Regulation of Expression of Cell-Adhesion Molecule H-Cadherin

Abstract

H-cadherin is a member of the cadherin superfamily of Ca2+ dependent cell adhesion molecules, the members of which are involved in diverse cellular processes. H-cadherin is an unusual member of the family that lacks a cytoplasmic domain, instead binding to the cell membrane via a glycophosphatidyl-inositol moiety. H-cadherin may mediate cell adhesion via a mechanism that is distinct from that of the classical cadherins. Few convincing studies exist that examine the mechanisms and factors capable of controlling H-cadherin expression. The present study describes the first reliable investigation into changes in H-cadherin mRNA and protein expression, and examines the structure of the promoter region of the gene. Studies of the 5' untranslated sequence identified two new transcription start sites, and confirmed the promoter activity of this region with luciferase reporter assays. These data suggested the presence of enhancer elements, and putative transcription factor binding sites in the sequence were screened for. Elements capable of binding estradiol (E2), progesterone (P4) and glucocorticoid receptors, as well as factors known to respond to epidermal growth factor (EGF) were identified and selected for further study. The regulation of H-cadherin expression was studied in the Hu09 human osteosarcoma cell line. H-cadherin has been implicated in bone development and fracture healing, and has been shown to be a prognostic marker in osteosarcoma. A novel two-step real-time quantitative RT-PCR (QRT-PCR) assay was developed and validated for use in measuring changes in H-cadherin mRNA levels, in response to physiological stimuli. A new commercially-available antibody was optimised for use in semi-quantitative Western blotting of cell lysates from cells treated with the same stimuli. These techniques provided data that suggest that serum contains positive regulators capable of upregulating transcription and translation of H-cadherin. In support of these results, P4 was found to upregulate H-cadherin transcription (P<0.02), and similarly for EGF, may alter localization and trafficking of protein. E2 is a positive regulator of translation of H-cadherin protein, but has differential effects in the presence of P4 (P<0.01). Together E2 and P4 act to upregulate both transcription and translation of H-cadherin as do EGF (P<0.03) and the synthetic steroid dexamethasone (P<0.04). From the above investigation, it was concluded that H-cadherin is regulated by hormones, glucocorticoids and EGF in a complex manner indicative of a role for this cell adhesion molecule in hormone and drug-induced changes in bone morphology and pathology.