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. 2012 Jul;18(7):1102-11.
doi: 10.1038/nm.2826.

A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis

Sang Jun Han  1 , Shannon M Hawkins, Khurshida Begum, Sung Yun Jung, Ertug Kovanci, Jun Qin, John P Lydon, Francesco J DeMayo, Bert W O'Malley
Affiliations

A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis

Sang Jun Han et al. Nat Med. 2012 Jul.

Abstract

Endometriosis is considered to be an estrogen-dependent inflammatory disease, but its etiology is unclear. Thus far, a mechanistic role for steroid receptor coactivators (SRCs) in the progression of endometriosis has not been elucidated. An SRC-1-null mouse model reveals that the mouse SRC-1 gene has an essential role in endometriosis progression. Notably, a previously unidentified 70-kDa SRC-1 proteolytic isoform is highly elevated both in the endometriotic tissue of mice with surgically induced endometriosis and in endometriotic stromal cells biopsied from patients with endometriosis compared to normal endometrium. Tnf⁻/⁻ and Mmp9⁻/⁻ mice with surgically induced endometriosis showed that activation of tumor necrosis factor a (TNF-α)-induced matrix metallopeptidase 9 (MMP9) activity mediates formation of the 70-kDa SRC-1 C-terminal isoform in endometriotic mouse tissue. In contrast to full-length SRC-1, the endometriotic 70-kDa SRC-1 C-terminal fragment prevents TNF-α-mediated apoptosis in human endometrial epithelial cells and causes the epithelial-mesenchymal transition and the invasion of human endometrial cells that are hallmarks of progressive endometriosis. Collectively, the newly identified TNF-α-MMP9-SRC-1 isoform functional axis promotes pathogenic progression of endometriosis.

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Figures

Figure 1
Figure 1
The endometriotic 70-kDa SRC-1 isoform. (a) Western blot analysis of SRC-1 expression in uterus of sham-treated mice and in the ectopic and eutopic endometrium of mice with SIE. Tubulin acts as a protein loading control. (b) The ratio of the 70-kDa fragment to intact SRC-1 in each type of endometrium (n=6/group). (c) Western blot analysis of SRC-1 expression pattern in the uterus of sham-treated mice, the eutopic endometrium of mice with SIE and the uterus of sham-treated SRC-1−/− mice. Tubulin acts as a protein loading control (d) Immunohistochemical analysis of SRC-1 expression pattern in uterus of SRC-1−/− mice, uterus of sham-treated mice and in the ectopic and eutopic endometrium of mice with SIE. (e) Western blot analysis of expression patterns of SRC-1, the 70-kDa SRC-1 isoform, ER-β and ER-α in cultured primary HESCs lines isolated from women without endometriosis (n=1) and from the ectopic lesions of women with endometriosis (n=6; lanes 1 to 6). Tubulin acts as a protein loading control. (f–h) The ratios of the SRC-1 isoform to tubulin (f), ER-β to tubulin (g) and ER-α to tubulin (h) in cultured primary HESC lines isolated from women without endometriosis (n=3) and from the ectopic and eutopic endometrium of endometriosis patients (n=10). * P<0.05, ** P<0.01 by Student's t test. Scale bars, 50 μm.
Figure 2
Figure 2
The SRC-1 gene had an essential role in the progression of endometriosis. (a) Wild-type ectopic lesions and SRC-1−/− ectopic lesions isolated from C57BL/6J recipient mice after the induction of endometriosis. (b) The volume analysis of wild-type and SRC-1−/− ectopic lesions (n=4/group). (c) The GFP images analysis of a wild-type and an SRC-1−/− ectopic lesion expressing GFP in mice with SIE. (d) The GFP pixel density of wild-type and SRC-1−/− ectopic lesions expressing GFP in mice with SIE. (e) Immunohistochemical analysis of COX-2, VEGFR and SRC-1 expression levels in wild-type ectopic lesions and SRC-1−/− ectopic lesions and quantification of COX-2 and VEGFR expression levels in each type of ectopic lesion (n=3/group). (f,g) Immunohistochemical analysis of expression levels of phospho-histone (H) 3 and activated caspase 3 in wild-type and SRC-1−/− ectopic lesions (f) or in wild-type and SRC-1−/− eutopic endometrium (g) and quantification of their expression levels in each type of ectopic lesion (n=3/group) and eutopic endometrium (n=9/group). PLC, percentage of labeled cells, **P<0.01 by Student's t test. Scale bars, 50 μm.
Figure 3
Figure 3
The endometriotic SRC-1 isoform was the SRC-1 C-terminal fragment. (a) Experimental scheme of the heterologous mouse model of endometriosis. (b) Western blot analysis of expression levels of ER-β, COX-2, VEGFR2 and Tubulin in mixed-cultured recombinant human endometrial cells and the ectopic lesions that developed from these cells. (c–e) Western blot analysis of SRC-1processing pattern in mixed-cultured recombinant human endometrial cells expressing F-SRC-1-M (Mixed-cultured:F-SRC-1-M), ectopic lesions developed with recombinant human endometrial cells expressing F-SRC-1-M (Ectopic:W/ FSRC-1-M), and parental ectopic lesions developed from parental human endometrial cells that did not express F-SRC-1-M (Ectopic: W/O F-SRC-1-M) using a Flag-specific antibody (c), a MYC-specific antibody (d) and an SRC-1-specific antibody (e). Tubulin acts as a protein loading control. WB; Western Blot analysis. *; nonspecific bands.
Figure 4
Figure 4
The TNF-α/MMP9 functional axis had an essential role in the progression of endometriosis and endometriotic SRC-1 isoform formation in endometriotic tissue. (a) Gelatin zymography analysis of MMP2 and MMP9 activity in uterus of sham-treated mice and eutopic endometrium of mice with SIE and ratio of MMP9 activity to tubulin in each type of endometrium (n=4/group). (b) Gelatin zymography analysis of MMP2 and MMP9 activity in primary HESCs isolated from women without endometriosis (W/O endometriosis) and from women with endometriosis (W/ endometriosis) and ratio of MMP9 activity to tubulin in each type of cells (n=3/group). (c) Gelatin zymography analysis of the MMP9 activity in the eutopic endometrium of C57BL/6J mice with SIE treated with vehicle or 2.5 mg kg−1 of MMP2/MMP9 inhibitor III (MMP2/9 In) and ratio of MMP9 activity to tubulin in each eutopic endometrium (n=4/group). (d) Ectopic lesions isolated from mice with SIE treated with vehicle or 2.5 mg kg−1 of MMP2/9 In, and volume analysis of each isolated ectopic lesion (n=4/group). (e) Western blot analysis of SRC-1 expression patterns in the eutopic endometrium of mice with SIE treated with vehicle or MMP2/9 In. and the ratio of the 70-kDa SRC-1 isoform to intact SRC-1 in each type of endometrium. (f) Gelatin zymography analysis of MMP9 activity in the eutopic endometrium of Tnf−/− mice and congenic wild-type mice with SIE and the ratio of MMP9 activity to tubulin in each type of eutopic endometrium (n=3/group) (g) Ectopic lesions isolated from congenic wild-type mice and Tnf−/− mice with SIE, and volume analysis of each isolated ectopic lesion (n=4/group). (h) Western blot analysis of expression patterns of SRC-1 in the eutopic endometrium of Tnf−/− mice and congenic wild-type mice with SIE and ratio of the 70-kDa SRC-1 isoform to intact SRC-1 in each type of eutopic endometrium (n=3/group). ** P<0.01, *** P<0.001 by Student's t test.
Figure 5
Figure 5
MMP9 directly cleaved human SRC-1 at P790-M791 in vitro and in vivo. (a–c) Western blot analysis of the resulting processed forms of SRC-1 by MMP9 with antibodies to Flag (a), MYC (b) and SRC-1 (c). (d–f). Western blot analysis of the resulting processed forms of the SRC-1(P790A) mutant by MMP9 with antibodies to Flag (d), MYC (e) and SRC-1 (f). (g) Experimental scheme for the transplantation of recombinant IHEECs stably expressing wild-type F-SRC-1-M or the F-SRC-1-M(P790A) into SCID mice to induce endometriosis. (h) Western blot analysis of SRC-1 expression pattern in ectopic lesions expressing F-SRC-1-M or the FSRC-1-M(P790A) with a MYC-specific antibody and the ratio of the 70-kDa SRC-1 isoform to intact SRC-1 in each ectopic lesion (n=3/group). *; Non-specific bands. (i) The processing pattern of SRC-1 to the endometriotic SRC-1 C-terminal isoform [791-1441 amino acids(aa)] by MMP9 in endometriotic tissues. bHLH, basic helix-loop-helix. RID, receptor interacting domain. AD, activation domain. Q, proline-rich domain. aa, amino acid. *** P<0.001 by Student's t test.
Figure 6
Figure 6
Endometriotic SRC-1 prevented TNF-α-induced cell death and increased EMT to improve the invasive capacity of human endometrial epithelial cells. (a) The crystal violet cell proliferation assay of parental IHEECS and recombinant IHEECS stably expressing full-length SRC-1 or the SRC-1 C-terminal isoform in the absence or presence of 10 ng ml−1 TNF-α for 2 d. (b–d) The viable cell numbers of the parental IHEECs (b) and the recombinant IHEECs expressing full-length SRC-1 (c) or the SRC-1 isoform (d) per field at the indicated time points. (e) Western blot analysis of expression levels of procaspase 8 (57 kDa) and its cleaved form (10 kDa) in parental IHEECS and recombinant IHEECS stably expressing full-length SRC-1 or the SRC-1 C-terminal isoform upon 10 ng ml−1 TNF-α treatment at the indicated time points and the ratio of the 10kDa isoform to the 50 kDa isoform in each type of cell at the indicated time points. (f) Western blot analysis of expression levels of apoptosis markers (tBid and activated caspase 3) in parental IHEECS and recombinant IHEECS stably expressing full-length SRC-1 or the SRC-1 C-terminal isoform upon 10 ng ml−1 TNF-α treatment at the indicated time points. (g) Immunoprecipitation (IP) of caspase 8 with parental IHEECs or IHEECs stably expressing full-length SRC-1 (SRC-1) or the SRC-1 C-terminal isoform (SRC-1 Iso) treated with vehicle or 10 ng ml−1 of TNF-α for 8 h. Western blot analysis of levels of the Flag-SRC-1 C-terminal isoform, SRC-1, Flag-SRC-1 and caspase 8 in immunoprecipitated pellets. *; Non-specific bands. (h) Transwell invasion assays of parental HEECS and recombinant IHEECS stably expressing full-length SRC-1 or the SRC-1 C-terminal isoform for 2 d and the average number of invasive cells per field at the indicated time points. (i) Western blot analysis of expression patterns of EMT markers (such as N-cadherin, Vimentin, Slug, Snail, β-catenin, and TCF8) in parental IHEECS and recombinant IHEECS stably expressing full-length SRC-1 or the SRC-1 C-terminal isoform upon 10 ng ml−1 TNF-α treatment at the indicated time points. (j) TNF-α/MMP9/SRC-1 isoform functional axis in human endometriotic epithelial cells. *** P<0.001 by Student's t test. Scale bars, 50 μm.
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