OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Abstract Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP 3 )–intracellular free calcium ([Ca 2+ ] i ) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP 3 level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ- 32 P]ATP to lysine-rich histone. [Ca 2+ ] i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP 3 formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca 2+ ] i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP 3 –[Ca 2+ ] i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.