title: Identification and analysis of the human hyaluronan synthase 1 gene promoter reveals Smad3- and Sp3-mediated transcriptional induction.
abstract: The ubiquitous mammalian extracellular matrix glycosaminoglycan hyaluronan (HA) plays a pivotal role in the regulation of cell phenotype in fibrosis and scarring. Transforming growth factor-beta 1 (TGF-β1) and interleukin-1 beta (IL-1β) up-regulate hyaluronan synthase (HAS) 1 and HAS2 in dermal fibroblasts and renal proximal tubular epithelial cells, and subsequent HA synthesis regulates cell phenotype. In the present study, we investigated the mechanism of HAS1 transcriptional up-regulation in response to these cytokines. We used 5'-rapid amplification of cDNA ends analysis to identify the 5' end of HAS1 transcripts, resulting in an increase of 26 nucleotides to the HAS1 exon 1 sequence of reference sequence NM_001523. Constitutive luciferase activity of upstream DNA sequences was shown in luciferase reporter assays, but our reporter vector signals were refractory to the addition of TGF-β1 and IL-1β. Using siRNAs to knockdown transcription factor mRNAs, we showed that TGF-β1 up-regulation of HAS1 transcription was mediated via Smad3 but not Smad2, while HAS1 induction by IL-1β was Sp3, not Sp1, dependent.
authors.affiliation: Section of Nephrology, Matrix Biology Research Group, Institute of Molecular and Experimental Medicine, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK.
authors.forename: Long
authors.initials: L
authors.lastname: Chen
authors.forename: Rachel D
authors.initials: RD
authors.lastname: Neville
authors.forename: Daryn R
authors.initials: DR
authors.lastname: Michael
authors.forename: John
authors.initials: J
authors.lastname: Martin
authors.forename: Dong Dong
authors.initials: DD
authors.lastname: Luo
authors.forename: David W
authors.initials: DW
authors.lastname: Thomas
authors.forename: Aled O
authors.initials: AO
authors.lastname: Phillips
authors.forename: Timothy
authors.initials: T
authors.lastname: Bowen
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics* / Q000502:physiology; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D000076002:Hyaluronan Synthases; D053583:Interleukin-1beta / Q000378:metabolism; D008156:Luciferases; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D051900:Smad3 Protein / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D053583:Interleukin-1beta; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; C497121:SP3 protein, human; D051900:Smad3 Protein; D051705:Sp3 Transcription Factor; D008156:Luciferases; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; D000076002:Hyaluronan Synthases