Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.
Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.
Baroni
Tiziano
T
Department of Experimental Medicine and Biochemical Sciences, University of Perugia, via del Giochetto, 06100 Perugia, Italy. tbaroni@unipg.it
Bellucci
Catia
C
Lilli
Cinzia
C
Pezzetti
Furio
F
Carinci
Francesco
F
Lumare
Eleonora
E
Palmieri
Annalisa
A
Stabellini
Giordano
G
Bodo
Maria
M
D016022:Case-Control Studies; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002675:Child, Preschool; D002971:Cleft Lip / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002972:Cleft Palate / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005109:Extracellular Matrix / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D005838:Genotype; D006801:Humans; D008297:Male; D009538:Nicotine / Q000494:pharmacology* / Q000633:toxicity; D018722:Nicotinic Agonists / Q000494:pharmacology* / Q000633:toxicity; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics
D018722:Nicotinic Agonists; D009538:Nicotine