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The Importance of the Canonical Wnt Signaling Pathway in the Porcine Endometrial Stromal Stem/Progenitor Cells: Implications for Regeneration.

ABSTRACT: The regenerative ability of the endometrium is strongly associated with the presence of adult stem/progenitor cells. Purposes of the present study were (1) to establish the presence of stem/progenitor cells in porcine endometrial stroma using a clonogenic assay and (2) to investigate whether the canonical Wnt pathway affects the potential of stem/progenitor cells to undergo self-renewal or differentiation. The utility of endometrial stromal clones as a model for stem/progenitor studies was evaluated based on these cells' increased expression of mesenchymal stem cell (MSC) marker genes, including CD29, CD73, CD90, and CD105, compared with primary cultured cells. Small molecules were introduced to activate (BIO) or inhibit (XAV939) the canonical Wnt pathway during stromal clone formation. Cloning efficiency assays revealed that activation of the Wnt/β-catenin pathway promoted formation of more differentiated small clones. Moreover, activation of the Wnt/β-catenin pathway decreased, whereas inhibition of the pathway increased MSC marker expression. Additionally, we confirmed the importance of canonical Wnt pathway stimulation in endometrial stromal cells through observing the appropriate changes in β-catenin cellular localization. These data indicate that modulation of the canonical Wnt pathway effects the process of regeneration in the porcine endometrium during the course of the estrous cycle.

SUBMITTER: Bukowska J

PROVIDER: S-EPMC4683563 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. METHODS:FOXF2, TGF-?1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-?1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/?-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/?-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. RESULTS:FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10?ng/ml TGF-?1 for 72?h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-?1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/?-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/?-catenin pathway, thereby altering TGF-?1-mediated ECM aggregation in endometrial stromal cells ex vivo. CONCLUSIONS:Regulation of the Wnt/?-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-?1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-?1-mediated fibrosis in primary HESCs.

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The Importance of the Canonical Wnt Signaling Pathway in the Porcine Endometrial Stromal Stem/Progenitor Cells: Implications for Regeneration.

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