Induction of Hyaluronan Synthase 2 by Human Chorionic Gonadotropin in Mural Granulosa Cells of Equine Preovulatory Follicles

Abstract

In contrast to other species, the preovulatory rise in gonadotropins in mares causes a remarkable expansion of the entire granulosa cell layer in vivo, suggesting that hyaluronan (HA) synthesis may be regulated in mural granulosa cells in this species. The objectives of this study were to clone and characterize equine hyaluronan synthase 2 (HAS2) and investigate the regulation of its transcript and of HA synthesis in equine follicles during human chorionic gonadotropin (hCG)- induced ovulation. Results showed that the equine HAS2 cDNA contains a 5'-untranslated region of 436 bp, an open reading frame of 1659 bp, and a 3'-untranslated region of 707 bp. The open reading frame encodes a 552-amino acid protein that is highly conserved (98-99% identity), compared with other known mammalian homologs. The regulation of HAS2 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h after an ovulatory dose of hCG and in corpora lutea obtained on d 8 of the estrous cycle. Results from semiquantitative RT-PCR/Southern blotting analyses revealed a transient induction of HAS2 during the ovulatory process. Levels of HAS2 transcripts were undetectable in follicles before hCG treatment (0 h), increased markedly after gonadotropin treatment (P < 0.05), but returned to undetectable levels in corpora lutea. Analyses performed on isolated preparations of theca interna and granulosa cells showed that the granulosa cell layer was the predominant site of HAS2 expression. An immunohistochemical approach showed that this induction of HAS2 transcript was accompanied by a dramatic increase in HA production after hCG treatment. The isolation and characterization of a 1.8-kb fragment of genomic sequence located immediately upstream of equine HAS2, and comparison with corresponding human and mouse genomic regions identified several conserved putative cis-acting elements. Thus, this study describes the primary structure of equine HAS2, demonstrates for the first time the regulation of HAS2 in mural granulosa cells during the ovulatory process in vivo and identifies a valuable model in which to study the molecular control of HAS2 gene expression.

Identification and Analysis of the Promoter Region of the Human Hyaluronan Synthase 2 Gene

“…Alignment of gDNA sequences surrounding the 5Ј terminus of AJ604570 for human, murine, and equine HAS2 loci illustrated the high similarity suggested previously (24). Fig.…”

“…DNA Sequence Alignment and Data Base Analysis-Alignments for the genomic sequences upstream of HAS2 from human (clone RP11-3G20, sequence AC104233), mouse (clone RP24 -147N23, sequence AC140799), rat (clone CH230 -228F12, sequence AC105559), and horse (AF508308) (24) were carried out using the ClustalW algorithm at the European Bioinformatics Institute (www.ebi.ac.uk/clustalw/index. html).…”

LIF Inhibits Osteoblast Differentiation at Least in Part by Regulation of HAS2 and Its Product Hyaluronan

“…(63,64) Increased HA secretion may thus be a common mechanism used by inflammatory cytokines to arrest osteoblast differentiation. The promoter sequences of all three HASs contain several putative transcription factor binding sites, (65)(66)(67)(68) and functional studies have confirmed the role of some regulators. For example, the p50/p65 NF-B complex regulates HAS1 expression in human fetal lung myofibroblasts in response to TNF-␣(54), and STAT-and RAR-response elements control expression of HAS2 in human keratinocytes.…”

4-Methylumbelliferone inhibits the phosphorylation of hyaluronan synthase 2 induced by 12-O-tetradecanoyl-phorbol-13-acetate

“…The thicker band migrated to the molecular weight of around 63 k, and the thinner band migrated more slowly. Since HAS2 consists of 552 amino acids (23), its molecular weight is presumed to be about 63 k (33). In MU-treated cells, the intensity of the HAS2 bands in the phosphorylated protein fraction was weakened ( Fig.…”