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Abstract
PURPOSE: Nonsyndromic cleft palate is a common craniofacial birth defect with an incidence
of 1 in 700 live births. Recent studies have shown that connective tissue growth factor
(CTGF/CCN2) has acted as a necessary downstream mediator of TGF-β-dependent mesenchymal
stem cell proliferation in palatogenesis. In our laboratory, in CTGF/CCN2 knockout
(KO) mice, we identified numerous craniofacial defects such as failure of secondary
palate formation. We investigated this novel cleft (CTGF/CCN2 KO) model’s anatomy,
histology, and cellular function compared to the wild-type (WT).
METHODS: Palates were isolated from CTGF/CCN2 KO and WT E14.5 day mice and mRNA expression
examined by qRT-PCR for alterations in known palatogenesis signaling pathways. Mesenchymal
cells were isolated from CTGF/CCN2 WT and KO palates and used for protein expression
analysis of genes that were found to have significantly altered mRNA expression. Mesenchymal
cells were used to assess for responsiveness to recombinant CTGF/CCN2 and recombinant
EGF.
RESULTS: mRNA isolated from palates demonstrated decreased EGFR expression in CTGF/CCN2
KO embryos. Mesenchymal cells isolated from CTGF/CCN2 KO palate demonstrates decreased
protein expression of EGFR consistent with the observed mRNA decrease. Recombinant
CTGF/CCN2 produced no effect on cellular proliferation. Recombinant EGF was found
to increase proliferation in WT cells but unable to stimulate proliferation in CTGF/CCN2
KO cells.
CONCLUSION: Defects in proliferation significantly contribute to the clefting phenotype
observed in the CTGF/CCN2 mouse model. CTGF/CCN2 interacts with several pathways involved
in proliferation. Evaluation of our model has revealed abnormalities in EGF signaling
in the knockout that lead to decreased proliferation. Further examination of these
pathways will contribute to our understanding of the cleft etiology and may elucidate
novel targets for clinical management of this birth defect.