Endocrine Abstracts

Regulation of many pituitary-specific genes involves calcium signalling, yet the mechanisms and signalling molecules involved have yet to be established. CaMK's are expressed in a wide variety of tissues, and are known to modulate transcriptional regulators such as MAPK's and CREB. It is unclear as to which anterior pituitary cell types express CaMK's, or which isoforms are present. We have examined the presence and localisation of type II and IV CaMK's in alphaT3-1, LbetaT2 (gonadotroph) and GH3 (somatotroph) cells. Fluorescent immunocytochemistry revealed expression of CaMKII in all three pituitary cell lines, with expression being localised to the nucleus in all cell lines. Western blotting confirmed the presence, size and specificity of CaMKII and CaMKIV immunoreactivity. Expression of both type II and IV isoforms was serum-dependent. When immunohistochemistry was performed on normal and adenomatous human pituitary sections, CaMKII was expressed in all tumour types (but poorly in growth-hormone secreting tumours), whereas CaMKIV expression was greatest in gonadotrophin-secreting tumours. Strong staining was seen for both type II and IV isoforms in normal human pituitary and rat testis positive controls. To assess the effects of CaMK activity on transcription of the human glycoprotein hormone alpha-subunit (alphaGSU) promoter, alphaGSU-luciferase reporter genes were transiently transfected into alphaT3-1 cells. Treatment of transfected cells with the specific CaMKII inhibitor, W-7, significantly inhibited basal promoter activity by 80% of control. Co-transfection of the alphaGSU-LUC promoter with constitutively active CaMKII and CaMKIV expression vectors significantly increased basal promoter activity by 4- and 5-fold, respectively. These observations reveal the presence of specific CaMK isoforms in anterior pituitary cells, and suggest a role for CaMK's in regulating alphaGSU transcription in gonadotrophs. Supported by the Joint Research Board, Barts Special Trustees & the North Thames RHA .