045 Characterization of the HAS 1 Promoter

Hyaluronan (HA) is a linear polysaccharide synthesized by three hyaluronan synthases (HAS1, 2, and 3) located on the plasma membrane. Evidence suggests that HA metabolism plays a significant role in the regulation of inflammation. More specifically, HA levels have been shown to rise in response to interleukin-1 (IL-1), a proinflammatory cytokine known to induce an increase in HAS1 transcript in adult dermal fibroblasts. To further examine the intricacies of HA metabolism, this study sought to characterize the HAS1 promoter and determine whether HAS1 regulation occurred at the level of transcription. This study characterized the genetic components needed for HAS1 promoter function, beginning with elucidation of the HAS1 transcription start site. 5′ RLM-RACE of purified adult dermal fibroblast mRNA specified a transcription start site 22 nucleotides further upstream when compared to previous published start sites (gi:9454515). A luciferase reporter assay of three PCR-amplified HAS1 promoter constructs detected that the core promoter was contained in the immediate 500 nt upstream of the HAS1 transcription start site (table). Promoter activity was also detected 1150 nt and 1600 nt upstream of the transcription start site, albeit at lesser values. This may be an indication of the existence of a repressor upstream of the 500 nt core promoter construct. In addition, to test the hypothesis that IL-1 regulates HAS1 at the level of transcription, fibroblasts were transfected with each promoter construct and examined for an IL-1 response. Promoter activity showed no significant increase. Initial nuclear run-on studies, however, indicated that the IL-1-induced increase in HAS1 transcripts may be under transcriptional regulation.   Vector 500 nt 1150 nt 1600 nt Luciferase Activity (RLU/s) 134 81051 7637 67 NIH, Grant GM58530, supported this study.