Understanding the mechanisms that regulate NO production in the endothelium can provide important insight into processes that initiate endothelial dysfunction and lead to the development of atherosclerosis. NO production is dynamically regulated by a number of humoral and mechanical factors. In this study, we investigated PECAM-1 regulation of basal eNOS activity. An unexpected finding of this study was the increased basal eNOS activity and NO production in PE-KO cells as well as PECAM-1−/−
mice. An attractive hypothesis to explain the basal regulation of eNOS by PECAM-1 is their reported physical association. However, reports of this interaction have been highly conflicting. One group showed that shear stress induces a transient increase in the association of PECAM-1 and eNOS6
, while other studies have shown just the opposite5
. Our own results suggest only a weak basal association of eNOS and PECAM-1 in static ECs (data not shown). In addition, ultra-structural and biochemical analyses suggest that eNOS resides within caveolae10, 35
, whereas PECAM-1 is found at a membrane network just below the plasmalemma at the cell borders that is distinct from caveolae36
. Furthermore, studies in HUVECs have shown no colocalization between PECAM-1 and caveolin-1 and that these two proteins do not co-migrate on sucrose gels36
. The lack of physical association between eNOS and PECAM-1 led us to investigate differences in eNOS protein interactions and localization as possible mechanisms of regulation.
It is well recognized that correct subcellular targeting of eNOS is critical for proper regulation of its activity and NO bioavailability; thus, tight control of eNOS targeting to different compartments appears to be essential. In this regard, our data point towards a requirement for tightly regulated levels of NOSTRIN expression within ECs. Overexpression of NOSTRIN can promote the translocation of eNOS from the plasma membrane to intracellular vesicles, with a concomitant reduction in eNOS enzyme activity12
. Conversely, decreased NOSTRIN expression also influences eNOS subcellular localization and may contribute to the increased NO levels observed in the PECAM-1 knockout. Interestingly, overexpression of the eNOS-binding partner, caveolin-1, leads to accelerated atherosclerosis formation in mice, partially through reduced NO production37
; while persistent eNOS activation secondary to caveolin-1 deficiency induces pulmonary hypertension38
. Thus, tight regulation of eNOS regulatory protein levels, including NOSTRIN and caveolin-1 expression, is required for proper eNOS function.
Here, we present exciting data to support a novel mechanism of eNOS regulation by PECAM-1. Our current working model is summarized in . The cytoplasmic tail of PECAM-1 acts as a scaffold for STAT3 and mediates its activation. A possible candidate for the activation of STAT3 are the Src-family kinases, as Src-mediated STAT activation has been previously reported39, 40
. Following activation, STAT3 dimerizes and translocates into the nucleus where it modulates expression of gene targets, including NOSTRIN. Once expressed, NOSTRIN facilitates eNOS trafficking and its correct subcellular localization. It is possible that the reduced NOSTRIN levels account for the difference in eNOS-caveolin-1 association, however this could be due to other undetermined mechanisms. Of note, it has been reported that eNOS, caveolin-1 and NOSTRIN form a ternary complex to facilitate eNOS translocation16
. Additionally, our data are consistent with the hypothesis that NOSTRIN might serve to stabilize the inhibitory effect of caveolin-1 on eNOS16
Figure 6 Model of PECAM-1-mediated NOSTRIN expression and eNOS trafficking. The cytoplasmic tail of PECAM-1 acts as a scaffold for STAT3 binding. Following activation, STAT3 translocates to the nucleus where it regulates NOSTRIN mRNA expression. NOSTRIN protein (more ...)
The signaling pathway identified here relates to the regulation of basal eNOS activity via PECAM-1. However, PECAM-1 is also known to regulate eNOS activation in response to the physiologic stimulus of shear stress5, 6, 25, 41
. PE-KO cells are unable to activate eNOS in response to shear stress, yet they activate eNOS in response to ionomycin, indicating a specific requirement for PECAM-1 in flow-induced eNOS activation ( and Supplemental Figure II
). It is worth noting here that PECAM-1 is also required for flow-induced activation of Akt and Src, two important upstream mediators of eNOS activity25, 41
. The role of shear stress in NOSTRIN-mediated eNOS regulation is currently under investigation.
Blood flow and the NO signaling pathway are both known modulators of cardiovascular development and physiology. Two recent studies have provided compelling evidence for an evolutionarily conserved, shear-stress- and NO-mediated pathway that also regulates hematopoiesis42–44
. PECAM-1is thought to be involved in flow mechanosensing, based on in vitro
and in vivo
experiments showing PECAM-1-dependentactivation of flow-mediated intracellular signaling pathways and vascular remodeling6, 45, 46
. Interestingly, PECAM-1 is required for NO-mediated dilation in response to shear stress in isolated skeletal muscle arterioles41
as well as in the mouse coronary circulation47
, thus underscoring the importance of both PECAM-1 and NO in flow-mediated remodeling. Previous studies have also identified the importance of eNOS in flow-mediated remodeling48
. We now reveal a sophisticated dual mode of eNOS regulation by PECAM-1; while PECAM-1−/−
ECs are unable to activate eNOS in response to shear stress, their basal eNOS activity and NO levels are, paradoxically, increased through STAT3-mediated transcriptional control of NOSTRIN.