Deletion of transcription factor AP‐2α gene attenuates fibroblast differentiation into myofibroblast

Excessive fibrosis underlies many critical organ dysfunctions.1, 2 Fibrosis emanates from fibroblast trans‐differentiation into myofibroblasts,3 marked by increased α‐smooth muscle actin (α‐SMA) expression and excessive collagen secretion, initiated as a reparative process of normal wound healing and tissue repair in response to injury.4 However, activated myofibroblasts accumulate within pathological lesions of various fibrotic disorders,5 including patchy and interstitial fibrosis in progressive heart failure and cardiac hypertrophy.6 Therefore, attenuation of differentiation to myofibroblasts is expected to mitigate fibrosis. We attempted to find a potential target to extenuate the fibroblast differentiation by analysing the transcription factors in human fibroblasts/myofibroblasts, as transcriptome changes occur in fibroblasts during differentiation.7 Here, we report a novel molecular target, transcription factor AP‐2α (TFAP2A), to reduce fibroblasts trans‐differentiation.


| INTRODUC TI ON
Excessive fibrosis underlies many critical organ dysfunctions. 1,2 Fibrosis emanates from fibroblast trans-differentiation into myofibroblasts, 3 marked by increased α-smooth muscle actin (α-SMA) expression and excessive collagen secretion, initiated as a reparative process of normal wound healing and tissue repair in response to injury. 4 However, activated myofibroblasts accumulate within pathological lesions of various fibrotic disorders, 5 including patchy and interstitial fibrosis in progressive heart failure and cardiac hypertrophy. 6 Therefore, attenuation of differentiation to myofibroblasts is expected to mitigate fibrosis. We attempted to find a potential target to extenuate the fibroblast differentiation by analysing the transcription factors in human fibroblasts/myofibroblasts, as transcriptome changes occur in fibroblasts during differentiation. 7 Here, we report a novel molecular target, transcription factor AP-2α (TFAP2A), to reduce fibroblasts trans-differentiation.
Mature RNA (miRNeasy Mini kit) was reverse transcribed using based on a Manual Selection of reference genes. The fold change/ regulation (2^(-ΔΔC T )) was calculated using ΔΔC T method [ΔC T was calculated between target gene and an average of reference genes (HKG), followed by ΔΔC T calculations (ΔC T (Test Group)-ΔC T (Control Group))].

| Quantitative real-time PCR
Total RNA was isolated from hVFs (miRNeasy Mini kit) and reverse transcribed (miScript RT II kit) with the supplied HiFlex buffer. qPCR was performed on the LightCycler 480 Instrument II, using the Power SYBR Green F I G U R E 1 Expression of TFAP2A gene in human ventricular (myo)fibroblasts (hVFs) is significantly decreased with decrease in trans-differentiation. Differentiation magnitude was assessed by the expression of α-SMA, a marker of differentiated myofibroblasts. (A) Immunoblot displaying high and low expressions of α-SMA in hVF lysates from each heart failure patients (HTV) ["red" label is patients with low differentiation], and control hVFs obtained from diseasefree trauma victims. Scattered plot displays the individual expression of α-SMA normalized to GAPDH. (B) Bar graph displays analysis of grouped samples of high-differentiation (HF-HD, n = 4) and low-differentiation (HF-HD, n = 4), based on the α-SMA/GAPDH ratio compared to the control (n = 3) group. (C) The Heat Map visualizing the fold changes in expression of genes in the Transcription factor qPCR Array between HF-LD and HF-HD group hVFs. Note that TFAP2A gene (well: G09) expression is significantly different between the two groups (n = 3). (D) Table provides the fold regulation (vs HF-LD) data used for the map associated with each gene. (E) Validation of TFAP2A expression in hVFs from HF-HD and HF-LD patients by qRT-PCR (2 -ΔCt ). a P = 0.014 vs Control; b P = 0.01 vs HF-HD; one way-ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, n = 5; unpaired t test F I G U R E 2 Deletion of TFAP2A gene significantly reduces TGF-β1-induced fibroblast differentiation. (A) CRISPR/Cas9-based gene editing in NIH/3T3 fibroblasts deleted the TFAP2A expression, as validated by pooled Real-time PCR data (2 -ΔCt ) of TFAP2A gene (normalized to B2M gene) and immunoblotting between NIH-3T3 (wild-type) and TFAP2A-knocked out (TFAP2A-KO) fibroblasts. Gene expression of α-smooth muscle actin (α-SMA), collagen (COL) 1A1 (COL1A1), COL2A1, and COL3A1 were quantitatively analysed by real-time PCR in wild-type and TFAP2A-KO fibroblasts. Incubation in TGF-β1(5 ng/ml for 48-72h) significantly increased the expression of α-SMA (B), COL1A1 (C), COL2A1(D) in the wild-type with muted effect in the TFAP2A-KO fibroblasts. While TGF-β1 did not have any significant effect on the COL3A1(E) expression in the wild-type, COL3A1 expression was significantly down-regulated both at basal level and following TGF-β1 treatment in the TFAP2A-KO fibroblasts. Immunoblot (F) and the bar graph(G) show that TGF-β1 significantly increased the α-SMA protein expression in the wild-type with muted effect in the TFAP2A-KO fibroblasts. TGF-β1 receptor type1 (TGFBR1) mRNA levels were increased in TFAP2A-KO (H) with no significant difference in type 2 (TGFBR2) (I) compared to wild-type fibroblasts. (J) Proliferation rate (doubling time) was not significantly different between the groups. a P < 0.05 vs wild-type, b P < 0.05 vs wild-type + TGF, c P < 0.05 vs TFAP2A-KO groups; n = 3, One-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05, unpaired t test.

| Proliferation
Both WT and KO fibroblasts were plated as stated before in tripli-

| RE SULTS AND D ISCUSS I ON
From left ventricle of human heart, fibroblasts were isolated and grouped into less differentiated (HF-LD) and highly differentiated (HF-HD) based on their α-SMA expression, compared to control hVFs as shown in Figure 1A,B. PCR array of human transcription factors uncovered that the TFAP2A expression, along with ELK1, was decreased with decrease in differentiation as visualized in the heat map ( Figure 1C) and fold regulation data ( Figure 1D) (n = 3). This decreased expression of TFAP2A in HF-LD fibroblasts compared to HF-HD myofibroblasts noticed in PCR array was validated by quantitative reverse transcriptase-PCR (n = 5) ( Figure 1E). Based on these data, we have suggested that TFAP2A is crucial for the trans-differentiation of fibroblasts into myofibroblasts. We applied CRISPR/Cas9-based gene editing to knockout TFAP2A from NIH/3T3 fibroblasts (Figure 2A)  Morphogenetic Protein (BMP) 6, a member of TGF-β superfamily, targets TFAP2A to positively regulate their growth. 12 In contrast, the basal proliferation of fibroblasts did not reduce following TFAP2A knockdown in our study. This is in accordance with the observation in another study where TFAP2A can induce cell cycle arrest 13 while reduced TFAP2A expression was suggested to impair p21cip-mediated growth arrest resulting in increased proliferation. 14 These properties found in the TFAP2A-KO fibroblasts suggest that TFAP2A could emerge as a useful molecular target to mitigate excessive fibrosis by inhibiting fibroblast differentiation. As evident from the isolated human cardiac fibroblasts from left ventricles, the decrease in TFAP2A expression when cardiac fibroblast differentiation is decreased, suggest that TFAP2A is crucial for the trans-differentiation of cardiac fibroblasts into myofibroblasts which can lead to excessive cardiac fibrosis underlying many cardiac dysfunctions. Therefore, selective inhibition of TFAP2A could develop as a novel therapeutic strategy to reduce cardiac fibroblast differentiation into myofibroblast, mitigate cardiac fibrosis and preserve cardiac function.

ACK N OWLED G EM ENTS
Aurora Health Care Cardiovascular Surgery Research Award (#570-5028) to GRR.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.

AUTH O R S' CO NTR I B UTI O N S
GRR initiated, designed, executed, analysed the study and wrote the manuscript; SE executed the real-time PCR and PCR array; CW and PH implemented the cell culture, immunoblotting and proliferation assays; FXD, LE, FR and AJ interpreted data and proof-read the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data sets are publicly available from the Dryad Digital Repository at https ://doi.org/10.6084/m9.figsh are.7898168.