Differential regulation of the ghrelin promoter in WRL68 and HEK293 cells
DP Macartney, BA Hughes, PM Stewart, MC Sheppard & AA Toogood
It is now evident that ghrelin is more than just a growth hormone (GH) secretagogue; it plays an important role in energy homeostasis, increasing food intake and fat deposition, has cardiovascular effects, and inhibits cell proliferation.
Ghrelin mRNA expression is widespread in human tissues, but little is known about the molecular mechanisms and signals that regulate gene expression at the transcriptional level. To address this we cloned and sequenced a 4kb region upstream of the human preproghrelin start codon. Comparison of the mouse and human sequences and analyses with MatInspector and TFSearch revealed a number of discreet regions of similarity between the two. Several sites of potential transcriptional importance including putative sites for SP1, AP1, and steroid response elements were highlighted. A deletion series spanning the 4kb region was constructed to study promoter activity, using a Dual luciferase assay, in a range of cell lines including WRL68 and HEK293 cells.
The 4kb region showed differential promoter activity in the two cell lines; in HEK293 cells activity was three times higher than in WRL68 cells (p<0.001). A construct containing a shorter 1.8kb promoter gave highest luciferase activity in both cell lines. Sequential deletion of -1800 to -887 resulted in a decrease of promoter activity, whereas deletion from -887 to -520 increased activity. Interestingly, cell type specific activity was observed in a construct containing -131bp; this construct showed similar activity to the 1.8kb region in WRL68 cells, but was less active in HEK293 cells (p=0.002).
These data demonstrate the differential activity of the ghrelin promoter in different cell lines and highlight discreet regions of transcriptional importance involved in repression, enhancement, and cell specific regulation.