1. Identification and analysis of the human hyaluronan synthase 1 gene promoter reveals Smad3- and Sp3-mediated transcriptional induction.
- Author
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Chen L, Neville RD, Michael DR, Martin J, Luo DD, Thomas DW, Phillips AO, and Bowen T
- Subjects
- Base Sequence, DNA Primers genetics, DNA, Complementary genetics, Electrophoresis, Agar Gel, Gene Expression Regulation physiology, Humans, Hyaluronan Synthases, Interleukin-1beta metabolism, Luciferases, Molecular Sequence Data, Nucleic Acid Amplification Techniques, RNA Interference, RNA, Small Interfering genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Extracellular Matrix Proteins genetics, Gene Expression Regulation genetics, Glucuronosyltransferase genetics, Promoter Regions, Genetic genetics, Smad3 Protein metabolism, Sp3 Transcription Factor metabolism
- 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-1β was Sp3, not Sp1, dependent., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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