Foxf2 and Smad6 co-regulation of collagen 5A2 transcription is involved in the pathogenesis of intrauterine adhesion

Abstract

The replacement of normal endometrial epithelium by fibrotic tissue is the pathological feature of intrauterine adhesion (IUA), which is caused by trauma to the basal layer of the endometrium. COL5A2 is a molecular subtype of collagen V that regulates collagen production in fibrotic tissue. Here, we investigated the roles of Foxf2 and Smad6 in regulating the transcription of COL5A2 and their involvement in the pathogenesis of IUA. Small interference-mediated Foxf2 (si-Foxf2) silencing and pcDNA3.1-mediated Smad6 (pcDNA3.1-Smad6) up-regulation were performed in a TGF-β1-induced human endometrial stromal cell line (HESC) fibrosis model. Assessment of collagen expression by Western blotting, immunofluorescence and qRT-PCR showed that COL5A2, COL1A1 and FN were significantly down-regulated in response to si-Foxf2 and pcDNA3.1-Smad6. Transfection of lentivirus vector-Foxf2 (LV-Foxf2) and pcDNA3.1-Smad6 into HESCs and qRT-PCR showed that Foxf2 promoted COL5A2 expression and Smad6 inhibited Foxf2-induced COL5A2 expression. Co-immunoprecipitation, chromatin immunoprecipitation and dual-luciferase reporter assays to detect the interaction between Foxf2 and Smad6 and their role in COL5A2 transcription showed that Foxf2 interacted with Smad6 and bond the same promoter region of COL5A2. In a rat IUA model, injection of ADV2-Foxf2-1810 and ADV4-Smad6 into the uterine wall showed that Foxf2 down-regulation and Smad6 up-regulation decreased fibrosis and the expression of COL5A2 and COL1A1, as detected by haematoxylin/eosin, Masson trichrome staining and immunohistochemistry. Taken together, these results suggested that Foxf2 interacted with Smad6 and co-regulated COL5A2 transcription in the pathogenesis of IUA, whereas they played opposite roles in fibrosis.

Keywords: Foxf2; Smad6; fibrosis; intrauterine adhesion.

Figure 1

Microarray sequencing for gene expression profiles of endometrial specimen (n = 5). (A) Heat map and Volcano plot representation of tissue microarray sequencing for gene expression profiles of IUA and normal control. Abbreviations: M, moderate IUA group; N, normal group; S, severe IUA group. (B) The relative mRNA expression of COL1A1, COL5A1, COL5A2, Foxf2 and Smad6 in each group. ^# P < .05, compared with normal group. *P < .05, compared with moderate IUA group

Figure 2

Human endometrial stromal cell lines (HESCs) were treated with TGF‐β1 (0, 1, 5, 10, 15 ng/mL) to select an optimal dosage to create a cell fibrosis model (n = 3). (A) Western blotting analysis of COL1A1, COL5A2 and FN in different concentration groups of TGF‐β1. (B) qRT‐PCR analysis of the mRNA expressions of COL1A1, COL5A2 and FN in different concentration groups of TGF‐β1. ^# P < .05, compared with control group (0 ng/mL). *P < .05, compared with 1 ng/mL group. **P < .05, compared with 5 ng/mL group

Figure 3

Foxf2 down‐regulation or (and) Smad6 up‐regulation have an anti‐fibrotic effects in TGF‐β1 stimulated cell fibrosis. HESCs were transfected with si‐Foxf2 or (and) pcDNA3.1‐Smad6 and then treated with TGF‐β1 (n = 3). (A) qRT‐PCR analysis of the COL5A2, COL1A1, Foxf2 and Smad6 expression in each group. Foxf2 and Smad6 mRNA expressions were obviously down‐regulated and up‐regulated by si‐Foxf2 and pcDNA3.1‐Smad6, respectively. The result showed that TGF‐β1 induced fibrosis, the expressions of COL5A2 and COL1A1 were increased by TGF‐β1, whereas down‐regulation of Foxf2 or (and) up‐regulation of Smad6 inhibited the expressions of COL5A2 and COL1A1 induced by TGF‐β1. **P < .05, compared with normal group. *P < .05, compared with pcDNA3.1 group. ^# P < .05, compared with TGF‐β1 and si‐negative control groups. ^## P < .05, compared with pcDNA3.1‐Smad6 group. (B) Western blotting analysis of the COL5A2, COL1A1, FN, a‐SMA, Foxf2 and Smad6 expressions in each group. (C) IF analysis of Foxf2 and COL5A2 expressions. The result showed that TGF‐β1 promoted COL5A2 expression, whereas down‐regulation of Foxf2 decreased COL5A2 expression induced by TGF‐β1. (D) IF analysis of Smad6 and COL5A2 expression. The result showed that TGF‐β1 promoted COL5A2 expression, whereas up‐regulation of Smad6 decreased COL5A2 expression induced by TGF‐β1

Figure 4

Foxf2 down‐regulation or/and Smad6 up‐regulation affects cell proliferation and cell cycle distribution induced by TGF‐β1 (n = 3). HESCs were transfected with si‐Foxf2 or (and) pcDNA3.1‐Smad6 and then treated with TGF β1 (A) EdU analysis of cell proliferation in each group. The result showed that TGF‐β1 increased EdU‐positive cell number, whereas down‐regulation of Foxf2 or (and) up‐regulation of Smad6 decreased EdU‐positive cell number induced by TGF‐β1. ** P < .05, compared with normal group. ^# P < .05, compared with TGF‐β1 and negative control groups. ^## P < .05, compared with pcDNA3.1 group. * P < .05, compared with pcDNA3.1‐Smad6 group. (B) Flow cytometry analysis of cell cycle distribution in each group. The result showed that TGF‐β1 promoted G0/G1 phase transition into S phase, whereas down‐regulation of Foxf2 or/and up‐regulation of Smad6 reversed the cell cycle changes induced by TGF‐β1. ** P < .05, compared with normal group. ^# P < .05, compared with TGF‐β1 and si‐negative control groups. * P < .05, compared with pcDNA3.1 group

Figure 5

Foxf2 and Smad6 co‐regulate COL5A2 transcription in the pathogenesis of HESC fibrosis. (A) Co‐IP analysis of the interaction between Foxf2 and Smad6. Cell lysates were immunoprecipitated with anti‐Foxf2 antibody and anti‐Smad6 antibody, respectively, and were subjected to subsequent immunoblotting with anti‐Smad6 or anti‐FoxfF2 antibody. The result confirmed that Foxf2 interacts with Smad6. (B) ChIP analysis of the potential binding sites at the promoter region of COL5A2 that Smad6 bond. DNA fragments immunoprecipitated by anti‐Smad6 antibody were quantified by qPCR using primers covering predicted binding site. The result showed that Smad6 may bind at the promoter region of COL5A2. (C) ChIP analysis of the potential binding sites at the promoter region of COL5A2 that Foxf2 bond. DNA fragments immunoprecipitated by anti‐Foxf2 antibody were quantified by qPCR using primers covering predicted binding site. The result showed that Foxf2 may bind at the promoter region of COL5A2. (D) LV‐Foxf2 was cotransfected with PGL3, PGL3‐COL5A2‐1, PGL3‐COL5A2‐2, PGL3‐COL5A2‐3 into 293T cells (n = 3). Dual‐luciferase assay was performed to detect the activity and showed that Foxf2 bond PGL3‐COL5A2‐3. (E) pcDNA3.1‐Smad6 was cotransfected with PGL3, PGL3‐COL5A2‐1, PGL3‐COL5A2‐2, PGL3‐COL5A2‐3 into 293T cells. Dual‐luciferase assay was performed to detect the activity and showed that Smad6 bond PGL3‐COL5A2‐3. (F) RT‐PCR analysis of the effect of Foxf2 and Smad6 on COL5A2 expression. The result showed Foxf2 promoted COL5A2 expression, but Smad6 inhibited Foxf2‐induced COL5A2 expression

Figure 6

The efficiency of ADV2‐Foxf2 and ADV4‐Smad6 in rat endometrium. (A) Fluorescence analysis of uterine frozen section after transfected with ADV2‐Foxf2 or ADV4‐Smad6 (n = 3). The ADV2‐Foxf2‐1810 carried red fluorescence, and the ADV4‐Smad6 carried green fluorescence. (B) qRT‐PCR analysis of Foxf2 and Smad6 expression in rat endometrium after transfected with ADV2‐Foxf2‐1810 or ADV4‐Smad6. The red histogram represented relative Foxf2 mRNA expression, and ADV2‐FOXF2‐1810 decreased Foxf2 mRNA expression significantly compared with normal and ADV2‐negative control groups (P < .05). The green histogram represented relative Smad6 mRNA expression. ADV4‐Smad6 up‐regulated Smad6 mRNA expression significantly compared with normal and ADV4‐negative control groups (P < .05)

Figure 7

The effect of Foxf2 down‐regulation or (and) Smad6 up‐regulation on anti‐fibrosis in rat IUA model (n = 10). (A) HE staining analysis of the number of glands in rat endometrium in each group. ^# P < .05, compared with sham operation group. * P < .05, compared IUA model and ADV‐negative control group. ** P < .05, compared with Smad6 up‐regulation therapy group. (B) Masson staining of analysis of the percentage of fibrosis area in each group. ^# P < .05, compared with sham operation group. * P < .05, compared with IUA model and ADV‐negative control group. ** P < .05, compared with Smad6 up‐regulation therapy group

Figure 8

The effect of down‐regulation of Foxf2 or (and) up‐regulation of Smad6 on collagen productions in rat IUA model (n = 10). (A) HE immunostaining analysis of COL5A2 expression in each group. (B) HE immunostaining analysis of COL1A1 expression in each group. (C) HE immunostaining analysis of Foxf2 expression in each group. (D) HE immunostaining analysis of Smad6 expression in each group. (E)The immunostaining was evaluated by semi‐quantitatively scored using the modified histochemical score (H‐score), and data were showed as mean ± SD. The result showed that down‐regulation of Foxf2 or (and) up‐regulation of Smad6 inhibited COL1A1 and COL5A2 expression in rat IUA model. ^# P < .05, compared with sham operation group. * P < .05, compared IUA model and ADV‐negative control group. ** P < .05, compared with Smad6 up‐regulation therapy group