Orofacial clefts are some of the most common birth disorders today. Typically, they are disfiguring, can affect respiration, speech, and eating, and require many surgeries to repair. The two main types of orofacial clefts are cleft lip with or without cleft palate (CL/P) and cleft palate (CP) alone. CL/P occurs in approximately 0.2 to 2.3 births per 1000 and CP in 0.1 to 1.1 per 1000 births . CL/P can affect the primary palate (PP), in which the four maxillary incisors are set at the front of the mouth, or both the PP and secondary palate (SP). The SP is posterior to the PP and forms the main barrier between the oral and nasal cavities. Similarly, CP can affect the SP or both the PP and SP.
Development of the SP begins around embryonic day (E) 12.0 in mice and in week six during human gestation. Due to developmental similarities between mice and humans, the mouse is an ideal model animal in which to study SP development. Initially, the SP exists as two shelves that arise from maxillary prominences on either side of the tongue. These shelves will grow downward alongside the tongue then quickly elevate when the tongue depresses, ultimately growing together around E14.0. With their medial edges in contact, the two shelves fuse together and the epithelial cells at the center form the midline epithelial seam (MES). By E15.5-E16.0, this MES has degraded and a solid, confluent SP remains (reviewed in [2, 3]). Development of the SP is a carefully coordinated event and requires the actions of many proteins, including transcription factors , growth factors and their receptors, and tissue re-modeling enzymes (reviewed in ). Any problem with shelf growth, elevation, tongue depression, shelf fusion, or MES degradation can result in a CP.
Among the proteins required for SP development are the matrix metalloproteinases (MMPs). These are a group of proteases with extra-cellular matrix substrates that require coordination of a zinc ion at the centre of the catalytic domain to be active. The MMPs are broadly classed into two categories: the secreted and the membrane-associated. They are synthesized intra-cellularly as pro-enzymes and activated via cleavage of their pro-domain in the Golgi network or extra-cellularly (reviewed in ). Treatment of in vitro SP cultures with a general chemical inhibitor of the MMPs results in impaired palate shelf fusion and persistence of the medial edge epithelium (MEE) where the shelves make contact . However, the MMP family contains 25 members and which one of the MMPs is playing a functional role in SP development is not clear. One candidate is MMP-25 (Membrane-type (MT) 6-MMP; Leukolysin), as a genetic analysis of human CP patients reveals a potential association between MMP-25 and CL/P .
MMP-25 is a membrane-associated MMP first cloned from leukocytes but later found in most tissues examined [9, 10]. This protein appears to have roles in both healthy and diseased systems. In healthy systems, MMP-25 can act as both an extra-cellular protease and an activator of some secreted pro-MMPs (reviewed in ); however, MMP-25 is also over-expressed in certain cancers and appears to play a role in their progression [12, 13].
Despite evidence to support a functional role for MMPs in SP development, no further investigations of individual MMPs and their role in SP formation have been conducted and thus the role of MMP-25 in SP development is currently unknown. To our knowledge, this study represents the first work done on MMP-25 in the mouse SP.
Using quantitative real-time PCR, in situ hybridization (ISH), western blot analysis, and immunohistochemistry (IHC), we show MMP-25 mRNA and protein are expressed at all stages of the developing mouse SP with a significant down-regulation at approximately E15.5 as the MES degrades and SP development comes to an end. Using an in vitro culture method and MMP-25-specific small, interfering RNA (siRNA), we show in vitro SP cultures treated with MMP-25 siRNA exhibit significantly decreased fusion success and increased persistence of the MEE. Lastly, addition of a TGF-β3-neutralizing antibody to in vitro SP cultures results in significantly decreased MMP-25 mRNA and protein expression, providing evidence that MMP-25 is a downstream target of TGF-β3 signaling.