Effects of a gamma‐secretase inhibitor of notch signalling on transforming growth factor β1‐induced urethral fibrosis

Abstract Urethral stricture (US) is a common disorder of the lower urinary tract in men caused by fibrosis. The recurrence rate of US is high; however, there are no effective therapies to prevent or treat urethral fibrosis. The pathogenesis of urethral fibrosis involves myofibroblast activation and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying this pathological activation are not completely understood. It has been demonstrated that Notch signalling contributes to the development of fibrosis and inflammation. However, whether this contributes to urethral fibrosis remains unclear. In this study, activation of Notch signalling was observed in patients with US. Additionally, it was noted that activation of Notch signalling promoted ECM production and myofibroblast activation in human urethral scar fibroblasts (HUSFs) treated with transforming growth factor (TGF) β1. However, the Notch inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) suppressed activation of Notch signalling as well as proliferation and migration of the TGFβ1‐treated HUSFs. Additionally, DAPT ameliorated TGFβ1‐induced urethral fibrosis in Sprague Dawley rats by suppressing ECM production, myofibroblast activation and the TGFβ signalling pathway. These findings demonstrate that Notch signalling may be a promising and potential target in the prevention or treatment of urethral fibrosis.


| INTRODUC TI ON
Urethral stricture (US) is a common disorder in men characterized by narrowing of the urethra, which is caused by urethral and corpus spongiosum fibrosis. 1 Complications of US include recurrent urinary tract infections and urinary retention. Currently, surgical strategies that are considered for US patients include urethral dilatation, urethrotomy and urethroplasty. However, stricture recurrence remains a challenge regardless of treatment. 2,3 Pathologically, lesions of the urethral tissue are chronically inflamed, and there is activation and accumulation of myofibroblasts, overexpression of collagen and excessive deposition of extracellular matrix (ECM), which lead to urethral fibrosis, and finally US. 4,5 Antifibrotic drugs are potentially used to enrich US treatment. However, the cellular and molecular mechanisms leading to urethral fibrosis remain unclear. Therefore, a better understanding of the mechanisms is crucial for exploring novel and precise pharmacological treatments to prevent urethral fibrosis.
Notch signalling, which is highly conserved in almost all animal species, is essential for regulating cell proliferation, differentiation and apoptosis. 6 Currently, four Notch receptors (Notch1-4) and two families of Notch ligands (Jagged and Delta-like) have been described. 7 Following activation of Notch signalling by binding of Notch ligands, Notch receptors are cleaved by the γ-secretase complex with subsequent release of the Notch intracellular domain (NICD). 8 Furthermore, translocation of NICD into the nucleus induces transcription of Notch signalling target genes such as Hes1. 9 This signalling activation can be inhibited by the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). 10 Abnormal activation of Notch signalling is involved in the pathogenesis of some human diseases, such as acute leukaemia, melanoma and systemic sclerosis. 11,12 Recent studies have demonstrated that Notch signalling contributes to the development of fibrotic diseases, including tissue fibrosis (pulmonary, liver, kidney and cardiac fibrosis). 13 Transforming growth factor β1 (TGFβ1) plays a crucial role in the pathogenesis of fibrosis. Accumulating evidence indicates a vital role of TGFβ1 in the pathophysiology of urethral fibrosis. 14,15 Moreover, TGFβ can stimulate the Notch ligand Jagged1 and further activate Notch signalling through mothers against decapentaplegic homolog 3 (Smad3)-dependent mechanisms. 16 Additionally, Notch signalling regulates TGFβ-induced cell migration and cell growth arrest in epithelial cells. 17 These mechanistic studies support crosstalk or interactions between TGFβ and Notch signalling. US involves fibrosis, and however, it is not clear whether Notch signalling promotes urethral fibrosis. Therefore, inhibition of Notch signalling may be a promising and potential therapeutic target in the management of urethral fibrosis.
In the present study, we investigated the potential role of Notch signalling in urethral fibrosis development and whether suppression of Notch signalling activation by DAPT can alleviate urethral fibrosis in vitro and in vivo. For the first time, we demonstrated that Notch signalling is activated in urethral fibrosis and plays an important role in ECM production and myofibroblast activation.

| Ethics statement
The protocols for the human and animal studies were approved by the Ethics Committee of Xi'an Jiaotong University (Xi'an, China).
Informed consent and approval were obtained from all participants.
The study of human tissues was performed according to the guidelines of the Research Committee of Xi'an Jiaotong University and the Declaration of Helsinki.

| Clinical tissues and primary cell culture
Normal and scar urethral tissues were obtained from six male patients with US undergoing primary urethroplasty at The Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China).
Normal urethral tissues were collected from the same patients ( Primary human urethral scar fibroblasts (HUSFs) were established and cultured as previously described. 18 Briefly, isolated samples were pre-treated with collagenase (3 ml, 30 mg/ml; Sigma-Aldrich) and seeded in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% foetal bovine serum (Gibco) and 1% penicillin-streptomycin at 37°C with 5% CO 2 . The cells surrounding the explants were passaged. HUSFs from passages 4-8 were used in the experiments.

| Immunofluorescence study
Human urethral scar fibroblasts were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton. The fixed cells were then incubated with primary antibodies against vimentin (1:200) and alpha smooth muscle actin (α-SMA, 1:200) (Cell Signaling Technology) primary antibodies overnight at 4°C. Fluorescein isothiocyanateconjugated goat anti-rabbit and Alexa Fluor-conjugated goat antibodies (Invitrogen) were used as secondary antibodies. Fluorescence confocal images were captured using a Zeiss Observer I fluorescence microscope (Zeiss).

| Cell growth assay
Human urethral scar fibroblasts were seeded in 96-well plates, after which their viability was measured in a WST-8 assay using a

| Cell migration assay
Pre-treated HUSFs were seeded in the upper chambers of 8 μM pore size inserts (BD Biosciences). The lower chambers were filled with complete medium containing 10% foetal bovine serum. The cells were incubated at 37°C for 24 h, and then, the inserts were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich).
The migrated cells were captured using a microscope (Leica DM IL inverted microscope, 10×) and analysed using ImageJ software (National Institutes of Health).

| Coimmunoprecipitation assay
Human urethral scar fibroblasts were starved in serum-free medium for 4 h and then treated with TGFβ1 (10 ng/ml) for 1 h. Triton X-100 cell lysates were immunoprecipitated with anti-NICD antibodies that were covalently coupled to Sepharose beads (Amersham Biosciences). The beads were then incubated at 4°C for 2 h.

| Cell transfection
Human urethral scar fibroblasts were transfected with lentiviral constructs expressing NICD at multiplicity of infection of 50 (GenePharma) according to the manufacturer's instructions.
Overexpression efficiency was evaluated using green fluorescent protein-positive cells under a fluorescence microscope and by Western blotting. All the treatments were administered as injections into the urethra of the rats. Urethral fibrosis was induced in rats in the TGFβ1 and TGFβ1 + DAPT groups with TGFβ1 local injection as described previously. 15 Briefly, all the rats were anaesthetized with intravenous pentobarbital (30 mg/kg) and fixed in the supine position. The ventral penile skin was then incised to visualize the urethra. Next, 10 µg TGFβ1 was injected into the urethral wall at the 3, 6, 9 and 12 o'clock positions with a 30-gauge needle. Rats in the control group were injected with saline. After 24 h, all the rats were continuously administered a second injection at the same position with either saline (control and TGFβ1 groups) or 1 mg DAPT (TGFβ1 + DAPT group).

| Rat model of urethral fibrosis and treatment
One month later, urethral tissues were harvested for the evaluation of urethral fibrosis. Glyceraldehyde 3-phosphate dehydrogenase was used as a proteinloading control.

| Quantitative reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA from clinical samples, HUSFs and rat urethral tissues was extracted using an RNA isolation kit (Takara Biochemical) or TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. A total of 2 µg of RNA was reverse transcribed using PrimeScript RT reagent kit (Takara Biochemical). RT-qPCR was performed using a Bio-Rad iQ5 PCR system (Bio-Rad). The primer sequences for each gene are listed in Table 2.

| Histologic analysis
The harvested rat urethral tissues were fixed in 4% paraformaldehyde solution and processed using routine methods. Cross sections of the urethral tissues were stained with Masson's trichrome (MT) and haematoxylin and eosin (H&E) as previously described. 19 Representative images were acquired using the CaseViewer software.

| Statistical analysis
All the data have been presented as mean ± standard error of the mean (SEM). A minimum of three independent replicates of each experiment were performed. GraphPad Prism 7.0 (GraphPad Software) was used for statistical analysis. Multiple groups were assessed using one-way analysis of variance. Differences in data were considered statistically significant at p < 0.05.

| Notch signalling is activated in the scar tissues of US patients
To determine whether Notch signalling is involved in scar tissue formation in US patients, we first analysed the expression of the four Notch receptors in the patients. Western blot analysis and quantification revealed that the protein levels of Notch1 and Notch3 were significantly higher in the US group than in the normal group ( Figure 1A,B). Although Notch2 was detectable, its protein levels did not differ between the US and normal groups ( Figure 1A,B). In contrast to Notch1, 2 and 3, Notch4 was barely detectable in either group ( Figure 1A,B). Furthermore, real-time PCR analysis showed that the mRNA levels of Notch1, 3 and 4 were significantly upregulated in the US group compared to their respective levels in the normal group ( Figure 1C). In contrast, the levels of Notch2 mRNA did not differ between the two groups.
The expression of Notch ligands and Notch target genes were also analysed to investigate the mechanisms that activate Notch signalling in US patients. We observed a significant expression of Notch ligands such as Jagged1, Jagged2, Delta3 and Delta4, except Delta1, in the US group compared to the normal group  Figure 1I). These results suggest that Notch signalling is activated in US scar tissue.

| Notch inhibitor DAPT suppresses activation of Notch signalling as well as proliferation and migration of TGFβ1-treated HUSFs
Human urethral scar fibroblast cultures were successfully estab-

| DAPT inhibits ECM production, myofibroblast activation and the TGFβ signalling pathway in TGFβ1treated HUSFs
To evaluate the effects of DAPT on ECM production and myofibroblast activation, we incubated TGFβ1-treated HUSFs with DAPT at an effective concentration based on the previous results ( Figure 2D,F).
Overexpression of ECM components and overactivation of myofibroblasts play a critical role in the pathogenesis of urethral fibrosis.
Western blot analysis showed intense bands for collagen I, collagen III, fibronectin and α-SMA following the TGFβ1 treatment; however, these were effectively attenuated by DAPT ( Figure 3A Figure 3D). Interestingly, DAPT had no effect on the protein and mRNA expression levels in the cells that were not treated with TGFβ1. Together, these data suggest that stimulation of HUSFs with TGFβ1 results in the production of ECM and active myofibroblasts; however, these effects are reversed by DAPT in TGFβ1-stimulated HUSFs.
It is reported that there is crosstalk between the Notch and TGFβ signalling pathway and that TGFβ/Smad is involved in the development of urethral fibrosis. Therefore, we explored whether the DAPT treatment affected this crosstalk in the HUSFs. As shown in Figure

| Activation of Notch signalling promotes ECM production and myofibroblast activation in TGFβ1treated HUSFs
We demonstrated that the effects of stimulating HUSFs with TGFβ1 could be suppressed by DAPT, which indicates that the process is regulated by a Notch-dependent pathway. To further confirm the effect of Notch signalling, HUSFs were transfected with lentiviral constructs overexpressing NICD (LV-NICD) to activate Notch signalling. As shown in Figure 4A, LV-NICD displayed a high transfection efficiency in the HUSFs, which was further confirmed by western blotting. Next, we cultured LV-NICD HUSFs treated with TGFβ1 to determine whether NICD overexpression contributes to aberrant production of ECM and activation of myofibroblasts. Western blot analysis showed a significant increase in p-Smad3, collagen I, collagen III, fibronectin and α-SMA protein levels in NICD-transfected HUSFs upon TGFβ1 stimulation compared to vector-transfected HUSFs ( Figure 4B,C). Importantly, NICD overexpression resulted in partial reversal of the ability of DAPT to inhibit ECM production and myofibroblast activation; however, this was not observed in the TGFβ1 + LV-Vector + DAPT and TGFβ1 + LV-NICD + DAPT cells ( Figure 4B,C). Similar effects were observed for the mRNA levels ( Figure 4D). Taken together, these results demonstrate that Notch signalling is essential for regulating ECM production and myofibroblast activation, indicating that Notch signalling participates in the development of urethral fibrosis. indicating that TGFβ1-induced urethral fibrosis can be alleviated by DAPT ( Figure 5C).

| DAPT counteracts TGFβ1-induced urethral fibrosis in rats by suppressing ECM production, myofibroblast activation and the TGFβ signalling pathway
We investigated whether Notch signalling is overactivated in TGFβ1-induced urethral fibrosis by analysing NICD expression by Western blotting. As shown in Figure 6A These data corroborate the histologic findings.

| DISCUSS ION
In this study, we showed that Notch signalling is activated in the scar tissues of US patients with a prominent expression of Notch receptors (Notch1 and Notch3), Notch ligands (Jagged1, Jagged2, It has been indicated that Notch signalling plays a vital role in the pathogenesis of fibrotic disorders. 20 In pathological scars, impaired Notch signalling results in the alleviation of hypertrophic scar formation. 21 Furthermore, it has been reported that Notch signalling is activated in patients with systemic sclerosis 12 and in an animal model of kidney fibrosis. 22 Therefore, Notch signalling has gained significance as a potential therapeutic target in fibrosis.
In the present study, we found that Notch signalling is activated in the scar tissues of patients with US. This important finding is a promising foundation based on which Notch signalling can be targeted to prevent the formation of US. Further, Notch ligand binding followed by γ-secretase-regulated cleavage is a crucial step in which Notch signalling activity can be pharmacologically mediated.
Inhibition of Notch signalling using a γ-secretase inhibitor has been reported to decrease the expression of Notch transcriptional target genes in cancer research. 23 Crosstalk or interactions between TGFβ signalling and Notch signalling have been described. Recent studies have shown that Notch signalling can be induced under hypoxic conditions or by TGFβ. 24 Moreover, recent work has indicated that the expression levels of Notch1 and Hes1 are increased in dermal fibroblasts following stimulation with TGFβ. 12 In the present study, we first  25 It has also been reported that pharmacological blockade of γ-secretase could result in decreased proliferation of systemic sclerosis fibroblasts. 12 Moreover, in a previous study, DAPT significantly decreased the proliferation of fibroblasts in pulmonary fibrosis. 26 In this context, the current findings can explain why Notch inhibitors can inhibit urethral fibrosis by their effect on fibroblast function.
In this study, we established a rat model of TGFβ1-induced urethral fibrosis, which is a well validated and described animal model, 19 4 Furthermore, α-SMA is a defining marker of myofibroblasts, which are essential cellular elements of fibrotic disease. 26 Consequently, the number of myofibroblasts correlates with the severity of fibrosis. 28 Our results showed that the Notch inhibitor DAPT an antifibrotic effect in urethral fibrosis by inhibiting ECM production and fibroblast differentiation, which is consistent with a previous finding that Notch inhibitors have an antifibrotic effect. 29 TGFβ signalling contributes to ECM production and fibroblast differentiation. 30  signalling has also been reported to directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism in airway subepithelial fibrosis. 33 Therefore, the precise mechanism underlying Notch downstream regulation of TGFβ/Smad signalling needs to be clarified in further experiments.
F I G U R E 6 DAPT counteracts TGFβ1-induced urethral fibrosis by suppressing ECM production, myofibroblast activation and the TGFβ signalling pathway. (A) Western blot analysis of NICD, p-Smad3, collagen I, collagen III, fibronectin and α-SMA protein expression in urethral tissues isolated from rats in the control, TGFβ1 and TGFβ1 + DAPT groups. (B) Quantification of the relative expression of NICD and p-Smad3 proteins in urethral tissues isolated from rats in the control, TGFβ1 and TGFβ1 + DAPT groups. (C) Quantification of the relative expression of collagen I, collagen III, fibronectin and α-SMA proteins in urethral tissues isolated from rats in the control, TGFβ1 and TGFβ1 + DAPT groups. (D) Real-time PCR analysis of COL1A1, COL3A1, fibronectin and α-SMA mRNA relative expression in urethral tissues isolated from rats in the control, TGFβ1 and TGFβ1 + DAPT groups. Data are expressed as mean ± SEM (n = 3). * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001

| CON CLUS ION
In conclusion, our results indicate that both Notch and TGFβ signalling contribute to urethral fibrosis and provide direct evidence that Notch signalling is activated in urethral fibrosis. Additionally, the Notch inhibitor DAPT significantly attenuates TGFβ1-induced urethral fibrosis by suppressing ECM production and fibroblast differentiation. Our current findings may provide new promising targets for inhibiting the development of urethral fibrosis.

ACK N OWLED G M ENTS
This study was supported by the National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.