Pelvic organ prolapse (POP) is characterized by abnormal protrusion of female pelvic organs involving the uterus, bladder, and vagina (reviewed in refs. 1
). Epidemiologic studies indicate that (a) vaginal birth, aging, and increased body mass index are major risk factors for the development of POP and (b) more than 1 pathology may be involved to exhibit full anatomical loss of support. Although approximately 11% of women have surgery for POP or urinary incontinence in their lifetimes (4
), to date, effective therapies to prevent progression of POP have not been established, thereby imposing profound social and financial burden to affected individuals (3
Despite a difference in anatomical position of pelvic organs relative to the body axis and pelvic floor, rodent models of POP have provided important tools to study underlying mechanisms of prolapse. In contrast to an observation that rectal prolapse is frequently associated with the presence of chronic inflammatory bowel disease (5
), POP has been found in animals with defective ECM proteins, including fibulin-3, fibulin-5, and lysyl oxidase-like–1 (LOXL-1) (an enzyme that predominantly catalyzes cross-linking of elastin) (6
). Interestingly, these proteins are abundantly expressed in the vaginal wall and involved in synthesis and assembly of elastic fibers. It is also known that uterosacral ligaments support the vaginal wall and that Hoxa11 is essential for formation of uterosacral ligaments in mice (9
-deficient mice exhibit increased mobility of the uterus; however, POP was not reported. Hence, it has been proposed that intact elastic fibers are required for maintenance of vaginal wall support. The specific role of elastic fibers in this process is not clear; however, because upregulation of matrix proteases in the vaginal wall either precedes or coincides with POP in these mice with elastic fiber deficiencies, a potential link between elastic fibers, matrix degradation, and development of POP is suggested (10
). The precise mechanisms that regulate matrix proteases in the vaginal wall, whether or not a tissue-specific mechanism exists, and how these mechanisms affect the integrity of the vaginal wall have not been fully elucidated.
Fibulins are a family of matricellular modular proteins characterized by the presence of repeated calcium-binding EGF-like motifs and a C-terminal fibulin domain and are involved in structural stability of the basement membrane and formation of elastic fibers (reviewed in refs. 11
). Among fibulins, fibulin-5 possesses potent elastogenic functions (reviewed in ref. 13
). It was suggested that fibulin-5 organizes elastic fiber assembly machinery on microfibrillar scaffolds by serving as an adaptor protein to structural components of elastic fibers and cross-linking enzymes, including elastin, fibrillin-1, emilin-1, and LOXL-1 (8
). Systemic elastic fiber phenotypes are evident early in life in Fbln5–/–
mice without overt inflammatory cell infiltration, suggesting that the underlying defect is primarily a developmental defect in elastogenesis rather than a progressive destruction of elastic fibers (14
). Interestingly, however, the POP phenotype in Fbln5–/–
mice is not observed until after puberty and is preceded by substantial upregulation of MMP-9 in the vaginal wall (10
). In addition, delayed manifestation and progressive worsening of the POP phenotype correlate with aging (7
Fibulin-5 contains a motif that is not shared by other fibulins: an evolutionally conserved arginine-glycine-aspartic acid (RGD) sequence known to mediate binding to cell surface integrin receptors (20
). Binding of the RGD-containing ECM proteins to integrins transmits environmental cues to cells, which activates intracellular signaling and induces cytoskeletal changes, as well as upregulates metalloproteases (ref. 21
, reviewed in ref. 22
, and ref. 23
). The RGD in fibulin-5 has been previously shown to facilitate adhesion of endothelial cells and inhibit transcription of MMP-7 in breast cancer cells (24
). We have also shown that the RGD-dependent fibulin-5–integrin interaction controls pancreas tumor growth by regulating ROS production and tumor angiogenesis (26
). Although it is initially speculated that the binding of fibulin-5 to integrins is necessary for tethering of newly synthesized elastic fibers to surrounding cells, the exact role of the RGD motif in elastic fiber assembly has not been elucidated. Therefore, we sought to determine the biological function of the RGD motif of fibulin-5, focusing on pathogenesis of POP. We hypothesized that disruption of the RGD motif would result in impaired elastic fiber assembly and dysregulation of MMP-9 in connective tissues of the pelvic floor.
Here, we demonstrate a dual role of fibulin-5 in regulation of matrix homeostasis in the vaginal wall. First, we show that fibulin-5 facilitates assembly of elastic fibers independent of integrins and that this fibulin-5–mediated elastogenesis is crucial for pelvic organ support. Second, we present evidence that fibulin-5 inhibits MMP-9 in an integrin-dependent manner. This function of fibulin-5 is important in the pathogenesis of POP because abnormal elastic fibers alone, without MMP-9 activation, are insufficient for full manifestation of the disease. Thus, fibulin-5 strengthens the vaginal wall and protects from prolapse development. Our finding that genetic ablation of MMP-9 significantly attenuated progression of POP in vivo provides the foundation for development of novel preventative and therapeutic strategies for POP in women.