Abstract
During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft palate fibroblast phenotype.
Highlights
The normal development of the upper jaw and of the palate starts at about the sixth week of intra-uterine life and requires growth and fusion of the medial nasal processes and maxillary processes to form the lip, while the fusion of the palatal shelves to form the secondary palate occurs later.Craniofacial malformations and in particular orofacial clefting are the most common birth defects that occur in humans
We found abnormal extracellular matrix (ECM) synthesis in cleft palate (CLP)-secondary palate (SP) fibroblasts, which secreted more FN and total and individual GAGs
The retinoic acid (RA) effects we found were not due to alterations in cell number, because RA does not affect cell growth in normal SP (N-SP) and CLP-SP primary human fibroblasts, our results suggest that RA disrupts normal ECM stoichiometry by changing the balanced expression of GAG and FN, altering normal palate development
Summary
The normal development of the upper jaw and of the palate starts at about the sixth week of intra-uterine life and requires growth and fusion of the medial nasal processes and maxillary processes to form the lip, while the fusion of the palatal shelves to form the secondary palate occurs later (tenth week).Craniofacial malformations and in particular orofacial clefting are the most common birth defects that occur in humans. The normal development of the upper jaw and of the palate starts at about the sixth week of intra-uterine life and requires growth and fusion of the medial nasal processes and maxillary processes to form the lip, while the fusion of the palatal shelves to form the secondary palate occurs later (tenth week). With or without cleft palate (CLP), and those that involve the palate only (CPO), are due to a failure in fusion of the facial processes and/or palatal shelves, and constitute 2 forms of oral-facial clefts considered separate birth defects involving many (but not all) of the same genetic and environmental causes [1]. The nonsyndromic CLP arises when nasal processes and/or palatal shelves fail to fuse because genetic abnormalities and/or a perturbed environment alter extracellular matrix (ECM) composition [2] and affect cell patterning, migration, proliferation [3], and differentiation [4].
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