Voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter.

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.     

Identification of the RA response element and transcriptional silencer in human <Emphasis Type="Italic">αCaMKII</Emphasis> promoter

The promoter of alpha subunit of the rat calcium/calmodulin-dependent protein kinase II (alphaCaMKII) gene was identified to contain an essential TATA element. Cell-based functional assay showed that the rat promoter displayed greater activity in neuronal cells than in non-neuronal cells. To characterize the human alphaCaMKII promoter, we have developed a promoter-reporter gene assay using different cell lines. A 2047 base pairs (bp) human alphaCaMKII gene promoter was cloned from human genomic DNA. Unlike the rat alphaCaMKII promoter, DNA sequence analysis showed that the human promoter was devoid of TATA element. We made series deletions of the promoter and fused the different sizes of the human promoter sequences to a luciferase reporter gene. The promoter-reporter constructs were transfected into human neuroblastoma SH-SY5Y, human neuroblastoma BE(2)-M17, and rat pheochromocytoma PC12 neuronal cell lines as well as human embryonic kidney HEK293 and human glioma U251 non-neuronal cell lines. The reporter gene assay demonstrated that the human alphaCaMKII promoter displayed high activity in the neuronal cell lines, while the activity was low in non-neuronal cell lines. All-trans retinoic acid (RA) enhanced the promoter activity in SH-SY5Y cells. Further analysis showed that there were two RA response elements located between +11 and +136 and -1911 to -593. In addition, we have identified a potent silencer at position -179 to -244 of the human alphaCaMKII promoter.