Adrenomedullin increases the expression of CLR and RAMP2 in microvascular endothelial cells
N Schwarz1, K Sidhu1, S Kapas2 & JP Hinson1
Adrenomedullin (AM) is a multifunctional peptide hormone which plays a significant role in vasodilation and angiogenesis, implicating it in hypertension as well as in carcinogenesis. AM exerts its effects via the Calcitonin Receptor-Like Receptor (CLR) complexed with either Receptor Activity Modifying Protein (RAMP) 2 or 3.
The aim of the present study is to investigate the biological actions, functions and possible target genes of AM in immortalized Human Microvascular Endothelial Cells (HMECs). We showed in this cell line the expression of AM receptor message and protein using PCR and western blotting respectively. Treatment of cells with AM elevated cAMP in a time (545 min) and dose (10−610−14M) dependent manner, as well as increasing phosphorylation of ERK 1/2 after 5 min of treatment. Even though the phosphorylated ERK 1/2 signal is translocated into the nucleus, this did not result in increased cell proliferation. However, using real-time PCR we were able to show for the first time that AM up-regulates mRNA expression of VEGF after 2 h of treatment as well as increasing mRNA levels of its own receptor (CLR/RAMP2) after 2 and 24 h of treatment. The biological function of this new finding is currently under investigation.
Additionally we established the correct transcription start site of RAMP2 using Rapid Amplification of cDNA Ends (RACE) PCR. Analysis of 1000 bp promoter region revealed a TATA-less promoter with several zinc fingers as well as an AP1 transcription site, suggesting possible utilisation of other signalling pathways. Taken together with the already published characterisation of the CLR promoter the comparison of potential transcriptional elements in both promoter regions may help to elucidate a common regulatory mechanism.