Inhibition of membrane depolarisation-induced transcriptional activity of cyclic AMP response element binding protein (CREB) by the dual-leucine-zipper-bearing kinase in a pancreatic islet beta cell line

• Published in: Diabetologia Vol. 49; no. 2; pp. 332 - 342
• Main Authors: Oetjen, E, Lechleiter, A, Blume, R, Nihalani, D, Holzman, L, Knepel, W
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 01-02-2006; Springer
• Subjects: Animals; Apoptosis; Beta cells; Biological and medical sciences; Blotting, Western; Calcineurin; Calcineurin - physiology; Cell Line; Cells, Cultured; CREB-Binding Protein - antagonists & inhibitors; CREB-Binding Protein - genetics; CREB-Binding Protein - physiology; Cyclic AMP response element binding protein; Cyclic AMP Response Element-Binding Protein - antagonists & inhibitors; Cyclic AMP Response Element-Binding Protein - genetics; Cyclic AMP Response Element-Binding Protein - physiology; Diabetes. Impaired glucose tolerance; Dual-leucine-zipper-bearing kinase; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Genes, Reporter; Glucose - pharmacology; Humans; Immunohistochemistry; Insulin - analysis; Insulin - genetics; Insulin-Secreting Cells - chemistry; Insulin-Secreting Cells - drug effects; Insulin-Secreting Cells - physiology; Islets of Langerhans - chemistry; Islets of Langerhans - physiology; MAP Kinase Kinase Kinases - genetics; MAP Kinase Kinase Kinases - metabolism; Medical sciences; Membrane depolarisation; Membrane Potentials; Mice; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Endocrinopathy; Enzyme; Membrane depolarisation; Transferases; Diabetes mellitus; Calcineurin; Langerhans islet; Cyclic AMP response element binding protein; Cyclic AMP; Leucine; Dual-leucine-zipper- bearing kinase; Response element; Beta cells; Binding protein; Cell line; Kinase; Aminoacid; β Cell; Inhibition; Endocrine pancreas
• ISSN: 0012-186X; 1432-0428
• DOI: 10.1007/s00125-005-0087-1

Aims/hypothesis The activation of the transcription factor cyclic AMP response element binding protein (CREB) by protein kinase A is inhibited by the human orthologue of the mitogen-activated protein kinase, dual-leucine-zipper-bearing kinase (DLK) in teratocarcinoma cells. However, pancreatic beta cells are electrically excitable and a major pathway regulating CREB in these cells is membrane depolarisation, leading to calcium influx and activation of the calcium/calmodulin-dependent protein phosphatase calcineurin. Therefore, the effect of DLK on CREB activity induced by membrane depolarisation was investigated in the beta cell line HIT. Materials and methods Reporter gene assays and biochemical techniques were used. Results RT-PCR, Western blot analysis and immunohistochemistry demonstrated the expression of DLK in HIT cells and primary mouse islets. In transient transfection experiments, DLK inhibited both GAL4-CREB activity induced by membrane depolarisation, and transcription directed by the CREB binding site, the cyclic AMP response element. Furthermore, DLK inhibited the transcriptional activity conferred by the CREB coactivator, CREB binding protein, both under basal conditions and after membrane depolarisation. DLK was also effective in response to glucose, the most potent physiological stimulus and known to cause membrane depolarisation of beta cells. Inhibition of calcineurin enhanced DLK activity, whereas overexpression of calcineurin reduced the inhibition by DLK of transcription directed by cyclic AMP response element after membrane depolarisation. Conclusions/interpretation These results demonstrate a calcineurin-sensitive inhibition by DLK of CREB activity after membrane depolarisation in pancreatic islet beta cells. This inhibition may, at least partially, be mediated at the coactivator level. The results thus suggest that DLK plays a role in the regulation of beta cell function, including insulin gene transcription and beta cell apoptosis.