a delicate balance between the two mutually antagonistic proteins p53 and NFκB is maintained for MyoFBs to survive, and CCN5 tips the balance in favor of the apoptotic protein p53. This study provides insight into the anti-fibrotic activity of CCN5 during the regression of CF.

Introduction

Heart failure (HF) remains a leading cause of mortality and morbidity worldwide. It is a chronic disease that is associated with adverse cardiac outcome [1, 2]. HF is often initiated after myocardial injuries that are caused by a variety of events, including myocardial infarction, hypertension, heart valve disease, and myocarditis, and is usually accompanied by inflammation and fibrosis [1-3]. Cardiac fibroblasts (FBs) play an important role in maintaining the function of cardiomyocytes through regulation of turnover of the extracellular matrix (ECM) proteins and communication with cardiomyocytes [4-6]. In addition to these supportive roles, FBs play a primary role in the progression of HF. Upon pathological stimuli, FBs undergo proliferation and trans-differentiation to myofibroblasts (MyoFBs) [7-9]. MyoFBs secret excessive amounts of ECM proteins, which are responsible for the increased ventricular stiffness and diastolic dysfunction observed in HF patients. Therefore, preventing FB trans-differentiation and/or reversing the differentiation of MyoFBs could be efficient strategies to treat HF. Effective anti-fibrotic therapies are currently unavailable.

The CCN family (CCN1-6) of matricellular proteins are associated with diverse cellular processes including fibrosis, angiogenesis, cell differentiation, and wound repair [10, 11]. Our group previously showed that CCN5, also known as WNT1-inducible signaling pathway protein 2 (WISP-2), inhibits cardiac fibrosis (CF) through inhibition of endothelial–mesenchymal transition and trans-differentiation of FBs to MyoFBs [12-16]. More strikingly, CCN5 reverses pre-established CF through induction of apoptosis in MyoFBs but not in cardiomyocytes or FBs [14]. However, the molecular mechanisms underlying the selective pro-apoptotic activity of CCN5 are largely unknown.

In this study, we attempted to further elucidate the role of CCN5 in MyoFB-specific apoptosis. FBs were freshly isolated from rat hearts, and MyoFBs were obtained by trans-differentiating by treatment with TGF-β. Our results suggest that a balance between pro-apoptotic p53 and anti-apoptotic NFκB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFκB. This work provides insight into the role CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies.

Materials and methods

Ethics statement

Animal experiments using Sprague-Dawley rats were granted by approval of the Institutional Animal Care and Use Committee (IACUC) of Gwangju Institute of Sciences and Technology (GIST-2020-056). The investigation conforms to the Guide for the Care and Use of Laboratory Anials published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Primary cardiac fibroblasts isolation procedures were performed under inhalational anesthesia with isoflurane gas (N2O:O2/70%:30%), and all efforts were made to minimize suffering. The eight-week-old male Sprague-Dawley rats were obtained from Daehan Biolink (Korea) and used for all isolation experiments.

Cell culture

Adult cardiac FBs were isolated from the hearts of male 300–350 gram Sprague Dawley rats. The heart was quickly removed from the chest, and the aorta was retrogradely perfused at 37°C for 3 min with calcium Tyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES pH 7.4, 10 mM 2, 3-butanedion monoxime, and 5 mM taurine) gassed with 100% O2. Enzymatic digestion was initiated by adding collagenase type II (300 U/mL; Worthington) and hyaluronidase (0.1 mg/mL; Worthington) to the perfusion solution. When the heart became swollen after 45 min of digestion, the ventricles were removed, cut into several chunks, added to a BSA solution, and physically separated in a shaker (60–70 rpm for 5 min at 37°C). The supernatants containing the fibroblasts were filtered through a cell strainer (100 μm pore size; BD Falcon) and gently centrifuged at 500 rpm for 3 min. The resultant supernatant was transferred into a new tube and centrifuged at 1,000 rpm for 10 min. FBs were suspended and cultured in DMEM (HyClone) supplemented with penicillin, streptomycin, and 10% FBS (HyClone). Cells were ready to use for further experiments 48 hours after seeding. Trans-differentiation of FBs was induced by treatment with 10 ng/mL TGF-β (Pepprotech, 10035B) [17] or 100 nM AngII (Sigma-Aldrich, #4474-91-3) for 48 hours. Both TGF-β and AngII are known to be potent profibortic molecule. An IKK inhibitor, IKK VII, was obtained from Millipore (#401486) and used at a concentration of 1 μM for 48 hours.

Conditioned media

COS-7 cells were cultured in DMEM (HyClone) supplemented with penicillin, streptomycin, and 10% FBS (HyClone). We synthesized a gene encoding CCN5 fused with a HA tag at its carboxy terminus and referred to it as CCN5-HA. 12 mL culture of COS-7 cells were transformed with 9 μg of pcDNA-CCN5-HA. The media were collected after 24 hours and referred to as CCN5-containing conditioned media (CM-CCN5). Control conditioned media (CM-Con) was similarly obtained but without the transformation with pcDNA-CCN5-HA. The concentration of CCN5-HA was typically 200~500 ng/mL when detected and calculated using anti-HA antibody (Roche, #11867423001). Blotting with an anti GAPDH antibody confirmed that the CM was not contaminated with cellular proteins.

siRNA knockdown analysis

Cardiac FBs were transfected with 30 nM of siRNA targeting rat p53 (Bioneer, 1774203), 25 nM of On-TARGET plus Rat Smad7 siRNA (Dharmacon, L-093737-02), or a control siRNA (Bioneer, SN-1002) using DharmaFECT Transfection reagent (Dharmacon, T-2001-02). Cells were treated with CM-CCN5 or CM-Con 48 hr after transfection.

Immunostaining

Cardiac FBs were seeded at 15,000 cells per well on 16 mm cover slips. When the cells reached 80% confluence, they were transfected with p53 siRNA, Smad7 siRNA, or a control siRNA. The cells were then treated with CM-CCN5 for 48 hours, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% BSA. Cells were incubated with an antibody against p53 (Cell Signaling Technology) or NFκB p65 (Santa Cruz), and were then incubated with secondary antibodies labelled with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Nuclei were stained with DAPI. Immunofluorescence was analyzed under a microscope equipped with a 60X objective lens and the appropriate filters (Olympus).

Terminal transferase dUTP nick end-labeling assay (TUNEL)

Cardiac FBs were knocked-out with the p53 and Smad7 siRNAs or a control siRNA. After treatment with CM-CCN5 or CM-Con, cells on glass coverslips were stained using the DeadEnd Fluorometric TUNEL kit (Promega). The presence of nicks in the DNA was confirmed using terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the addition of labeled dUTPs (6). TUNEL-positive cells were captured using a Fluoview FV 1000 confocal laser scanning microscope.

Western blotting

RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100) with Protease Inhibitor Cocktail Set III (Merck Millipore, 535140) was used to solubilize cells. The cell lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23227), and concentrations were normalized against bovine serum albumin. The proteins from the quantified cell lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, IPVH00010). The transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against α-SMA (Sigma-Aldrich, A5228), SMAD7 (Invitrogen), caspases (Antibody Sampler Kit, Cell Signaling), phospho–NFκB p65 (Ser 536) (Santa Cruz Biotechnology), NFκB p65 (Santa Cruz Biotechnology), cleaved PARP, PARP, phospho-IκBα, or IκBα (Cell Signaling Technology) overnight at 4°C. After washing the blots with Tris buffered saline (TBS) with 0.1% Tween 20, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (Thermo Scientific, 31430) and washed again. The protein band signal was detected using a chemiluminescence solution (Dogen, DG-WP100). GAPDH was used as a normalizing control to validate the expression levels of the target proteins.

Smad7 reporter gene assay

Rat MyoFBs were plated at 3x105 cells/well in 6-well culture plates and transfected with the Gluc-On Promoter reporter clone human Smad7 (GeneCopoeia; HMRM377850-PG04), pcDNA3.0-hCCN5, or an empty pcDNA3.0 vector using Lipofectamine 2000 (Invitrogen). After 48 hours, cell culture medium was collected, and luciferase assay signaling detection was performed using the Secrete-Pair Gaussia Luminescence Assay Kit (GeneCopoeia; LF061).

Statistical analysis

Student’s t-test and One-Way Analysis of Variance (ANOVA) were used for statistical analysis to determine the significance of the data. An asterisk (*P<0.05) or a double asterisk (**P<0.01), (***P<0.001) indicate significant probability.

Results

The p53 and NFκB signaling pathways are up-regulated in MyoFBs

Freshly prepared rat cardiac FBs were trans-differentiated to MyoFBs by treating them with the pro-fibrotic cytokine TGF-β for 48 hours. Western blotting of the cell extracts revealed that the protein level of p53 and the phosphorylation levels of p53 at serine 15 and serine 392 were elevated in MyoFBs compared to FBs. The protein and phosphorylation levels for NFκB p65 were also elevated in MyoFBs compared to FBs (Fig 1A). These data suggest that a pro-apoptotic (p53) and an anti-apoptotic (NFκB) signal are simultaneously elevated during trans-differentiation of FBs and that they remain balanced in MyoFBs. To test whether this phenomenon is specific to TGF-β–mediated differentiation, FB trans-differentiation was induced by the treatment with another pro-fibrotic cytokine angiotensin II (AngII). Western blotting revealed that similar patterns of up-regulation for both p53 and NFκB were induced in the trans-differentiated MyoFBs (Fig 1B). Freshly prepared FBs undergo trans-differentiation over the course of cell passaging even without any exogenous pro-fibrotic stimuli. Under our experimental conditions, we found that trans-differentiation of FBs was indeed induced over the course of passaging. The protein levels of p53 and NFκB concomitantly increased over the course of cell passaging (Fig 1C).

Fig 1

The p53 and NFκB signaling pathways are up-regulated in MyoFBs.

Freshly prepared FBs were treated with (A) 10 ng/mL TGF-β or (B) 100 nM AngII, or (C) were subcultured. Cells lysates were then used for western blotting analysis. The expression levels of p53, phosphorylated p53 (p-p53 S15 or p-p53 S392), NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), vimentin, and α-smooth muscle actin (α-SMA) were monitored by western blotting. GAPDH was used as a loading control. For each group, three independent cultures were prepared and treated (n = 3).

These data indicate that p53 and NFκB are simultaneously induced in MyoFBs compared to FBs, and this phenomenon is associated with trans-differentiation of FBs under various circumstances.

p53 is involved in CCN5-mediated MyoFB apoptosis

To examine the role of p53 in MyoFBs, we utilized a siRNA that is designed to target p53 transcripts (p53 siRNA). MyoFBs were treated with either a control siRNA or the p53 siRNA, and were incubated in either control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for 48 hours. Preparation of the conditioned media are described in the Materials and Methods section. Western blotting was then performed to examine the effects. Consistent with the data shown in Fig 1, the expression level of p53 increased and was further elevated when the MyoFBs were incubated in CM-CCN5. The protein levels of the executioner caspases, caspase 3 and caspase 7, and the levels of cleavage of these caspases were elevated after the cells were cultured in CM-CCN5. In addition, the cleavage of poly (ADP-ribose) polymerase (PARP-1) also increased in cells incubated in CM-CCN5. These data indicate that CCN5 induces apoptosis in MyoFBs, which is consistent with our previous results [14] (Fig 2A). Pre-incubation with the p53 siRNA blocked the expression of p53 in MyoFBs cultured either in CM-Con or CM-CCN5, and concomitantly blocked the expression and cleavage of caspase 3 and caspase 7 and the cleavage of PARP-1 (Fig 2A). TUNEL assay and immunostaining for p53 were performed simultaneously. In line with the western blotting results, incubation in CM-CCN5 induced the appearance of TUNEL-positive apoptotic cells, which was significantly blocked when the cells were pre-incubated with the p53 siRNA (Fig 2B).

Fig 2

p53 is involved in CCN5-mediated apoptosis of MyoFBs.

Freshly prepared FBs were transfected with 30 nM p53 siRNA and trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours. (A) Cell lysates were then used for western blotting analysis of p53, caspase 3 (Cas3), cleaved caspase 3 (c-Cas3), caspase 7 (Cas7), cleaved caspase 7 (c-Cas7), Poly (ADP-ribose) polymerase (PARP), and cleaved PARP (cPARP). GAPDH was used as a loading control. (B) Cells were co-stained with TUNEL (green) and p53 (red). Hoechst dye was used for nuclear staining. TUNEL-positive apoptotic cells were counted and plotted. Scale bar is 50 μm. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01.

These results indicate that CCN5 induces apoptosis in MyoFBs through the activity of p53, a pro-apoptotic protein.

NFκB protects MyoFBs from apoptosis

We previously showed that when cells are treated with CCN5, NFκB is excluded from the nucleus [14], where it acts as an anti-apoptotic factor. Therefore, we examined the role of NFκB in MyoFBs using IKK VII, a selective inhibitor of IκB kinase. Treatment with IKK VII completely abolished the phosphorylation of IκBα and consequently diminished the phosphorylation level and the expression level of NFκB p65. This IKK VII-mediated inhibition of NFκB signaling was associated with activation of caspases 3 and 7 and cleavage of PARP-1 even when the MyoFBs were cultured in CM-Con (Fig 3A). TUNEL assay further confirmed that IKK VII induced MyoFB apoptosis without incubation in CM-CCN5. Co-treatment with IKK VII and CM-CCN5 did not synergistically enhance the level of apoptosis (Fig 3B).

Fig 3

NFκB protects MyoFBs from apoptosis.

Freshly prepared FBs were trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours in the presence or absence of 1 μM IKK VII, a selective IκB kinase inhibitor. (A) Cell lysates were used for western blotting analysis of NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), IκB, phosphorylated IκB (p-IκB), caspase 3 (Cas3), cleaved caspase 3 (c-Cas3), caspase 7 (Cas7), cleaved caspase 7 (c-Cas7), Poly (ADP-ribose) polymerase (PARP), and cleaved PARP (cPARP). GAPDH was used as a loading control. (B) Cells were co-stained with TUNEL (green) and p53 (red). Hoechst dye was used for nuclear staining. TUNEL-positive apoptotic cells were counted and plotted. Scale bar is 50 μm. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01.

These data suggest that NFκB plays a critical role in preventing apoptosis in MyoFBs and that CCN5 induces apoptosis through inhibition of the NFκB signaling pathway. These results and those from the previous section collectively suggest that a delicate balance between p53 and NFκB maintains MyoFB survival, and that CCN5 tips this balance over in favor of p53-induced apoptosis through inhibition of NFκB.

CCN5 inhibits NFκB signaling through elevation of SMAD7

We previously observed that CCN5 inhibits the TGFβ-SMAD signaling pathway by elevating the expression level of SMAD7, an inhibitory SMAD [13, 14]. Interestingly, SMAD7 potentiates apoptosis in epithelial cells through inhibition of NFκB [18, 19]. Therefore, we hypothesized that SMAD7 is involved in the observed CCN5-mediated inhibition of NFκB. Western blotting analysis revealed that the expression level of SMAD7 robustly increased after cells were cultured in CM-CCN5. The levels of p53 and phosphorylated p53 increased, and the levels of phosphorylated IκB and NFκB p65 decreased. Pre-incubating the MyoFBs with a siRNA for SMAD7 (SMAD7 siRNA) completely blocked the CCN5-mediated increase in the level of SMAD7 and eliminated the effects of CCN5 on p53 and NFκB. In addition, pre-incubation with the SMAD7 siRNA inhibited the cleavage of caspases 3 and 7 and PARP-1 (Fig 4A). TUNEL assay also revealed that pre-incubation with the SMAD7 siRNA inhibited CCN5-mediated apoptosis of MyoFBs, whereas the control siRNA exhibited no effects (Fig 4B).

Fig 4

CCN5 inhibits NFκB signaling through elevation of SMAD7 levels.

Freshly prepared FBs were transfected with 25 nM SMAD7 siRNA and were trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours. (A) Cell lysates were used for western blotting analysis of SMAD7, p53, phosphorylated p53 (p-p53 S15 or p-p53 S392), NFκB p65, phosphorylated NFκB p65 (p-NFκB p65), IκB, phosphorylated IκB (p-IκB), caspase 3 (Cas3), cleaved caspase 3 (c-Cas3), caspase 7 (Cas7), cleaved caspase 7 (c-Cas7), Poly (ADP-ribose) polymerase (PARP), and cleaved PARP (cPARP). GAPDH was used as a loading control. (B) Cells were co-stained with TUNEL (green) and p53 (red). Hoechst dye was used for nuclear staining. TUNEL-positive apoptotic cells were counted and plotted. Scale bar is 50 μm. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01.

These data suggest that SMAD7 plays a critical role in CCN5-mediated MyoFB apoptosis.

CCN5 regulates the transcription of SMAD7

CCN5 acts as a transcriptional co-repressor and regulates the expression of TGFβ receptor II [16]. We hypothesized that CCN5 is involved in the transcriptional regulation of other genes, including SMAD7. Quantification of western blotting results showed that after incubating the MyoFBs in CM-CCN5, the expression level of SMAD7 increased approximately 2.5-fold (Fig 5A). Quantitative RT-PCR also revealed that culturing cells in CM-CCN5 caused the level of SMAD7 transcripts to increase approximately 3-fold (Fig 5B). We generated a reporter plasmid that drives the expression of secreted luciferase under the control of the SMAD7 enhancer and promoter regions. This reporter plasmid was transformed into MyoFBs. Co-transformation with CCN5-expressing plasmids significantly increased the activity of luciferase in the culture medium. Treatment with TGF-β, which served as a positive control, also increased the activity of luciferase to a similar extent.

Fig 5

CCN5 regulates the transcription of SMAD7.

Freshly prepared FBs were transfected with 25 nM SMAD7 siRNA and were trans-differentiated to MyoFBs by treating them with 10 ng/mL TGF-β for 48 hours. MyoFBs were then cultured in control conditioned media (CM-Con) or CCN5-containing conditioned media (CM-CCN5) for a further 48 hours. (A) Cell lysates were used for western blotting analysis of SMAD7. (B) Total RNA was prepared from cell lysates and used for qRT-PCR analysis. (C) A reporter construct for monitoring transcription from the SMAD7 promoter was constructed. This reporter construct and CCN5-expressing plasmids were co-transformed into MyoFBs. Luciferase activity was measured 48 hours after transformation. The result is the ratio of luminescence intensities (RLU, Relative light Unit) of the Gluc over SEAP (S). Compare the normalized Gluc activity (RLU/S) of all samples. TGF-β was used as a positive control, and empty plasmid was used as a negative control. For each group, three independent cultures were prepared and treated (n = 3). *P<0.05, **P<0.01, ***p<0.001.

These data suggest that CCN5 directly regulates the expression of SMAD7 at the transcriptional level.

Discussion

CF is associated with diverse cardiac conditions including myocardial infarction, hypertensive heart diseases, and diabetic cardiomyopathy. CF initially provides a protective mechanism with proper ECM protein deposition, which is beneficial for wound healing and tissue regeneration. However, when uncontrolled and sustained, CF causes excessive accumulation of ECM proteins and eventually leads to further cardiac complications such as diastolic dysfunction and arrhythmia [20-23]. Therefore, CF is one of the biggest concerns in the cardiovascular research community.

We previously found that the matricellular protein CCN5 inhibits CF through blocking the TGF-β–SMAD signaling pathway [13]. More strikingly, we found that CCN5 reverses pre-established CF through inducing apoptosis in MyoFBs but not in cardiomyocytes or FBs [14]. CCN5 accelerates the removal of MyoFBs through inducing the intrinsic pathway of apoptosis [14]. NFκB is involved in fibrosis in various organs [24-26] and it also possesses an anti-apoptotic activity in various contexts [27-30]. We previously showed that NFκB is involved in the regulation of trans-differentiation and apoptosis in MyoFBs [14]

In this study, we showed that NFκB and the pro-apoptotic protein p53 are up-regulated in MyoFBs that are generated in vitro in diverse ways (Fig 1). NFκB has dual roles in fibrosis, namely, (i) inducing fibrosis through activation of the TGF-β signaling pathway [24-26], and (ii) keeping MyoFBs viable though inhibition of apoptosis (this study). We showed that CCN5 induces p53-mediated apoptosis in MyoFBs (Fig 2) through negatively regulating NFκB (Fig 3). It is intriguing to note that a thin line of balance between pro-apoptotic and anti-apoptotic proteins is established in MyoFBs and that small changes in this balance can quickly lead to removal of MyoFBs. This phenomenon might reflect the transient nature of MyoFBs.

CCN5 consistently and robustly increases the expression level of SMAD7 in FBs and MyoFBs. SMAD7 potentiates the apoptosis of epithelial cells through inhibition of the NFκB signaling pathway [14]. In addition, SMAD7 sensitizes breast and gastric cancer cells to TNF-induced apoptosis through down-regulation of the NFκB signaling pathway [31-33]. Consistent with these reports, we showed that SMAD7 is involved in the down-regulation of NFκB in MyoFBs (Fig 4). On the basis of our findings, we conclude that CCN5 up-regulates SMAD7, which inhibits NFκB, to activate p53-mediated selective apoptosis of MyoFBs. CCN5 functions as a transcription cofactor that negatively regulates the expression of TGF-β receptor II [16]. In this study, we showed that CCN5 positively regulates the expression level of SMAD7 at the transcriptional level (Fig 5). Therefore, CCN5 can act as a transcriptional activator or inhibitor depending on the context.

Targeted apoptosis of MyoFBs in other contexts has previously been reported. In the MyoFBs of scleroderma, an autoimmune disease characterized by multi-organ fibrosis, the pro-apoptotic BH3-only protein BIM and anti-apoptotic protein BCL-XL are kept in balance to ensure the survival of MyoFBs. The BH3 mimetic drug ABT-263 (navitoclax) prevents BCL-XL from checking BIM so that BIM can induce targeted apoptosis in MyoFBs [34, 35]. The anti-cancer drug elesclomol induces targeted apoptosis of MyoFBs in hypertrophic scars, although its underlying molecular mechanism of action is unclear [36]. Future studies should investigate whether BH3 mimetic drugs or elesclomol can induce selective apoptosis in MyoFBs derived from heart tissue.

Collectively, this study shows that survival of MyoFBs in the heart is dependent on a delicate balance between p53 and NFκB, and that CCN5 induces targeted apoptosis in MyoFBs through SMAD7-mediated inhibition of the NFκB signaling pathway.

(PDF)

Click here for additional data file.

6 Oct 2021

17 Nov 2021

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the submitted manuscript authors evaluated the mechanism of CCN5 mediated apoptosis of myofibroblasts involving p53 and NFkB. Further, SMAD7 was identified as the downstream target of CCN5 using luciferase reporter construct and reported to be positively regulated by CCN5 at transcriptional level. The manuscript is written with clarity and includes sufficient detail in each section. The experimental design is logical and most of the experiments were conducted in satisfactory manner. There are few minor things that authors should address:

1. In figure 1a loading control (GAPDH) seems to be high in FB+TGFβ samples. Authors are encouraged to quantify the western blots by densitometry to confirm if there is any overexpression of P53 and p-p53 (both S392 and S15).

--> We have now quantitative graphs in figures 1~4.

2. Authors could have used high magnification images for Tunnel assay.

--> We have now higher mag images with scale bars.

3. The statement in the last paragraph of the introduction “cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-�  ” is little confusing. It seems cardiac MyoFBs were transdifferentiated, though cardiac FBs were differentiated. Authors should review the statement.

--> We revised the sentence to read " In this study, we attempted to further elucidate the role of CCN5 in MyoFB-specific apoptosis. FBs were freshly isolated from rat hearts, and MyoFBs were obtained by trans-differentiating FBs by treatment with TGF-�  ."

Reviewer #2: The Manuscript "The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB" by Nguyen et al is the extension of their earlier work where they have shown that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, authors have the elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. Authors have demonstrated that the role of CCN5 in MyoFB-specific apoptosis using cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β. They found that a balance between pro-apoptotic p53 and anti-apoptotic NFkB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFkB. In the end, the authors have claimed that this work provides insight into the role of CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies.

There are several concerns that need to be addressed.

General comments:

1. In the Abbreviations section, "E" of extracellular should be written in the capital later (line-47)

--> Corrected.

2. Extend the abbreviations "CCN5-5HA" (line- 114)

--> We inserted a sentence that explains CCN5-HA in line 117~118, which read " We synthesized a gene encoding CCN5 fused with a HA tag at its carboxy terminus and referred to it as CCN5-HA."

3. Animal Ethics Statement- should be written properly. It is written very haphazardly

--> We inserted a sentence in lines 93~95, which read " The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996)."

Specific comments:

1. In the result section, as the authors mention in the materials and methods, they used GAPDH to validate the expression level of target proteins but they did not normalize the target protein with GAPDH (All western blot figures except fig.5)

--> We have now quantitative graphs in figures 1~4.

2. The authors did not mention the molecular weight of any proteins (All figures)

--> All western blots now have molecular sizes marked.

3. In the result section, the authors use two type samples (FB and FB+ TGFβ2) but the western blot result showed the three bands of each group. If all three bands indicate the same group, then please mention it properly.

--> For two groups (FB, FB+ TGFβ2), we had three independent cultures. Each lane in western blots represent samples of these three independent cultures. We added a sentence, " For each group, three independent cultures were prepared and treated (n=3)." at the ends of each figure legends.

4. In Figure1A, the authors mention TGFβ2 but in Figure Legends and result section they mention only TGF-β. Please correct this.

--> In Figure 1A, TGF-β2 is now chaged to TGF-β.

5. What is the rationale of 30mM p53 siRNA dose selection because the authors did not explain the selection?

6. Again, the authors used 10ng/ml TGFβ and 100 nM AngII to transdifferentiate FB to MyoFBs but they did not explain why they used this concentration?

7. Similarly, they did not explain the dose selection of NFkB inhibitor (1µM)?

10. Line-245, the authors used siRNA of SMAD7 but they did not mention the concentration, however, they mentioned the concentration (25mM SMAD7 siRNA) in the representative figure-4A.

--> For the issues 5,6,7,10, we now indicate the concentration and more detailed procedure in materials and methods section as well as figure legends. The indicated concentrations were those that were recommended by manufacturors or the best ones when tested in our lab.

8. In the result section, sudden introduce that they used profibrotic proteins (TGFβ and AngII) to induced FB into MyoFB but they should be included in material and methods also.

--> This is now included in material and methods section.

9. In line-216, the authors should use words significantly blocked when the cells were pre-incubated with the p53 siRNA

--> We added a word "significantly" in line 219.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

5 Apr 2022

29 Apr 2022

Reviewer #1: In the submitted manuscript authors evaluated the mechanism of CCN5 mediated apoptosis of myofibroblasts involving p53 and NFκB. Further, SMAD7 was identified as the downstream target of CCN5 using luciferase reporter construct and reported to be positively regulated by CCN5 at transcriptional level. The manuscript is written with clarity and includes sufficient detail in each section. The experimental design is logical and most of the experiments were conducted in satisfactory manner. There are few minor things that authors should address:

1. In figure 1a loading control (GAPDH) seems to be high in FB+TGFβ samples. Authors are encouraged to quantify the western blots by densitometry to confirm if there is any overexpression of P53 and p-p53 (both S392 and S15).

--> We have now quantitative graphs in figures 1~4.

2. Authors could have used high magnification images for Tunnel assay.

--> We have now higher mag images with scale bars.

3. The statement in the last paragraph of the introduction “cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β” is little confusing. It seems cardiac MyoFBs were transdifferentiated, though cardiac FBs were differentiated. Authors should review the statement.

--> We revised the sentence to read " In this study, we attempted to further elucidate the role of CCN5 in MyoFB-specific apoptosis. FBs were freshly isolated from rat hearts, and MyoFBs were obtained by trans-differentiating FBs by treatment with TGF-β."

Reviewer #2: The Manuscript "The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB" by Nguyen et al is the extension of their earlier work where they have shown that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, authors have the elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. Authors have demonstrated that the role of CCN5 in MyoFB-specific apoptosis using cardiac MyoFBs freshly isolated from rat hearts and trans-differentiated by treatment with TGF-β. They found that a balance between pro-apoptotic p53 and anti-apoptotic NFκB is established in MyoFBs and that CCN5 tips over the balance to favor p53, which results in apoptosis through SMAD7-mediated inhibition of NFκB. In the end, the authors have claimed that this work provides insight into the role of CCN5 in reversing CF and a basis for the development of anti-fibrotic therapies.

There are several concerns that need to be addressed.

General comments:

1. In the Abbreviations section, "E" of extracellular should be written in the capital later (line-47)

--> Corrected. (Line 50)

2. Extend the abbreviations "CCN5-5HA" (line- 114)

--> We inserted a sentence that explains CCN5-HA in line 121~122, which read " We synthesized a gene encoding CCN5 fused with a HA tag at its carboxy terminus and referred to it as CCN5-HA."

3. Animal Ethics Statement- should be written properly. It is written very haphazardly

--> We inserted a sentence in lines 93~95, which read " The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996)."

Specific comments:

1. In the result section, as the authors mention in the materials and methods, they used GAPDH to validate the expression level of target proteins but they did not normalize the target protein with GAPDH (All western blot figures except fig.5)

--> We have now quantitative graphs in figures 1~4.

2. The authors did not mention the molecular weight of any proteins (All figures)

--> All western blots now have molecular sizes marked.

3. In the result section, the authors use two type samples (FB and FB+ TGFβ2) but the western blot result showed the three bands of each group. If all three bands indicate the same group, then please mention it properly.

--> For two groups (FB, FB+ TGFβ2), we had three independent cultures. Each lane in western blots represent samples of these three independent cultures. We added a sentence, " For each group, three independent cultures were prepared and treated (n=3)." at the ends of each figure legends.

4. In Figure1A, the authors mention TGFβ2 but in Figure Legends and result section they mention only TGF-β. Please correct this.

--> In Figure 1A, TGF-β2 is now changed to TGF-β.

5. What is the rationale of 30mM p53 siRNA dose selection because the authors did not explain the selection?

6. Again, the authors used 10ng/ml TGFβ and 100 nM AngII to transdifferentiate FB to MyoFBs but they did not explain why they used this concentration?

7. Similarly, they did not explain the dose selection of NFkB inhibitor (1µM)?

10. Line-245, the authors used siRNA of SMAD7 but they did not mention the concentration, however, they mentioned the concentration (25mM SMAD7 siRNA) in the representative figure-4A.

--> For the issues 5,6,7,10, we now indicate the concentration and more detailed procedure in materials and methods section as well as figure legends. The indicated concentrations were those that were recommended by manufacturers or the best ones when tested in our lab.

8. In the result section, sudden introduce that they used profibrotic proteins (TGFβ and AngII) to induced FB into MyoFB but they should be included in material and methods also.

--> This is now included in material and methods section.

9. In line-216, the authors should use words significantly blocked when the cells were pre-incubated with the p53 siRNA

--> We added a word "significantly" in line 233.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 May 2022

The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB

PONE-D-21-25986R2

Dear Dr. Park,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Dhyan Chandra, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Submitted filename: PONE_comments.docx

Click here for additional data file.

22 Jul 2022

PONE-D-21-25986R2

The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFkB

Dear Dr. Park:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Dhyan Chandra

Academic Editor

PLOS ONE