Expression of ectonucleotide pyrophosphatase-1 in end-plate chondrocytes with transforming growth factor beta 1 siRNA interference by cyclic mechanical tension

Abstract

Background Ectonucleotide pyrophosphatase/phosphodiesterase (ENPP)-1 is a membrane-bound protein that catalyzes the hydrolysis of extracellular nucleoside triphosphates to monophosphate and extracellular inorganic pyrophosphate (ePPi). Mechanical stimulation regulates ENPP-1 expression. This study sought to investigate the changes in ENPP-1 expression after stimulation using cyclic mechanical tension (CMT). Methods Rat end-plate chondrocytes were cultured and subjected to CMT (at 3%, 6%, and 9% elongation) for 20, 40, and 60 minutes to observe changes in the expression of ENPP-1. To investigate the pathway, end-plate chondrocytes were exposed to 10 ng/ml of transforming growth factor beta 1 (TGF-β1), TGF-β1 siRNA, or a specific extracellular signal-regulated kinase (ERK)1/2 inhibitor, U0126, in addition to CMT. Changes in ENPP-1 expression were measured by reverse transcription PCR (RT-PCR) and Western blotting. Results We observed the largest increase in ENPP-1 expression following 3% elongation CMT stimulation. ENPP-1 expression was also increased when end-plate chondrocytes were exposed to 10 ng/ml of TGF-β1, but decreased after TGF-β1 knockdown with siRNA. ERK1/2 phosphorylation was activated after 3% elongation for 40 minutes, and the stimulatory effect of TGF-β1 on ENPP-1 mRNA and protein expression was inhibited by the suppression of the ERK1/2 pathway using U0126. Conclusion CMT increases the expression of ENPP-1 in end-plate chondrocytes in a manner likely dependent on TGF-β1 induction by the ERK1/2 signaling pathway.