Matrix metalloproteinase-25 has a functional role in mouse secondary palate development and is a downstream target of TGF-β3

Abstract

BackgroundDevelopment of the secondary palate (SP) is a complex event and abnormalities during SP development can lead to cleft palate, one of the most common birth disorders. Matrix metalloproteinases (MMPs) are required for proper SP development, although a functional role for any one MMP in SP development remains unknown. MMP-25 may have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the MMP-25 gene. We report on the gene expression profile of MMP-25 in the developing mouse SP and identify its functional role in mouse SP development.ResultsMMP-25 mRNA and protein are found at all SP developmental stages in mice, with the highest expression at embryonic day (E) 13.5. Immunohistochemistry and in situ hybridization localize MMP-25 protein and mRNA, respectively, to the apical palate shelf epithelial cells and apical mesenchyme. MMP-25 knockdown with siRNA in palatal cultures results in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium. MMP-25 mRNA and protein levels significantly decrease when cultured palate shelves are incubated in growth medium with 5 μg/mL of a TGF-β3-neutralizing antibody.ConclusionsOur findings indicate: (i) MMP-25 gene expression is highest at E12.5 and E13.5, which corresponds with increasing palate shelf growth downward alongside the tongue; (ii) MMP-25 protein and mRNA expression predominantly localize in the apical epithelium of the palate shelves, but are also found in apical areas of the mesenchyme; (iii) knockdown of MMP-25 mRNA expression impairs palate shelf fusion and results in significant medial edge epithelium remaining in contacted areas; and (iv) bio-neutralization of TGF-β3 significantly decreases MMP-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development and are the first to identify a role for a single MMP in mouse SP development.

Transforming growth factor-β (TGF-β) pathway abnormalities in tenascin-X deficiency associated with CAH-X syndrome

“…This result lends support to the Western blot findings, since it is known that excess TGF-β3 leads to increased pSmad1 expression. [12] However, secreted TGF-β1, TGF-β2, and BMP-4 were not different between groups (p > 0.05, data not shown). The possibility that the secreted TGF-β3 could potentiate an auto-feedback loop on the CAH-X fibroblasts was tested; however, no effect was seen in the fibroblasts (data not shown).…”

“…[18] In mouse models of cleft palate, it was shown that increased TGF-β3 in tissue and palatal fibroblasts led to increased signaling of pSmad1 and MMP-25 and MMP-13 (collagenase-3), which are also important in palate formation. [12, 13]…”

“…[20] In support of the BMP pathway findings, we found that CAH-X dermal fibroblasts secreted significantly more TGF-β3, which is known to induce pSmad1 expression. [12] TGF-β3 is essential for palatal confluence and mutations in TGFB3 are linked to cleft palate in mice. [18, 21] Rare variants in TGFB3 have been linked to nonsyndromic cleft palate in humans[22] and a mutation in TGFB3 has been shown to be disease causing in a human patient presenting with myopathy, joint laxity, distal contractures, bifid uvula, and other features overlapping with TGF-β spectrum disorders.…”

Association of MMP3 and TIMP2 promoter polymorphisms with nonsyndromic oral clefts

“…Considerable biological evidence implicates MMPs as key molecules during palatogenesis for their role in extracellular matrix remodeling (Morris-Wiman et al, 2000; Blavier et al, 2001; Brown et al, 2002; Brown and Nazarali, 2010; de Oliveira Demarchi et al, 2010). In this study, we investigated 45 polymorphisms in 14 MMP and TIMP genes relevant for craniofacial development as candidates for oral clefts in a large case-control dataset from Brazil.…”

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…MMP-25 gene expression is high during the development of mouse secondary palate, wherefore MMP-25 is the only MMP showing a functional role (Brown and Nazarali, 2010).…”