Abstract 17035: A Central Role of SIAH in DCC-Dependent Cardioprotection Provoked by Netrin-1/NO
We and others have previously shown that the axon guiding molecule netrin-1 can also function as a potent angiogenic and cardioprotective agent (PNAS 06, J Mol Cell Cardiol 10). Remarkably, within a very short time of 2 hrs, netrin-1 is able to feed-forwardly upregulate its large transmembrane receptor DCC that is required for nitric oxide (NO) production to mediate cardioprotection. In the present study we investigated novel mechanisms underlying NO upregulation of DCC, which is believed to be important in maintaining sustained increase in NO production to potentiate cardioprotection. Exposure of aortic endothelial cells to NO donor MAHMA NONOate resulted in a time-dependent increase in DCC protein expression, which occurred as quickly as 5 min after treatment (1.72±0.22 fold vs. 0 min, p=0.029), and the response maximized at 30 min [15 min (1.98±0.21, p=0.009), 30 min (2.28±0.30, p=0.013), and 60 min (1.31±0.07, p=0.011)]. NO donor also reduced ubiquitination of endogenous DCC, implicating a role of protesome pathway. Indeed, MG132 (20 μM, n=3) treatment increased DCC protein abundance. Of note, co-transfection with E3 ubiquitin ligase SIAH1 or SIAH2, but not with their dominant negative forms, dose-dependently diminished DCC protein levels. SIAH1 or SIAH2 interacted with DCC physically, and increased its level of ubiquitination. SIAH1 or SIAH2 is able to compensate for each other when one is suppressed, as RNAi for SIAH1 led to upregulated SIAH2, and vice versa. Intriguingly, NO donor time-dependently abrogated SIAH1 expression, which corresponded well with elevated DCC abundance. In additional experiments, C57BL/6 mice were subjected to in vivo transfection of combined SIAH1 and SIAH2 siRNA, prior to being exposed to ischemia/reperfusion (I/R) injury using a Langendorff perfusion system. Interestingly, SIAH RNAi was able to reduce infarct size determined by TCC staining. In summary, these data seem to demonstrate a central role of SIAH in mediating NO-dependent inhibition of DCC degradation, resulting in a secondary feed-forward loop of DCC/ERK/eNOS/NO activation in response to netrin-1, which is beneficial in potentiating NO-dependent cardioprotection.
- © 2013 by American Heart Association, Inc.