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 matrixmediated 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 TM 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.
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-B 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. Hyaluronan (HA)3 is a linear non-sulfated glycosaminoglycan found commonly in the vertebrate extracellular matrix and which has a variety of functions during and following development (1-6). HA is synthesized by the HA synthase (HAS) enzymes that are encoded by the corresponding multigene family HAS1, -2, -3a, and -3b (7-10), and its importance in the extracellular matrix is underlined by the expanding range of pathological contexts in which modified or aberrant HA metabolism appears to play a role. These include malignancy, osteoarthritis, and pulmonary and vascular disorders, along with other immune and inflammatory diseases (11)(12)(13)(14)(15)(16)(17)(18)(19). HA has also been implicated in regenerative processes such as wound healing (e.g. and as a key mediator of the immune process (19).Under homeostasis in the healthy kidney, the expression of HA in the cortical interstitium is low, with high levels found only in the renal papilla. Following acute ischemic injury, interstitial inflammation, or during progressive renal fibrosis, however, there is greatly increased peritubular expression of both HA and the cell surface HA receptor CD44 in the cortex (23)(24)(25)(26)(27)(28). This suggests that alterations in HA synthesis and turnover may be involved either in the maintenance of homeostasis...
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