FoxM1 promotes Wnt/β‐catenin pathway activation and renal fibrosis via transcriptionally regulating multi‐Wnts expressions

Abstract The activation of Wnt/β‐catenin pathway plays a pivotal role in promoting renal fibrosis. The activation of Wnt/β‐catenin pathway relies on the binding of Wnts to Frizzled receptors on cell membrane. However, the factor regulating Wnts production remains unclear. Here, we demonstrated that transcriptional factor FoxM1 was significantly increased in obstructed kidneys and patients' kidneys with fibrosis. The up‐regulation of FoxM1 mainly distributed in tubular epithelial cells. Pharmacological inhibition of FoxM1 down‐regulated multi‐Wnts elevation in UUO mice and attenuated renal fibrosis. In cultured renal tubular epithelial cells, overexpression of FoxM1 promoted 8 Wnts expression, while knock‐down on FoxM1‐suppressed multi‐Wnts including Wnt1, Wnt2b and Wnt3 expression induced by Ang II. Chromatin immunoprecipitation PCR confirmed that FoxM1 bound to Wnt1, Wnt2b, Wnt3 promoters and luciferase assay further identified that the transcriptions of Wnt1, Wnt2b and Wnt3 were regulated by FoxM1. Thus, our findings show that multi‐Wnt family members were regulated by transcriptional factor FoxM1. FoxM1 might be a key switch for activating β‐catenin pathway and renal fibrosis. Therefore, FoxM1 might be a potential therapeutic target in manipulating renal fibrosis.

| 1959 XIE Et al. renin-angiotensin system, infiltration of myeloid inflammatory cells, sustained inflammation in local tissue and activation of interstitial fibroblasts, have been reported to contribute to the progression of renal fibrosis. [2][3][4] It has been recently well documented that the activation of Wnt/β-catenin signalling pathway is implicated in renal fibrosis. 5,6 Once activated, β-catenin translocates into nucleus and acts as a transcriptional co-activator to enhance multiple fibrotic gene expressions. The activation of Wnt/β-catenin signalling pathway relies on the bindings of Wnts to Frizzled receptors on cell membrane.
Forkhead Box M1 (FoxM1), a member of the Forkhead family, is a representative proliferation-related transcription factor. 13 FoxM1 is known as an important factor in regulating cell cycle transition and chromosome segregation. [14][15][16] FoxM1 has been found to be expressed in alveolar epithelial cells. 17 Recent study indicated that FoxM1 provoked injured proximal tubular proliferative repair in acute kidney injury (AKI). 18 Bioinformatic analysis results showed that there are computative binding sites of FoxM1 on all human Wnts promoter regions and 13 of 19 rat Wnts promoter regions. In unilateral ureteral obstruction (UUO)-induced renal fibrosis mice, our preliminary study result showed that FoxM1 was persistently increased in the fibrotic kidneys.
Based on the clue, the current study clarified that FoxM1 was an essential regulator of Wnts. Persistent increase of FoxM1 up-regulated the expression of multi-members of Wnts. Increased Wnts, as a consequence, activated β-catenin signalling pathway in renal tubular epithelial cells and then promoted renal fibrogenesis. The up-regulation of FoxM1 was also observed in biopsy samples with renal fibrosis.
UUO mice were established as previously described. After anesthetized, the left ureter was exposed and ligated. The sham group was operated in the same procedure, except the left ureter ligation. Mice were administered with either DMSO or FoxM1 inhibitor Siomycin A via the i.p. injection at a dose of 6.25 mg/kg bodyweight at designated time points (days 1,5,9,13)

| Cell culture
NRK-52E, a rat renal tubular epithelial cell line, was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China).

The cells were cultured in DMEM media supplemented with 10%
FBS at 37°C in an atmosphere of 5% CO 2 . After starved overnight in a serum-free medium, cells were treated with Ang II (Millipore).
FoxM1 inhibitor Siomycin A (Sigma-Aldrich) was added to cells at 0.1-1 μmol/L for 0.5 hour prior to Ang II.

| Western blot
Kidney tissue or cells were lysed in lysis buffer containing 2% SDS following centrifugation. The supernatant was separated on SDSpolyacrylamide gels. Then, proteins were electrophoretically trans- F I G U R E 1 FoxM1 was elevated in tubular epithelial cells from UUO or FA-induced fibrotic kidneys. A, Representative Western blot of FoxM1 in UUO mice kidneys. B, Quantitative data for FoxM1 in mice kidneys at 3, 7 and 14 d after UUO (n = 6). **P < .01 vs Sham. C, Representative Western blot of FoxM1 in FA mice kidneys. D, Quantitative data for FoxM1 in mice kidneys at 28 d after FA treatment (n = 9). **P < .01 vs Con. E, Immunohistological staining of FoxM1 in sham and UUO mice at 14 d after surgery. Scale Bar: 50 μm (left panels) and 25 μm (right panels). F, Co-immunofluorescence staining of FoxM1 (green) with AQP1 (red) and DAPI (blue). Scale bar: 50 μm. G, Immunohistological staining of FoxM1 (Scale bar: 50 μm) and Masson staining (Scale bar: 100 μm) in kidney biopsies from CKD patients.

| RNA extraction and real-time quantitative PCR
Total RNA was extracted using the TRIzol reagent (Life Technologies). For mRNA quantification, cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara) and quantitative PCR was performed on Applied Biosystems 7300 Plus using SYBR Green PCR master mix (Toyobo). The primers sequences were listed in Supplemental Table S2. The relative abundance of target mRNA was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

| Renal histology and Immunohistochemistry staining
Mouse kidneys fixed in 10% formalin were embedded into paraffin and cut into 4-μm thick sections. HE and Masson's trichrome staining were conducted following a protocol described previously.
The tubular injury was assessed by HE staining based on tubular changes including tubule dilation, epithelial swelling, loss of brush borders and protein cast formation. 19 Masson's trichrome staining was used to evaluate interstitial fibrosis. Fibrotic areas in 5 discretionary fields of each mouse were quantified. The average percentage of fibrotic area relative to the total area was regarded as fibrotic score. The mouse kidneys and human kidney biopsy sections were stained using specific anti-FoxM1 antibody (ab1807.10; Abcam).

| Construction of Vectors
Rat FoxM1 gene was cloned into pcDNA3.1 as described previously. 10 Wnt1, Wnt2b and Wnt3 promoter fragments were amplified from rat genomic DNA with appropriate primers. Mutated Wnt1, Wnt2b and Wnt3 promoter regions were constructed by deleting FoxM1 binding site on the corresponding promoter region. The PCR products were cloned into the luciferase vector pGL3-basic. Adenoviral expression vector GV314 encoding rat FoxM1 gene (Ad-3 × FLAG-EGFP-FoxM1) was constructed according to the manufacturer's protocol. Transduction was confirmed via FoxM1 expression. All constructs were verified by sequencing.

| Isolation of nuclear proteins
Cells were pre-treated with and without 1 μmol/L of Siomycin A for 0.5 hour prior to Ang II. Then, cells were harvested and conducted nuclear protein extraction using the Nuclear and Cytoplasmic Protein Extraction Kit (P0028; Beyotime) based on the manufacturer's instructions. The nuclear fraction was dissociated using nuclear lysis buffer and subjected to Western blot.

| Luciferase assay
The luciferase assay was performed as described previously. Briefly, human embryonic kidney (HEK) 293T cells were co-transfected of FoxM1 overexpression plasmid and β-galactosidase (β-gal) plasmid with the wild-type Wnt1, Wnt2b, Wnt3 or mutant Wnt1, Wnt2b and Wnt3 luciferase reporter plasmid using Lipofectamine 3000 reagent. After cultured for 48 hours, the cells were harvested, and luciferase activity was measured with the Luciferase Assay Kit (Promega). Relative luciferase activity was calculated as the ratio of Luc/β-gal activity.

| Chromatin immunoprecipitation assay
The assay was performed with EZ-Chromatin immunoprecipitation (ChIP)TM assay kit (17-371; Millipore) according to the manufacturer's protocols. In brief, NRK-52E cells were transfected with adenovirus harbouring FoxM1 for 48 hours. The crosslink was performed by adding 1% formaldehyde to the lysate for 10 minutes at room temperature and terminated by glycine. After sonicated, the DNA fragments in cell lysates were subjected to immunoprecipitation using rabbit anti-FLAG antibody (14793; Cell Signaling Technology) or normal rabbit IgG (as a control). The reverse crosslinked complexes were purified and amplified by real-time qPCR. PCR primers to analyse Wnt1, Wnt2b and Wnt3 were listed in Supplemental Table S3. Fold enrichment of bound area was defined by DNA amount relative to the total DNA.

| Statistical analysis
Data are shown as the mean ± SEM. The analysis of data was performed by SPSS 19.0 (Chicago, IL, USA). Comparisons between groups were performed by one-way ANOVA with post hoc analysis using Tukey's test. P < .05 was considered as statistically significant.

| FoxM1 was increased in UUO or FA-induced fibrotic kidneys
The expression of transcriptional factor FoxM1 in fibrotic mice was detected by Western blot. The result showed that FoxM1 was significantly increased at 3 days after UUO surgery and sustained to the end of experiment (14 days) ( Figure 1A,B). The elevation of FoxM1 was also observed in FA-induced renal fibrosis animals ( Figure 1C,D).
Immunohistochemistry result showed that FoxM1 was mainly distributed in renal tubules ( Figure 1E). To identify the tubular segment specificity of FoxM1 expression in the kidneys, we performed double-immunostaining for FoxM1 (green) and aquaporin-1 (AQP1, red), a marker of proximal tubule. The result indicated that the increased FoxM1 was predominantly localized in nucleus of proximal renal tubules ( Figure 1F).
To identify the relevance of FoxM1 in clinic, the expression of fibrosis. Immunohistochemistry result showed that the expression of FoxM1 was also principally located in the nucleus of tubular epithelial cells ( Figure 1G).

| Selective FoxM1 inhibitor Siomycin A significantly alleviated fibrosis in UUO mice
To identify the effect of FoxM1 on renal fibrosis, Siomycin A, a selective FoxM1 inhibitor, was injected intraperitoneally into UUO mice. HE and Masson staining results showed that the injection of

| Overexpression of FoxM1-induced mRNA and protein expression of Wnts in renal tubular epithelial cells
The  Table S1). To identify whether FoxM1 regulated the expression of Wnts, an adenovirus vector harbouring

| Knock-down on FoxM1 suppressed Ang IIinduced the expression of Wnts and fibrosis-related gene expressions in vitro
To verify the result observed in vivo, cultured renal tubular epithelial cells were treated with Ang II, the major effector in renin-angiotensin system. 20 As showed in Figure 4A

| FoxM1 inhibitor Siomycin A suppressed Ang II-induced Wnts and fibrosis-related gene expressions in vitro
Consistent with the results from FoxM1 knock-down, pharmacologi-

| FoxM1 augmented β-catenin nuclear distribution via enhancing Wnts transcription in renal tubular epithelial cells
To identify the role of FoxM1 in β-catenin activation, the nuclear β-catenin was detected by Western blot. Exposure of the renal tubular epithelial cells to Ang II enhanced β-catenin nuclear distribution, whereas pre-treatment with Siomycin A evidently blunted β-catenin nuclear translocation ( Figure 6A,B). Overexpression of FoxM1 also induced a significant increase in β-catenin nuclear distribution ( Figure 6C).
ChIP-qPCR assay was performed to determine whether FoxM1 directly regulates Wnts gene transcription. Compared with IgG, the precipitated Wnt1, Wnt2b and Wnt3 promoter DNAs were dramatically increased ( Figure 6D). To further confirm the binding of FoxM1, original or mutant Wnt1, Wnt2b and Wnt3 promoter luciferase plasmids were co-transferred into HEK 293T cells with FoxM1 overexpression plasmid or empty vector for 48 hours.
When wide-type (WT) Wnt1, Wnt2b or Wnt3 promoter luciferase plasmid was co-transfected with FoxM1 overexpression vector, luciferase activity was significantly increased while the mutation of Wnt1, Wnt2b or Wnt3 promoter region by deleting FoxM1 binding site abolished the effect ( Figure 6E). The data indicated that FoxM1 was able to bind to the promoter region of Wnt1, Wnt2b and Wnt3 and positively regulated the expression of those Wnts.

| FoxM1 inhibition alleviated the Wnts and fibrotic-related gene expressions in UUO mice
To observe the effects of FoxM1 on expressions of Wnts and renal fibrosis-related genes, UUO mice were treated with FoxM1 inhibitor Siomycin A. The result showed that Siomycin A did not exhibit obvious effects on the expression of FoxM1 in sham-operated kidneys, but dramatically abrogated the increase in FoxM1 in obstructed kidneys ( Figure 7A,B). As expected, Siomycin A significantly attenuated the increase in Wnt1, Wnt2b and Wnt3 in UUO mice ( Figure 7C,D). Similarly, Siomycin A administration did not exhibit obvious influence on β-catenin activation, snail and E-cadherin in sham-operated kidneys, but significantly suppressed the increase in β-catenin activation, snail and the decrease in E-cadherin in UUO kidneys ( Figure 7E,F). Besides, immunostaining confirmed the Wnt1 expression was increased in UUO mice and mainly distributed in the renal tubules ( Figure 7G).
These data supported the idea that FoxM1 promoted renal fibrosis via Wnts secretion and subsequently initiated β-catenin pathway activation.
F I G U R E 6 FoxM1 augmented nuclear distribution of β-catenin and directly regulated Wnts transcription in renal tubular epithelial cells. A, Representative Western blot of β-catenin in nuclear fractions. NRK-52E were treated with Ang II (10 −6 M) for 48 h with or without FoxM1 inhibitor Siomycin A. B, Quantitative data for β-catenin in nuclear fractions (n = 3). **P < .01 vs Con. # P < .05 vs Ang II. C, Representative immunofluorescence images of FoxM1 (green), active β-catenin (red) and DAPI (blue). Cells were infected with Ad-FoxM1 or Ad-null for 48 h. Bar = 50 μm. D, Chromatin immunoprecipitation assay was conducted with antibody against IgG or Flag and normalized to the input DNA. Amplifications of Wnt1, Wnt2b and Wnt3 were performed by qPCR (n = 3). *P < .05, **P < .01 vs IgG. E, HEK 293T cells were co-transfected with the plasmids expressing FoxM1, β-gal and luciferase promoter containing Wnt1, Wnt2b, Wnt3 WT or Wnt1, Wnt2b and Wnt3 MU promoter sequence. Luciferase activity was detected 48 h after transfection (n = 3). **P < .01 vs Wnt1 WT, *P < .05 vs Wnt2b WT or Wnt3 WT. Ad-FoxM1, recombinant adenovirus containing rat FoxM1 gene; Ad-null, empty adenovirus vectors; Con, control; DAPI, 4′, 6-diamidino-2-phenylindole; MU, luciferase promoter containing mutant promoter sequences of Wnts; WT, luciferase promoter containing wild-type promoter sequences of Wnts; β-gal, β-galactosidase It has been well documented that the activation of Wnt/βcatenin plays a pivotal role in promoting renal fibrosis. As described in previous study, pharmacological inhibition of β-catenin in tubular epithelium suppressed the progression of renal fibrosis under pathological circumstances. 5,6 Activation of β-catenin requires molecular Wnts binding to their receptors. 9 Conditional expression of Wnt1 in the proximal tubules effectively activated fibroblasts and promoted renal fibrosis. 22 In human proximal tubular cells, overexpression of Wnt1 apparently activated β-catenin nuclear translocation and promoted cellular senescence. 23,24 A latest study illustrated tubule-specific deficiency of Wnts sufficiently reduced renal fibrosis. 25,26 All these studies indicate the renal tubular epithelial cells are an important source of Wnts in kidneys.

| D ISCUSS I ON
Nineteen Wnt family members have been identified. In the present study, our result showed that almost all Wnts expressions were increased in UUO-induced fibrotic kidneys, which was consistent with the previous observations. 10 This result also suggested subduction on any Wnt production and secretion may not be able to effectively block the progression of renal fibrosis. Thus, finding out the master switch on Wnt/β-catenin signalling pathway becomes essential in manipulating renal fibrosis.
Here, we identified that FoxM1 was a key transcriptional regulator of multi-Wnts. The expression of FoxM1 was increased in both UUO and FA-induced fibrotic kidneys. The distribution of Wnts expression. Moreover, FoxM1 was able to regulate β-catenin transcription 27,28 and facilitate β-catenin nuclear translocation. [29][30][31] These studies indicated that the effect of FoxM1 in promoting renal fibrosis may not completely rely on regulating Wnts expressions.
The results in the study suggest that FoxM1 is a pivotal regulator in promoting renal fibrosis, but what is line on the upstream of FoxM1 still need to be elucidated.
In conclusion, our data demonstrated that up-regulated transcriptional factor FoxM1 stimulates multi-Wnts transcription and then promotes renal fibrosis via activating Wnt/β-catenin signalling pathway. FoxM1 might be a master switch to turn on the abnormal activation of Wnt/β-catenin signalling pathway under pathological conditions.

ACK N OWLED G EM ENTS
This research was financially supported by the National Natural

CO N FLI C T O F I NTE R E S T
All the authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Hongyan Xie: Resources (lead); Software (lead); Supervision

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used in the current study are available from the first author and corresponding author on reasonable request. F I G U R E 7 Siomycin A inhibited Wnt/β-catenin pathway activation and fibrotic-related gene expressions in UUO mice. A and C, Representative Western blot B and D, Quantitative analysis of renal FoxM1, Wnt1, Wnt2b and Wnt3 expression at 14 d following UUO (n = 6). **P < .01, ***P < .001 vs Sham. # P < .05, ## P < .01 vs UUO. E, Representative Western blot F, Quantitative analysis of renal active β-catenin, Snail and E-cadherin expression at 14 d following UUO (n = 6). *P < .05, **P < .01 vs Sham. # P < .05 vs UUO. G, Representative immunohistological staining for Wnt1 in sham and UUO mice. Scale bar: 50 μm. UUO, unilateral ureteral obstruction