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.
Highlights
Responding multigene family HAS1, -2, -3a, and -3b [7,8,9,10], 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
HAS2 appears to be the most frequently induced isoform in the context of clinical nephrology, and increased HAS2 transcription, together with raised levels of HA synthesis, have been reported in renal human proximal tubular epithelial HK-2 cells cultured in vitro in glucose concentrations similar to those found in diabetic nephropathy [29]
The Electrophoretic mobility shift assays (EMSAs) profiles seen after incubation with HK-2 nuclear extracts, and labeled a, were characteristic of binding by the transcription factors Sp1 and Sp3 [36, 46, 47]
Summary
Responding multigene family HAS1, -2, -3a, and -3b [7,8,9,10], 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. Interstitial inflammation, or during progressive renal fibrosis, 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 or in the development of pathological events, but the role of HA remains unclear. Analysis of the HAS2 proximal promoter region in silico [35] identified a range of putative upstream transcription factor-binding sites (TFBSs). The prefix mut- denotes an oligonucleotide conferring point mutations of wild-type HAS2 promoter sequence and altered nucleotides are shown in upper case; con- denotes a consensus sequence for the respective TFBS
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