The human hyaluronan synthase genes: putative mediators of renal fibrosis

Abstract

Introduction  The glycosaminoglycan hyaluronan (HA) is a key component of the vertebrate extracellular matrix and is synthesized by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasizes the relevance of HA metabolism in clinical nephrological processes such as renal fibrosis (Strutz 2001) and peritoneal mesothelial wound healing (Yung et al. 2000). In the present study, the genomic sequences and organization of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data and subsequently supported by in vitro data. The region immediately upstream of each HAS gene was then screened for promoter activity.Materials and methods  The in silico methods used in the present study have been described in detail elsewhere (Williams et al. 2002). The programmes BLAST and BLAST2 were used to identify genomic DNA sequences containing the reference cDNA sequences for HAS1, HAS2 and HAS3. Intron/exon boundary sequences were confirmed by sequencing of PCR products from genomic DNA, and each PCR‐amplified promoter region was cloned and analysed by luciferase assay (Hoogendoorn et al. 2003).Results  The HAS1 gene comprised five exons, with the translation start site situated 9 bp from the 3′‐end of 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 and the translation start site located at nucleotide 1 of exon 2. Luciferase analysis of approximately 500 bp of the flanking genomic sequence of each HAS gene showed constitutive promoter activity.Discussion  Excessive HA synthesis in the renal cortex is characteristic of pathological processes such as the inflammation and early matrix expansion that precede renal fibrosis (Strutz 2001). The regulation of expression of the human HAS genes is therefore of great interest. We have used in vitro and in silico methods to deduce the genomic structures of the human HAS genes. Furthermore, we have demonstrated that the sequences immediately upstream of these genomic loci are transcriptionally active. We are continuing with more detailed analysis of these promoters, the data from which will help to evaluate the role of the HAS genes in renal fibrosis.