ABSTRACT
BACKGROUND: Transforming growth factor-ß is a multifunctional and pleiotropic factor with decisive role in tissue repair. In this context, we have shown previously that TGF-ß inhibits the proliferation of fetal human skin fibroblasts but stimulates that of adult ones. Given the dynamic reciprocity between fibroblasts, growth factors and extracellular matrix (ECM) in tissue homeostasis, the present study aims to investigate the role of fibronectin and collagen in the proliferative effects of TGF-ß on fetal and adult cells. METHODS: Human fetal and adult skin fibroblasts were grown either on plastic surfaces or on surfaces coated with fibronectin or collagen type-I, as well as, on top or within three-dimensional matrices of polymerized collagen. Their proliferative response to TGF-ß was studied using tritiated thymidine incorporation, while the signaling pathways involved were investigated by Western analysis and using specific kinase inhibitors. RESULTS: Fetal skin fibroblast-proliferation was inhibited by TGF-ß, while that of adult cells was stimulated by this factor, irrespective of the presence of fibronectin or collagen. Both inhibitory and stimulatory activities of TGF-ß on the proliferation of fetal and adult fibroblasts, respectively, were abrogated when the Smad pathway was blocked. Moreover, inhibition of fetal fibroblasts was mediated by PKA activation, while stimulation of adult ones was effected through the autocrine activation of FGF receptor and the MEK-ERK pathway. CONCLUSIONS: Fetal and adult human skin fibroblasts retain their differential proliferative response to TGF-ß when cultured in the presence of fibronectin and unpolymerized or polymerized collagen. GENERAL SIGNIFICANCE: The interplay between TGF-ß and ECM supports the pleiotropic nature of this growth factor, in concordance with the different repair strategies between fetuses and adults. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.
Subject(s)
Collagen/pharmacology , Fetus/cytology , Fibroblasts/cytology , Fibronectins/pharmacology , Skin/cytology , Transforming Growth Factor beta/pharmacology , Adult , Animals , Cattle , Cell Proliferation/drug effects , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/metabolism , Gels , Humans , Male , Mitogens/pharmacology , Phosphorylation/drug effects , Polymerization/drug effects , Rats , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/drug effects , Smad Proteins/metabolismABSTRACT
BACKGROUND: Mutations in the GCH1 gene are associated with childhood onset, dopa-responsive dystonia (DRD). Correct diagnosis of DRD is crucial, given the potential for complete recovery once treated with L-dopa. The majority of DRD associated mutations lie within the coding region of the GCH1 gene, but three additional single nucleotide sequence substitutions have been reported within the 5' untranslated (5'UTR) region of the mRNA. The biologic significance of these 5'UTR GCH1 sequence substitutions has not been analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Luciferase reporter assays, quantitative real time PCR and RNA decay assays, combined with bioinformatics, revealed a pathogenic 5'UTR GCH1 substitution. The +142C>T single nucleotide 5'UTR substitution that segregates with affected status in DRD patients, substantially attenuates translation without altering RNA expression levels or stability. The +142C>T substitution disrupts translation most likely by creating an upstream initiation start codon (uAUG) and an upstream open reading frame (uORF). CONCLUSIONS/SIGNIFICANCE: This is the first GCH1 regulatory substitution reported to act at a post-transcriptional level, increasing the list of genetic diseases caused by abnormal translation and reaffirming the importance of investigating potential regulatory substitutions in genetic diseases.