RGS2 Regulates cAMP Production in Mechanically Stressed Human PDL Cells

Objective: Using a microarray approach for gene expression induced in periodontal ligament (PDL) cells under compressive force, we recently identified regulator of G-protein signaling (RGS) 2. RGS proteins are known to turn off G-protein signaling. Signal transduction via G-proteins involves the synthesis of cAMP. The aim of this study was to investigate the role of RGS2 as a regulator of cAMP production in the PDL cells under mechanical stress. Methods: PDL cells were derived from the ligament tissues of human premolar teeth, isolated and maintained in alpha minimal essential medium containing 10% fetal bovine serum. Cells were then embedded and cultured in a three-dimensional collagen gel system and subjected to compressive force (6 g/cm²) for 1, 3, 6, 12, 24, 48 and 72 hours. The intracellular cAMP production was measured using enzyme immunoassay and the expression of RGS2 was assessed by real-time PCR and Western blot. To investigate the interrelationship between cAMP and RGS2, PDL cells were treated with 2',5'-dideoxiadenosine (DDA) (1, 10 and 20µM), an inhibitor of adenylyl cyclase, or antisense-oligonucleotide (10µM) to RGS2, and RGS2 expression or cAMP production was analyzed, respectively. Results: Compressive force time-dependently elevated the cAMP production. The expression of RGS2 mRNA was elevated up to 24 h, and the RGS2 protein levels were markedly increased at 24 h of compression. Inhibition of cAMP by DDA inhibited the RGS2 expression in the PDL cells under mechanical stress. Blockage of RGS2 by antisense-oligonucleotide resulted in a 2.0-fold increase in the intracellular cAMP production at 24 h. Conclusions: These results indicate that cAMP enhanced the RGS2 expression and that the inactivation of G-protein signaling by RGS2 decreased the production of cAMP. Thus, RGS2 negatively regulates the production of cAMP in the PDL cells under mechanical stress.