title: Retinoic acid, GABA-ergic, and TGF-beta signaling systems are involved in human cleft palate fibroblast phenotype.
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-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.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy. tbaroni@unipg.it
authors.forename: Tiziano
authors.initials: T
authors.lastname: Baroni
authors.forename: Catia
authors.initials: C
authors.lastname: Bellucci
authors.forename: Cinzia
authors.initials: C
authors.lastname: Lilli
authors.forename: Furio
authors.initials: F
authors.lastname: Pezzetti
authors.forename: Francesco
authors.initials: F
authors.lastname: Carinci
authors.forename: Ennio
authors.initials: E
authors.lastname: Becchetti
authors.forename: Paolo
authors.initials: P
authors.lastname: Carinci
authors.forename: Giordano
authors.initials: G
authors.lastname: Stabellini
authors.forename: Mario
authors.initials: M
authors.lastname: Calvitti
authors.forename: Eleonora
authors.initials: E
authors.lastname: Lumare
authors.forename: Maria
authors.initials: M
authors.lastname: Bodo
mesh_terms: D002452:Cell Count; D048708:Cell Growth Processes / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002675:Child, Preschool; D002972:Cleft Palate / Q000473:pathology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology*; D005353:Fibronectins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D008297:Male; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D053782:Transforming Growth Factor beta3 / Q000235:genetics / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D005353:Fibronectins; C546273:GABRB3 protein, human; D006025:Glycosaminoglycans; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D018125:Receptors, Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; D014212:Tretinoin; D005944:Glucosamine