Molecular Landscape of Pelvic Organ Prolapse Provides Insights into Disease Etiology

Abstract

Pelvic organ prolapse (POP) represents a major health care burden in women, but its underlying pathophysiological mechanisms have not been elucidated. We first used a case-control design to perform an exome chip study in 526 women with POP and 960 control women to identify single nucleotide variants (SNVs) associated with the disease. We then integrated the functional interactions between the POP candidate proteins derived from the exome chip study and other POP candidate molecules into a molecular landscape. We found significant associations between POP and SNVs in 54 genes. The proteins encoded by 26 of these genes fit into the molecular landscape, together with 43 other POP candidate molecules. The POP landscape is located in and around epithelial cells and fibroblasts of the urogenital tract and harbors four interacting biological processes-epithelial-mesenchymal transition, immune response, modulation of the extracellular matrix, and fibroblast function-that are regulated by sex hormones and TGFB1. Our findings were corroborated by enrichment analyses of differential gene expression data from an independent POP cohort. Lastly, based on the landscape and using vaginal fibroblasts from women with POP, we predicted and showed that metformin alters gene expression in these fibroblasts in a beneficial direction. In conclusion, our integrated molecular landscape of POP provides insights into the biological processes underlying the disease and clues towards novel treatments.

Keywords: TGFB1; exome chip study; genetics; metformin; pelvic organ prolapse; single nucleotide variants (SNVs).

Figure 1

Molecular landscape of pelvic organ prolapse (POP). The POP landscape is located in and around epithelial cells and fibroblasts of the female urogenital tract. Four main biological processes operate in the landscape: (1) The first and most important process in the landscape is EMT. Under normal conditions, epithelial cells are connected to their environment through cell-cell interactions as well as cell-ECM interactions. Among the cell-cell interactions are epithelial cadherin (E-cadherin/CDH1)-based adherens junctions that stabilize epithelial cell-cell adhesion. EMT is characterized by a ‘cadherin switch’, which is initiated when epithelial cells start producing less CDH1 and more of the mesenchymal marker N-cadherin (CDH2) (not shown). This results in loss of epithelial cell-cell adhesion and the epithelial cells gradually transforming into mesenchymal cells, which then further differentiate into fibroblasts. EMT involves the key transcription factor SMAD3 and is negatively regulated by three sex hormone-bound receptors—the estrogen receptors ESR1 and ESR2 and the progesterone receptor (PGR)—that each upregulate CDH1 expression and hence prevent EMT. Conversely, TGFB1 induces EMT as it downregulates CDH1 expression. (2) Proteins of the major histocompatibility complex II (MHC II)—i.e., HLA-DQA1 and HLA-DQB1—are expressed in epithelial cells of the female reproductive tract, where they are involved in regulating the activity of the immune response through presenting foreign antigens to circulating T lymphocytes (not shown). TGFB1 regulates the expression of these two HLA proteins, in part through upregulating the expression of beta-2-microglobulin (B2M), an EMT-inducing extracellular protein that mediates antigen presentation. (3) Further, the ECM provides support to epithelial cells and fibroblasts, and modulation of the ECM is essential for many pathophysiological processes such as tissue growth, wound healing, and fibrosis. The ECM is composed of different molecules, including elastin (ELN) and collagen (COL) fibers, as well as proteins that crosslink or regulate the degradation of ELN and COL (e.g., LOX and LOXL1), various matrix metalloproteinases (MMPs) and their regulators, TIMP1 and TIMP2. Other important ECM proteins are ECM1, FBLN5, LAIR2, and LAMC1. TGFB1 is involved in ECM remodeling through regulating the expression of most ECM proteins in the landscape. (4) Since fibroblasts are responsible for the synthesis and secretion of the main ECM components through SMAD3—which, as indicated above, also plays a major role in EMT—fibroblast survival and apoptosis (and hence proper functioning) is another important landscape process. TRAIL, a cytokine of the tumor necrosis factor (TNF) family, regulates fibroblast survival and apoptosis through its receptors and signaling involving pro-apoptotic proteins such as NFKB and CASP3. As with the three other main processes, TGFB1 is a key regulator, e.g., through regulating the expression of TRAIL-R1 and CASP3 and activating NFKB. In addition, extracellular AGE (or advanced glycation end product) molecules binding to their receptor RAGE results in NFKB activation, CASP3 inhibition, and translocation of SMAD3 to the nucleus, which results in the AGE-RAGE complex stimulating the apoptosis of fibroblasts. Lastly, nuclear proteins such as HIF1A and PARP1 are also important regulators of fibroblast survival and apoptosis.

Figure 2

Metformin downregulates TGFB1-induced gene expression in fibroblasts from women with POP. Cells derived from prolapsed tissues from premenopausal women (n = 4) were stimulated for 24 h with TGFB1 [0.1 ng/mL] and then challenged with Metformin [2 mM] for another 24 h. Real-time PCR was used to measure three TGFB1-regulated genes from the landscape, i.e., the collagen genes COL1A1 and COL3A1 and elastin (ELN) and the two housekeeping genes YWHAZ and HPRT1. (A) shows the percentage (%) of downregulated gene expression in TGFB1-stimulated fibroblasts (with the dotted line corresponding to 100% gene expression in cells treated with TGFB1 [0.1 ng/mL]). One outlier was identified using Grubbs’ test [18] and removed from the ELN gene expression data set (n = 3). (B–D) show the expression of COL1A1, ELN, and COL3A1 in the fibroblasts from each individual woman with POP normalized to the housekeeping genes according to the Pfaffl model [19], respectively.