Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype
Copyright policy )Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype
During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-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-beta 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-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (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-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta 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-beta signaling systems could be involved in human cleft palate fibroblast phenotype.
- University of Milan Italy
- Alma Mater Studiorum University of Bologna Italy
- University of Perugia Italy
- University of Ferrara Italy
Male, Glucosamine, Cell Survival, Cell Count, Cell Growth Processes, Fibroblasts, RETINOIC ACID; GABA; TGF-BETA; CLEFT PALATE; DIFFERENTIAL EXPRESSION, Receptors, GABA-A, Fibronectins, Cleft Palate, Phenotype, Transforming Growth Factor beta3, Gene Expression Regulation, Child, Preschool, Humans, Female, RNA, Messenger, Receptors, Transforming Growth Factor beta, Cells, Cultured, Glycosaminoglycans, Signal Transduction
Male, Glucosamine, Cell Survival, Cell Count, Cell Growth Processes, Fibroblasts, RETINOIC ACID; GABA; TGF-BETA; CLEFT PALATE; DIFFERENTIAL EXPRESSION, Receptors, GABA-A, Fibronectins, Cleft Palate, Phenotype, Transforming Growth Factor beta3, Gene Expression Regulation, Child, Preschool, Humans, Female, RNA, Messenger, Receptors, Transforming Growth Factor beta, Cells, Cultured, Glycosaminoglycans, Signal Transduction
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