Downregulation of GATA6 in mTOR-inhibited human aortic endothelial cells: effects on TNFα-induced VCAM-1 expression and monocytic cell adhesion.

Increased expression of vascular cell adhesion molecule-1 (VCAM-1) on aortic endothelium is an early marker of atherogenesis, promoted in part by elevated inflammatory cytokines such as TNFα. Mammalian target of rapamycin (mTOR) is a ubiquitous signaling molecule that has been considered to contribute to diverse cellular processes through mTOR complex 1 (mTORC1) or complex 2 (mTORC2). This study aimed to elucidate the role of mTOR signaling in TNFα-induced VCAM-1 expression by arterial endothelium. Primary human aortic endothelial cells (HAEC) were treated with low-dose (0.1ng/mL) TNFα and VCAM-1 expression measured by real time quantitative PCR, Western blot, and flow cytometry. Inhibition of mTOR through siRNA-mediated depletion, or treatment with chemical inhibitors rapamycin or torin 1, suppressed VCAM1 transcription which translated to inhibition of VCAM-1 surface expression by HAEC and concomitant decreased adhesion of monocytes. A promoter luciferase assay and chromatin immunoprecipitation indicated that mTOR regulated VCAM1 transcription through a mechanism involving transcription factor GATA6. Activation of PKCα and an increase in microRNA(miRNA)-200a-3p expression, caused by mTOR inhibition but not disruption of mTORC1 or mTORC2 singly or together, decreased TNFα-induced GATA6 expression and its enrichment at the VCAM1 promoter. In conclusion, mTOR inhibition activates PKCα independently of disruption of mTORC1 and/or mTORC2, which challenges the conventional wisdom regarding mTOR signaling. Moreover, mTOR signals through transcriptional and post-transcriptional mechanisms to elicit maximal cytokine-induced endothelial inflammation that precedes atherosclerosis.