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Matrix Metalloproteinases have a Role in Palatogenesis
N.L. Brown,
S.J. Yarram,
J.P. Mansell and
J.R. Sandy*
Division of Child Dental Health, University of Bristol Dental School, Lower Maudlin Street, Bristol, BS1 2LY, UK;
Correspondence: *corresponding author, Jonathan.Sandy{at}bristol.ac.uk
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ABSTRACT
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Mammalian palatogenesis depends on palatal shelf elevation, medial edge epithelium (MEE) breakdown, and mesenchyme flow. These all require matrix remodeling, which is controlled in part by the family of matrix metalloproteinases (MMPs). We used an organ culture system to examine the effect of a general MMP inhibitor (BB3103) on mouse palatogenesis. Palates cultured in 20 µM BB3103 contained no active MMP-2, and only one palate fused from a sample size of 15. In this single palate, MMP-3 was present at higher levels than in palates that failed to fuse. MMP-3 is known to be involved in epithelial mesenchymal transformation (EMT), and its persistence may explain why this palate fused. This implies a role for MMPs in normal palatogenesis, and disruption of their activity may result in cleft palate.
Key Words: palate • MMP-2 • MMP-3
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INTRODUCTION
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Normal development of the mammalian palate is a complex co-ordinated sequence of events which, if disrupted, results in clefting. In man, cleft lip and palate account for about 65% of all congenital anomalies (Slavkin, 1995). The cause of clefting is not known, but environmental and genetic factors are involved. Secondary palatogenesis (Fig. 1 ) commences with the maxillary processes of the first branchial arch orientated downward. At a precise developmental stage, these vertical palatal shelves elevate to a horizontal position above the dorsum of the tongue (Ferguson, 1988). This movement occurs in a matter of minutes or hours (Brinkley, 1980), any delay may result in failure of fusion. What happens to MEE is clearly important, since persistence will prevent fusion of palatal shelves and continued mesenchyme flow. Three mechanisms to explain the loss of MEE have been proposed: Epithelial cells may undergo transformation to mesenchyme, migrate to oral and nasal epithelia, or undergo apoptosis (Martinez-Alvarez et al., 2000; Sun et al., 2000).
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Figure 1. Hematoxylin and eosin (H&E)-stained sections showing palate development in vivo alongside palates cultured in vitro. Scale bar represents 0.1 mm. The tongue (T), palatal shelves (P), nasal septum (NS), Meckel's cartilage (M), and medial edge epithelium (MEE) are labeled. (A) Vertical orientation of palatal shelves. (B) Horizontal orientation of palatal shelves, no contact. (C) Palatal shelves in contact, medial edge epithelium present. (D) Palatal shelves fused, mesenchyme continuous.
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The process of shelf elevation involves a combination of intrinsic and extrinsic factors, and it is known that remodeling of the extracellular matrix (ECM) is likely to be important. The proteases responsible for ECM turnover may be implicated in cleft palate. A wide range of proteolytic enzymes are involved in ECM catabolism, including the MMPs. The role of MMPs in palatogenesis is not yet known. We and others have shown a temporal variation in MMPs and TIMPs during palate formation (Morris-Wiman et al., 1999, 2000; Mansell et al., 2000). There is also variation in the spatial distribution, which may correlate with re-organization and transformation of the palatal epithelium and functions such as shelf elevation. Most recently, Blavier et al. (2001) have confirmed this view, suggesting that proteolytic degradation of the ECM is a necessary step for palatal fusion. At least one null mutant exists to reinforce this concept. Newborn EgfR -/- mice have a high incidence of cleft palate, and although palatal shelf explants fuse, the midline epithelial seam persists (Miettinen et al., 1999). The MMP, stromelysin-1, may be pivotal in this process, since induction of its expression results in cleavage of cadherin, loss of epithelial phenotype, and a stable EMT conversion (Lochter et al., 1997). We believe that the polygenetic input into palate formation may disrupt several common mechanisms, and matrix turnover is an example. In this study, we examined the potential role of MMPs in the development of the palate.
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MATERIALS & METHODS
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All reagents were purchased from Sigma (Poole, Dorset, UK) unless otherwise specified.
Animal Maintenance
CD1 mice were maintained on a reverse light/dark cycle, and males were placed with females for 2 hrs during the dark phase. If mated, females were isolated and, if pregnant, killed by cervical dislocation 13.0 days post-coitum +/-2 hrs. This ensured accuracy and consistency in obtaining day 13 embryos for organ culture. For histology, days 14 and 15 embryos were also obtained to demonstrate in vivo palatogenesis. Our animal use protocol was reviewed and approved by an institutional review board.
Dissection and Culture of Palates
After death of the dam, day 13 embryos were removed from the uterus and separated from their amniotic sacs in preparation for organ culture according to a method previously described (al-Obaidi et al., 1995). Briefly, palates were dissected from the embryos with 2 incisions, the first at the level of the oral cavity removing the tongue and mandible, the second at the level of the embryo's eye. This method of dissection ensured that palatal shelves were held in their normal position relative to the nasal floor. Palates were cultured in sialinized roller tubes with silicon rubber septae (3 palates per tube) containing 8 mL of standard culture medium, consisting of Biggers BGJ medium (Gibco BRL, Paisley, UK) supplemented with 0.6% (w/v) bovine serum albumin and 850 µM ascorbic acid. The tubes were sealed and purged with 95% O2/5% CO2 for 3 min before being placed on a roller device (34 RPM) at 37°C. Experiments were based on littermate embryos to minimize variability. Palates were cultured for 72 hrs with gas purges every 24 hrs and change of medium after 48 hrs. Palates were also removed at timed intervals during culture (0, 21, 30, 45, 53, and 72 hrs) for MMP-2 assessment. Following culture, palates were analyzed for fusion and prepared for histological analysis or gelatin gel zymography.
The palates were cultured with either standard media, or media with addition of BB3103 (British Biotech), retinoic acid (5 µM), or DMSO, providing vehicle and positive and negative controls as well as an experimental group for each litter.
Histology
Cultured palates were fixed in formal saline for 24 hrs and prepared for histology on a Shandon processor 2LE with graded industrial methylated spirits, xylene, and paraffin wax baths. On completion, wax was removed from the palates by immersion in 100% xylene for 4 hrs. Specimens were left to air-dry overnight. A light microscope at low magnification with an external light source was used to assess fusion. Palates were then embedded in paraffin wax, sectioned on a microtome (2-µm-thick sections), and either stained with hematoxylin and eosin for histological analysis or prepared for immunohistochemistry. Palates were graded as not fused (no shelf contact, or contact with no MEE breakdown) or fused (MEE breakdown with mesenchymal continuity).
Zymography
For zymography, palatal shelves were dissected from the cranial base and homogenized in 10 mM HEPES buffer at pH 7.4 to a concentration of 50 mg/mL. Homogenates were centrifuged at 5000 RPM for 10 min at 4°C, and the supernatant was used for MMP-2 analysis. A protein assay (Bio-Rad, Hertfordshire, UK) was used to measure the protein concentration for each sample, and the equivalent of 5 µg protein was subjected to electrophoresis in 8% SDS-polyacrylamide gels (acrylamide from Bio-Rad) co-polymerized with 0.5 mg/mL gelatin (Acrylamide from Bio-Rad). On completion of electrophoresis, the gels were bathed in 2.5% (v/v) Triton X-100 for 10 min and incubated at 37°C for 24 hrs in 50 mL of 0.5 M NaCl, 0.05 M CaCl2, 0.05 M Trizma base, pH 7.8, supplemented with p-amino phenyl mercuric acetate (APMA) at a final concentration of 200 µM. APMA activated latent MMP-2 following electrophoresis, allowing for visualization of the 72-kDa latent isoform of MMP-2 on a gelatin gel as well as the 62-kDa active isoform. To confirm that clarified zones were attributed to MMP-2 activity, we incubated the gels in proteolysis buffer supplemented with the MMP inhibitor BB3103. The resultant gel had no clearance zones. Furthermore, protease activity could be eliminated from samples following adsorption with gelatin but not casein agarose. Gels were stained for 15 min with Coomassie blue and then bathed in an aqueous solution of 25% methanol (BDH, Poole, UK) and 9% acetic acid (BDH) for 30 min prior to being scanned with an AGFA studiostar flatbed scanner. Images were visualized with the use of Adobe Photoshop 3.0.
Immunohistochemistry
Sections of paraffin-embedded palates (2 µm thick) were incubated on polylysine-coated slides (BDH) overnight at 37°C. We removed wax by bathing the palates in xylene for 6 min, followed by 15 min in 3% (v/v) hydrogen peroxide. MMP-3 primary antibody was a kind gift from Professor G. Murphy and Dr. J. Gavrilovic, University of East Anglia, and was diluted with normal rabbit serum (DAKO, Cambridgeshire, UK) to a working concentration of 31.4 µg/mL. Sections were bathed in primary antibody for 1 hr, biotin-labeled secondary antibody for 30 min (sheep anti-rabbit immunoglobulin, DAKO), streptavidin horseradish peroxidase for 30 min (DAKO), 3,3'-diaminobenzidine chromogen (DAKO) for 6 min, and hematoxylin for 50 sec. Each step was separated by thorough washes with phosphate-buffered saline. Finally, slides were mounted with coverslips and viewed with conventional light microscopy.
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RESULTS
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Organ Culture
A comparison of in vivo and in vitro palate development is shown in Fig. 1 . The morphology and tissue structure of the palate are similar.
MMP-2
The pro and active forms of MMP-2 increased during mouse palatogenesis in vivo (Fig. 2a). The increase in active MMP-2 was also seen in cultured palates (Fig. 2b), suggesting that the culture model reflected in vivo the changes of MMP-2 levels seen during palatogenesis. In a pilot study to establish potential concentrations of BB3103 which might cause clefting, 6 palates were cultured with increasing concentrations of the MMP inhibitor (Table). At each concentration of BB3103, a single palate was homogenized for MMP-2 analysis and the remainder processed for histology. The Table shows that no inhibition of palatal fusion was seen with BB3103 concentrations less than 10 µM. Single palates were also cultured at 15 and 20 µM BB3103, and Fig. 2c demonstrates the effect of the MMP inhibitor in culture on MMP-2 levels within the palate. There was complete inhibition of the active form of MMP-2 in palatal shelves cultured with 20 µM BB3103, although the 72-kDa latent MMP-2 remained. At concentrations less than 10 µM BB3103, active MMP-2 was still present. Following this pilot study and the resultant zymogram for MMP-2, a concentration of 20 µM BB3103 was used to examine the incidence of palatal clefting in culture with MMP inhibition. A second pilot study was used to determine power. At 20 µM BB3103, the frequency of fusion of palatal shelves decreased from 78% to 11%, comparable with the frequency of fusion of shelves cultured in 5 µM retinoic acid (negative control). Fisher's exact test gave 78% power at the 5% level, with n = 9 in each group. Our pilot data therefore enabled us to calculate appropriate sample sizes to test our hypothesis on the importance of the MMPs in palatogenesis. Fifteen palates were required per group, and final results show that, at 20 µM BB3103, the frequency of fusion of palatal shelves decreased from 80% to 6.6% (Table). Fisher's exact statistical test gave a p value of 0.00013. This was comparable with the frequency of fusion of shelves cultured with the negative control, 5 µM retinoic acid, which induced clefting (13% of palates fused). The palates cultured with 20 µM BB3103 yielded 12 with no contact between palatal shelves and 2 which came into contact but without MEE breakdown. Only one palate showed evidence of MEE breakdown, with a high arched morphology and narrow shelves. The mesenchyme in this latter palate was partly continuous. The failure of fusion in the presence of 20 µM BB3103 was not due to the toxicity of BB3103. This was confirmed by incubation of palatal mesenchymal cells in culture medium supplemented with 20 µM BB3103 for two days with no resultant change in cellularity (assessed with MTS-PMS).
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Figure 2. Gelatin gel zymograms for MMP-2 assessment. Each lane represents a single palate. A 5-µg quantity of protein was loaded per sample. Pro and active (72 and 62 kDa) MMP-2 bands were present on all gels. MMP-9 was not detected by gelatin gel zymography at any of these stages of palate development. (A) MMP-2 levels during in vivo murine palate development. Each of the key stages of palatogenesis is represented in duplicate by embryonic day 13, 14, and 15 palates. Pro and active levels of MMP-2 increase from embryonic day 13. (B) MMP-2 levels during in vitro murine palate development. Elevated active MMP-2 levels with increasing time in culture, suggesting that the in vitro model of palate development was representative of in vivo palatogenesis with respect to MMP-2. (C) MMP-2 levels during in vitro murine palate development with BB3103, a synthetic MMP inhibitor. Reduction of active MMP-2 (62 kDa) to levels undetectable with gelatin gel zymography at 20 µM BB3103.
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MMP-3
MMP-3 was immunolocalized in vivo subjacent to the MEE following contact of the palatal shelves (data not shown). A similar pattern of MMP-3 staining was seen in vitro (Fig. 3a). In those palates cultured with BB3103 and immunostained for MMP-3, much higher levels were seen at the MEE in the single fused palate (Fig. 3b), compared with the 2 which had merely come into contact (Fig. 3c).
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Figure 3. MMP-3 expression during in vitro palatogenesis, with immunohistochemistry. Scale bar represents 0.1 mm. (A) MMP-3 was expressed within the medial edge epithelium during its breakdown. MMP-3 staining was minimal preceding and following this event, similar to the pattern of staining seen in vivo (data not shown). (B) MMP-3 expression in the palate cultured with 20 µM BB3103 that fused. High expression of MMP-3 leads to medial edge epithelium breakdown and palatal shelf fusion in the presence of the inhibitor. (C) MMP-3 expression in a palate cultured with 20 µM BB3103 that attained contact without fusion. Medial edge epithelium breakdown was inhibited. MMP-3 was less prominent in palates that failed to fuse.
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DISCUSSION
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Matrix movement is likely to be pivotal in several areas of development. In the palate, where the maxillary processes have to extend initially to lie dorsolaterally to the tongue, then elevate and fuse, the matrix is likely to be modified. Homozygous mouse models exist for numerous MMP classes, including MMP-2, MMP-3, MMP-7, MMP-9, and MMP-12 (Itoh et al., 1997; Shapiro, 1997), but none displays oro-facial clefting. These models do not have sharply defined phenotypes or even die, which highlights the complexity of the MMP family and their functions. If one enzyme is knocked out, another may become more highly expressed to compensate for the loss (Woessner, 1998). Our results support recent observations by Blavier et al. (2001) on the importance of MMPs in palatogenesis, and previous reports imply that palatogenesis requires proteolytic degradation of ECM and MMPs as a necessary step for palatal fusion. Our findings also support those of Morris-Wiman et al. (1999 2000), who found an immunohistochemical increase in MMP-2 during murine palatogenesis. Furthermore, our findings (Figs. 3a, 3b, 3c) indicate that MMP-3 is localized to the area in and around the MEE. MMP-3 induces EMT in mammary epithelial cells (Lochter et al., 1997) and may be responsible for the initiation of MEE breakdown. Interestingly, in the single palate cultured with 20 µM BB3103 that showed evidence of fusion, MMP-3 was more abundant around the MEE than in palates cultured in the same medium without any evidence of MEE breakdown. This implies that MMP-3 induces epithelial breakdown at the MEE if present in adequate amounts. We would also comment on the organ culture systems used. Blavier et al. (2001) used the Trowell palate culture system, where the tips of individual palatal shelves are dissected from the embryo and placed in apposition for the study of MEE breakdown. A 1-µM quantity of BB3103 was able to prevent MEE breakdown, but the experiments reported by us relate MMP levels to palatal shelf re-orientation and growth as well as MEE breakdown. The palate culture method we have used, initially described by al-Obaidi et al. (1995), is possibly a more realistic model of palatogenesis. Palatal shelf growth and MEE breakdown were evident in the majority of palatal shelves cultured with up to 7 µM BB3103. Our data showed significant inhibition of MMPs in culture (including MMP-2) when 20 µM BB3103 was used, and this prevented palatal contact and therefore fusion. This cannot be attributed to the inhibition of MMP-2 alone; other MMPs would also be inhibited by this concentration of hydroxamate, but this does confirm that MMPs have a role in normal palatogenesis. Zymography (Fig. 2c) implies that production of the latent form of the enzyme was not inhibited by BB3103, but probably its activation was. This may have resulted from the inhibition of MT1-MMP, which is known to activate MMP-2 and MMP-13 (Knäuper and Murphy, 1998). Interestingly, Blavier et al. (2001) implicate down-regulation of MMP-13 and TIMP-2 as the causative factors in clefting of TGFβ3 -/- palates. Unwittingly, by using a concentration of BB3103 that inhibits MMP-2, we may also have indirectly inhibited the activation of MMP-13 by blocking the function of MT1-MMP. Our findings confirm previous observations and suggest that both gelatinase A and stromelysin-1 have an important role in palate development.
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ACKNOWLEDGMENTS
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This work was funded by the Craniofacial Society of Great Britain and Ireland and the Wellcome Trust for Nathan Brown (REF 013504). Dr. J. Gavrilovic and Professor G. Murphy, University of East Anglia, kindly donated the MMP-3 antibody.
Received for publication December 6, 2001.
Revision received September 12, 2002.
Accepted for publication September 30, 2002.
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Journal of Dental Research, Vol. 81, No. 12,
826-830 (2002)
DOI: 10.1177/154405910208101206
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ABSTRACT
INTRODUCTION