UC Riverside

Endothelial cells play an active role in maintaining vascular health. Their response to different flow patterns and circulating molecules initiate signaling cascades that determine endothelial phenotypes and ultimately vascular health or disease. One key molecule that initiates endothelial pro-health cascades is the AMP-activated protein kinase (AMPK). Responding to the onset of cellular stress, AMPK not only maintains the cellular energy status by activating catabolic pathways, while shutting down anabolic ones, it also promotes endothelial differentiation and quiescence while attenuating inflammation. However, the precise signaling events that result in AMPK's beneficial effects remain elusive.

Here, we show that both Poly (ADP-ribose) polymerase-1 (PARP-1) and nucleolin (NCL) are phosphorylated by AMPK. PARP-1 is an abundant nuclear protein that once activated, uses NAD+ to "PARylate" itself as well as other protein targets. It serves multiple functions in the cell including DNA repair, epigenetics, mitosis, and transcription by binding to TTGATATAAAT sequences within the genome. The B-cell lymphoma 6 (Bcl-6) intron 1 contains a PARP-1 binding sequence. PARP-1 binds to this sequence it the intron in its inactive state, but upon activation by AMPK phosphorylation, it dissociates allowing transactivation of Bcl-6 protein. Bcl-6 acts as a co-repressor for VCAM-1, MCP-1, and MCP-3, which inhibits the recruitment of macrophages to the endothelium thus attenuating the local inflammatory response. In addition to the phosphorylation of PARP-1, AMPK regulates the transcriptome through the phosphorylation of nucleolin.

We also demonstrate that AMPK regulates endothelial health phosphorylation of nucleolin. Nucleolin is a ubiquitous, multifunctional protein that traffics to multiple cellular locations. It facilitates ribosome biogenesis in the nuceolus, transcription in the nucleus, mRNA stability in the cytoplasm, and angiogenesis at the cell surface. Upon phosphorylation of nucleolin by AMPK, nucleolin transiently translocates to the nucleus where it transactivates KLF2 expression. KLF2 is a zinc finger containing shear stress responsive transcription factor that regulates the majority of the transcriptome in response to pulsatile flow. Additionally, KLF2 is involved in the differentiation of endothelial cells and maintains endothelial quiescence.

Taken together, AMPK regulates the PARP-1 anti-inflammatory and nucleolin/KLF2 pathways which promote endothelial health.