RESUMO
The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.
Assuntos
Glucuronosiltransferase/genética , Glicosiltransferases , Proteínas de Membrana , Repetições de Microssatélites/genética , Regiões Promotoras Genéticas , Transferases , Proteínas de Xenopus , Animais , Sequência de Bases , Éxons , Humanos , Hialuronan Sintases , Íntrons , Camundongos , Dados de Sequência Molecular , Polimorfismo Genético , Homologia de SequênciaRESUMO
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.
Assuntos
Glucuronosiltransferase/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Sequência de Bases , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Hialuronan Sintases , Interleucina-8/genética , Túbulos Renais Proximais/citologia , Dados de Sequência Molecular , NF-kappa B/genética , Neuroblastoma , Regiões Promotoras Genéticas/fisiologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Transcrição Gênica/fisiologiaRESUMO
Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2 and -3. The regulation of HA synthesis has been implicated in a variety of extracellular matrix-mediated and pathological processes, including renal fibrosis. We have recently described the genomic structures of each of the human HAS genes. In the present study, we analyzed the HAS2 promoter region. In 5'-rapid amplification of cDNA ends analysis of purified mRNA from human renal epithelial proximal tubular cells, we detected an extended sequence for HAS2 exon 1, relocating the transcription initiation site 130 nucleotides upstream of the reference HAS2 mRNA sequence, GenBank accession number NM_005328. A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM_005328 demonstrated the constitutive promoter activity of sequences directly upstream of the repositioned transcription initiation site but not of the newly designated exonic nucleotides. Using reverse transcription-PCR, expression of this extended HAS2 mRNA was demonstrated in a variety of human cell types, and orthologous sequences were detected in mouse and rat kidney. Alignment of human, murine, and equine genomic DNA sequences upstream of the repositioned HAS2 exon 1 provided evidence for the evolutionary conservation of specific transcription factor binding sites. The location of the HAS2 promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts as well as in developmental models in which HAS2 null animals have an embryonic lethal phenotype.