ITCH-Mediated Ubiquitylation of ITGB3 Promotes Cell Proliferation and Invasion of Ectopic Endometrial Stromal Cells in Ovarian Endometriosis

Abstract

Post-translational modification of proteins is involved in the occurrence of endometriosis (EM); however, the role of ubiquitination modification in EM remains unclear. Integrin β3 (ITGB3) is one of the β-subunits of integrins, which plays a key role in tumor progression. In this study, we investigated the roles of ITGB3 and ITCH, one of the ubiquitin E3 ligases, in ectopic endometrial stromal cells (ESCs) and EM. Primary ectopic ESCs and normal ESCs were isolated and purified. Western blot was used to detect the expression of ITGB3 and ITCH in ESCs. The interaction between ITGB3 and ITCH in ESCs was investigated by the co-immunoprecipitation and ubiquitylation analysis. With or without the overexpression of ITCH and/or ITGB3, the proliferation and invasion of ectopic ESCs were detected by the CCK8 assay and transwell migration assay, respectively. We found that ITGB3 is upregulated in ectopic ESCs from patients with EM. ITCH interacts with ITGB3 by co-immunoprecipitation, and ITCH-overexpressing significantly increased the ubiquitination of ITGB3. The data of the CCK8 assays showed that ITGB3 overexpression significantly promoted cell proliferation of ectopic ESCs at 12, 24, 48, and 72 h. The transwell migration assays showed that ITGB3 overexpression significantly enhanced the invasive ability. However, ITCH had the opposite effects in both assays. Our findings indicate that ITCH-mediated ubiquitylation of ITGB3 regulates the proliferation and invasion ability of ectopic ESCs in EM.

Keywords: ITCH; ITGB3; endometriosis; ubiquitination.

Figure 1

Immunostaining identification of ectopic ESCs and normal ESCs. Intracellular expression of cytoskeleton proteins Vimentin and CK19 was measured with immunohistochemistry in ectopic ESCs and normal ESCs. It was indicated that intracellular expression of Vimentin was positive, whereas that of CK19 was negative. The images of representative morphology are shown. Scale bar: 25 μm.

Figure 2

ITGB3 is upregulated in ectopic ESCs from patients with EM. Western blot analyses detecting ITGB3 relative expression levels (A). ITGB3 relative expression levels in the normal ESCs and the ectopic ESCs, * p < 0.05, compared to the normal group (B). Predicated human E3 ubiquitin ligases interact with ITGB3 and are shown in UbiBrowser alongside the brief information of ITCH (C). ITCH relative protein expression levels in the normal ESCs and the ectopic ESCs (D); * p < 0.05 compared with the normal group (E). Data are expressed as mean ± SD, and data between two groups were compared by two-tailed Student’s t-test.

Figure 3

ITCH interacts with ITGB3. Cell lysates were subjected to IP with anti-ITCH, anti-ITGB3, or anti-IgG antibody (A). After infection with ITCH-overexpressing (ITCH-OE) plasmids in ectopic ESCs, the ITCH expression was measured by Western blot (B,C) and real-time PCR (D); * p < 0.05 compared with NC. After overexpressing of ITCH in ectopic ESCs, the ITGB3 expression was measured by Western blot (E,F); * p < 0.05 compared with NC. The effect of ITCH-OE on the ubiquitination of ITGB3 in ectopic ESCs was detected by IP and Western blot (G). NC represents negative control groups.

Figure 4

ITGB3 and ITCH regulate the proliferation of ectopic ESCs. After infection with ITGB3-overexpressing (ITGB3-OE) plasmids in ectopic ESCs, the ITGB3 expression was measured by Western blot (A,B) and real-time PCR (C); * p < 0.05 compared with NC. Cell growth was evaluated using a CCK8 assay (D). Results are presented as mean ± SD of three samples in triplicate, and mean comparisons were performed using two-way ANOVA followed by Tukey’s test. * p < 0.05 compared with NC, ** p < 0.01 compared with NC, ^# p < 0.05 compared with ITCH-OE group, and ^Δ p < 0.05 compared with ITGB3-OE group. NC represents negative control groups.

Figure 5

ITGB3 and ITCH regulate the invasion of ectopic ESCs. After infection with ITGB3-OE and/or ITCH-OE plasmids, the migration of ectopic ESCs was detected by transwell migration assay (A,B), Scale bar: 25 μm. *** p < 0.001 compared with NC, ^## p < 0.01 compared ITCH-OE group, and ^ΔΔΔ p < 0.001 compared with ITGB3-OE group. Data are expressed as mean ± SD, and data between two groups were compared by two-tailed Student’s t-test. NC represents negative control groups.