title: Sp1 and Sp3 mediate constitutive transcription of the human hyaluronan synthase 2 gene.
abstract: The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.
authors.affiliation: Institute of Nephrology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, Wales, UK.
authors.forename: Jamie
authors.initials: J
authors.lastname: Monslow
authors.forename: John D
authors.initials: JD
authors.lastname: Williams
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Fraser
authors.forename: Daryn R
authors.initials: DR
authors.lastname: Michael
authors.forename: Pelagia
authors.initials: P
authors.lastname: Foka
authors.forename: Ann P
authors.initials: AP
authors.lastname: Kift-Morgan
authors.forename: Dong Dong
authors.initials: DD
authors.lastname: Luo
authors.forename: Ceri A
authors.initials: CA
authors.lastname: Fielding
authors.forename: Kathrine J
authors.initials: KJ
authors.lastname: Craig
authors.forename: Nicholas
authors.initials: N
authors.lastname: Topley
authors.forename: Simon A
authors.initials: SA
authors.lastname: Jones
authors.forename: Dipak P
authors.initials: DP
authors.lastname: Ramji
authors.forename: Timothy
authors.initials: T
authors.lastname: Bowen
mesh_terms: D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D000076002:Hyaluronan Synthases; D016209:Interleukin-8 / Q000235:genetics; D007687:Kidney Tubules, Proximal / Q000166:cytology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics; D009447:Neuroblastoma; D011401:Promoter Regions, Genetic / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: D023081:CCAAT-Binding Factor; D016209:Interleukin-8; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases