Knockdown of Hoxa11 In Vivo in the Uterosacral Ligament and Uterus of Mice Results in Altered Collagen and Matrix Metalloproteinase Activity1

Abstract

Homeobox (HOX) genes are evolutionarily conserved genes encoding transcription factors that regulate mammalian embryonic growth and development of the urogenital tract. In both humans and mice, HOXA11 persists in the adult reproductive tract and is thought to play an important role in maintaining tissue developmental plasticity by regulating the expression of genes involved in extracellular matrix metabolism in the reproductive organs. Previously, we have shown that HOXA11 is necessary for development of the uterosacral ligaments in mice and is deficient in women with pelvic organ prolapse. Therefore, we hypothesized that Hoxa11 regulates the synthesis and/or metabolism of collagens in the uterosacral ligaments and uterus, and tested this by establishing an in utero and peritoneal Hoxa11 gene knockdown system in C57/BL6 mice using vectors bearing Hoxa11 short hairpin RNA. Specific knockdown of Hoxa11 transcripts and protein levels were confirmed versus control vectors. Protein and mRNA expression of collagen types I and III exhibited significant decreases following Hoxa11 knockdown according to Western blot analysis and real-time PCR. Tissue inhibitor of matrix metalloproteinase 1 (MMP1) expression also exhibited a significant decrease. Gelatinase zymography confirmed increases in pro-MMP2 and MMP9, as well as activated MMP2, following Hoxa11 knockdown. These results reveal that Hoxa11 knockdown in the uterosacral ligaments and uterus increases extracellular matrix degradation. More importantly, it suggests a mechanism in the weakening of the pelvic floor support in women, because decreased HOXA11 gene expression has been reported to be associated with decreased collagen and increased MMP2 expression in the uterosacral ligaments of women with pelvic organ prolapse.

Recent studies of genetic dysfunction in pelvic organ prolapse: The role of collagen defects

“…It is proposed that HOXA11 regulates the expression of genes involved in extracellular matrix metabolism. 55 Allen-Brady et al, 56 used genomewide association analysis on 2976 women and identified six single nucleotide polymorphisms (SNP) linked to POP. SNPs are very common mutations, occurring when a different nucleotide is substituted for the normal nucleotide in a DNA sequence.…”

Tissue Remodeling in the Female Reproductive Tract—A Complex Process Becomes More Complex: The Role of Hox Genes

“…Ma and colleagues demonstrate that both TIMP1 and Timp2 decrease in the Hoxa11 KD mice. However, Timp1 was not affected, suggesting that TIMP1 was regulated at a posttranscriptional level [7].…”

“…Thus, the study by Ma and others [7] represents a significant step in our understanding of female reproductive tissue remodeling. Their approach using a Hoxa11 KD [7] mouse model, with similar pelvic anatomy to humans [29], provides the foundation for elucidating the complex pathways of tissue remodeling that are critical to our understanding of female reproductive tract disorders. An important question to answer is: are the changes in collagens, MMP2, Timp2, and MMP9 an effect of attempted repair after injury or a cause of POP because of collagen destruction in the uterosacral ligament [12,26]?…”

“…Interestingly, pro-MMP2 is recruited to the cell surface and activated by the membrane-bound MT-MMPs and therefore probably has very local collagenase activity [24]. The study by Ma et al [7] utilizes gelatin zymography to demonstrate that pro-MMP2 and pro-MMP9 and active MMP2 are increased in Hoxa11 KD mice. In gelatin zymography, the active forms of the MMPs are separated from their latent forms by molecular mass, as the active forms of the enzymes do not possess the 10-kDa inhibitory N-terminal domain and are therefore smaller molecules [27].…”

“…1) occurs when changes in Hox/HOX gene expression modulate expression of ECM molecule receptors and matrix-degrading enzymes and their inhibitors, which then further alters the expression of Hox/HOX genes and ultimately results in additional alterations in the ECM [28]. Thus, the study by Ma and others [7] represents a significant step in our understanding of female reproductive tissue remodeling. Their approach using a Hoxa11 KD [7] mouse model, with similar pelvic anatomy to humans [29], provides the foundation for elucidating the complex pathways of tissue remodeling that are critical to our understanding of female reproductive tract disorders.…”