SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis

Abstract

Background: Endometriosis is a widespread benign gynecological disorder. The signal transducer and activator of transcription 3 (STAT3) signaling pathway plays an important role in the pathogenesis of endometriosis through regulating proliferation and invasion of endometrial stromal cells. Furthermore, the protein tyrosine phosphatase (PTP), SH2 domain-containing phosphatase 1 (SHP-1), negatively regulates STAT3 activation. However, regulation of the SHP-1-STAT3 pathway in the pathogenesis of endometriosis remains unclear.

Methods: Cell proliferation and invasion were assessed by Cell Counting Kit-8 (CCK-8) assay and Transwell analysis, respectively, to investigate the role and regulation of the SHP-1-STAT3 pathway in the proliferation and invasion of endometrial stromal cells. Expression of Smad ubiquitin regulatory factor 1 (SMURF1), SHP-1, matrix metalloproteinase 2 (MMP2), MMP9, STAT3, and phospho-STAT3 (p-STAT3) level in patients with endometriosis were measured by Western blotting and/or immunohistochemical staining. The interaction between SMURF1 and SHP-1 was investigated by co-immunoprecipitation and ubiquitylation analysis.

Results: The present study demonstrated that downregulation of SHP-1 expression in patients with endometriosis was negatively correlated with SMURF1 expression. SMURF1, an E3 ubiquitin ligase, activated the STAT3 pathway via ubiquitylation and degradation of SHP-1. Furthermore, SMURF1 promoted cell proliferation and invasion of endometrial stromal cells by activating STAT3 signaling and expression of its downstream targets, MMP2 and MMP9, whereas SHP-1 demonstrated an inverse effect. Additionally, SHP-1 inhibited SMURF1-mediated cell invasion and proliferation of endometrial stromal cells.

Conclusions: Our findings indicate that SMURF1-mediated ubiquitylation of SHP-1 regulates endometrial stromal cell proliferation and invasion during endometriosis.

Keywords: Endometriosis; SH2 domain-containing phosphatase 1 (SHP-1); Smad ubiquitin regulatory factor 1 (SMURF1); invasion; ubiquitylation.

Figure 1

SHP-1 expression levels in ectopic endometrium from patients with endometriosis. (A,B) Representative SHP-1 protein expression in ectopic endometrium from women with endometriosis (EM1–4) or in normal endometrium from women without endometriosis (Nor1–4) was measured by Western blotting. Results are presented as mean ± SD of 12 samples in triplicate and means comparisons were performed using unpaired t-test. ***, P<0.001 vs. Nor groups.

Figure 2

Expression of predicated E3 ligases and correlation analysis in ectopic endometrium from patients with endometriosis. (A) Top 20 predicated human E3 ubiquitin ligases interact with SHP-1. Five high confidence interactions between SHP-1 and human E3 ubiquitin ligases are shown. Representative SMURF1 (B,C), CBL (B,D), MDM2 (B,E), and SYVN1 (B,F) expression levels in ectopic endometrium from women with endometriosis (EM1–4) or in normal endometrium from women without endometriosis (Nor1–4) were measured using Western blotting. Results are presented as mean ± SD of 12 samples in triplicate and means comparisons were performed using unpaired t-test. (G) Pearson correlation scatter plot in ectopic endometrium from patients with endometriosis (n=12). ***, P<0.001 vs. Nor groups.

Figure 3

SMURF1, SHP-1, and STAT3 expression and p-STAT3 levels in ectopic endometrial stromal cells from patients with endometriosis. (A,B) SMURF1, SHP-1, and p-STAT3 levels in ectopic endometrium from women with endometriosis (EM) or in normal endometrium from women without endometriosis (Nor) were measured by IHC. Scale bar: 25 µm. (C) Intracellular expression of cytoskeleton proteins CK19 and vimentin was measured with immunocytochemistry. Scale bar: 10 µm. (D) Stromal cells expressed vimentin and CK19 were measured by flow cytometric analysis. (E,F,G) Representative SMURF1, SHP-1, and STAT3 expression as well as p-STAT3 levels in endometrial stromal cells from ectopic endometrium of women with endometriosis (EMS1–3) or that from normal endometrium of women without endometriosis (Nor1–3) was measured by Western blotting. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using two-way ANOVA followed by Tukey’s test (B,F) or unpaired t-test (G). ***, P<0.001 vs. Nor groups. IHC, immunohistochemistry; ANOVA, analysis of variance.

Figure 4

SMURF1 binds to and promotes ubiquitylation of SHP-1. (A) Co-immunoprecipitation showed that SMURF1 interacted with SHP-1. (B) The subcellular localizations of SMURF1 (green) and SHP-1 (red) were measured by immunofluorescence. DAPI (blue) was used for nuclear counter stain. Scale bar: 10 µm. (C-F) Ectopic endometrial stromal cells were infected with pLVX-Puro-SMURF1 or pLKO.1-SMURF1-shRNA in the absence or presence of 10 µM MG132, and SMURF1 and SHP-1 expression was measured by qPCR and Western blotting. (G) Ectopic endometrial stromal cells infected with pLVX-Puro-SMURF1or blank vector were treated with CHX (0.1 mg/mL) and harvested at the indicated times. Cells were lysed and the lysates were then blotted with the indicated antibodies. (H) Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, and SHP-1 was immunoprecipitated and immunoblotted using the indicated antibodies. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (C-F) or two-way (G) ANOVA followed by Tukey’s test. *, P<0.05, **, P<0.01, ***, P<0.001 vs. vector, shNC or 0 h groups; ^###, P<0.001 vs. SMURF1-OV (SMURF1 overexpression) groups. qPCR, quantitative polymerase chain reaction; CHX, cycloheximide; ANOVA, analysis of variance.

Figure 5

Silencing of SMURF1 and/or SHP-1 regulates the proliferation and invasion of ectopic endometrial stromal cells. Ectopic endometrial stromal cells were infected with pLKO.1-SMURF1-shRNA, pLKO.1-SHP-1-shRNA, or pLKO.1-scramble shRNA as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. *, P<0.05, ***, P<0.001 vs. shNC; ^##, P<0.01, and ^###, P<0.001 vs. SMURF1-shRNA groups. qPCR, quantitative polymerase chain reaction; MMP, matrix metalloproteinase; CCK-8, Cell Counting Kit-8; ANOVA, analysis of variance.

Figure 6

Overexpression of SMURF1 and/or SHP-1 regulates the proliferation and invasion of normal endometrial stromal cells. Normal endometrial stromal cells were infected with pLVX-Puro-SMURF1, pLVX-Puro-SHP-1, or blank pLVX-Puro as a negative control, and the expression of SHP-1, MMP2, MMP9, and STAT3 as well as p-STAT3 levels were determined by qPCR (A) or Western blot analysis (B,E-G), and cell proliferation (C) and invasion (D) were measured by CCK-8 and Transwell analysis, respectively. Scale bar: 10 µm. Results are presented as mean ± SD of three samples in triplicate and means comparisons were performed using one-way (A,B,D,G) or two-way (C,F) ANOVA followed by Tukey’s test. ***, P<0.001 vs. blank vector; ^###, P<0.001 vs. SMURF1-OV (SMURF1 overexpression) groups. qPCR, quantitative polymerase chain reaction; MMP, matrix metalloproteinase; CCK-8, Cell Counting Kit-8; ANOVA, analysis of variance.