The paradoxical world of protein O-GlcNAcylation: a novel effector of cardiovascular (dys)function

This editorial refers to ‘O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway’ by V.V. Lima et al., pp. 614–622, this issue.

Post-translational modification of proteins is a common mechanism for the modulation of protein function, location, and turnover, and protein phosphorylation is probably the most widely studied form of post-translational modification. However, there is an increasing interest in modification of serine/threonine residues by the O-linked attachment of the monosaccharide β-N-acetyl-glucosamine (O-GlcNAc); this is referred to as protein O-GlcNAcylation, to contrast it with traditional N- and O-glycosylation within the secretory pathways. Similar to phosphorylation, O-GlcNAc modification of proteins alters their function, activity, subcellular localization, and stability. Furthermore, since the same residues can be targets for both phosphorylation and O-GlcNAcylation, there is clearly a potential for direct interactions between them (Figure 1A).¹

Figure 1.

(A) O-linked attachment of the GlcNAc (G) moiety to nuclear and cytosolic proteins is catalysed by O-GlcNAc transferase (OGT) and the removal catalysed by O-GlcNAcase (OGA), a process that is similar to and can interact with phosphorylation (P). (B) Summary of the positive and negative effects of O-GlcNAc signalling in the cardiovascular system as described in more detail in the following reviews.^7–9

The study by Lima et al.² focuses on the intersection of endothelin (ET-1) signalling and regulation of protein O-GlcNAcylation and their effects on vascular reactivity. They showed that both ET-1 treatment and augmentation of O-GlcNAc levels increased vascular reactivity to phenylephrine, and both of these effects could be blocked by inhibition of the RhoA/Rho-kinase pathway. Of particular interest, ET-1 itself increased the levels of O-GlcNAc on vascular proteins, and this was blocked by pharmacological inhibition or knockdown of O-GlcNAc transferase (OGT), which catalyses O-GlcNAc synthesis. The inhibition of the ET-1-induced increase in O-GlcNAc also attenuated the activation of RhoA/Rho-kinase, which raises the intriguing possibility that O-GlcNAcylation of vascular proteins contributes to the regulation of ET-1 action. This study complements the previous work by the same authors in which they demonstrated that ET-1 augments O-GlcNAc in a time-dependent manner and that DOCA-salt rats have higher levels of O-GlcNAc protein in the vasculature.^3,4

Together, these findings suggest that increased O-GlcNAc levels in the vasculature are a contributing factor to hypertension and that this could be mediated via activation of the RhoA/Rho-kinase pathway. These novel observations have potentially profound implications for our understanding of the regulation of vascular function in both normal and pathophysiological conditions; however, they also leave a number of key questions unanswered. Cellular O-GlcNAc levels increase in response to numerous stress stimuli,⁵ which raises the question as to whether vascular dysfunction is a stressor that induces O-GlcNAcylation and contributes to further dysfunction via modulation of other signalling pathways. Or, as suggested by Lima et al.,² perhaps O-GlcNAc plays a direct role in mediating vascular dysfunction in response to activation by agonists such as ET-1. Although it is clear that the activation of O-GlcNAc levels has effects on vascular function that are similar to those of ET-1 and that blocking the increase in O-GlcNAc attenuates ET-1 activation of the RhoA/Rho-kinase pathway, the mechanism(s) by which ET-1 increases O-GlcNAc levels remain(s) to be determined. Since inhibition or knockdown of OGT inhibits the effects of ET-1, this would suggest that ET-1 acts by increasing O-GlcNAc synthesis; however, it is currently not known whether the activation of the ET-1 receptor leads to increased OGT activity or increased flux through the hexosamine biosynthesis pathway, leading to an increase in the levels of UDP-GlcNAc, the precursor for O-GlcNAc synthesis. Insulin stimulates the formation of an OGT–insulin receptor complex, resulting in phosphorylation, O-GlcNAcylation, and increased activity of OGT;⁶ therefore, there is precedence for receptor-mediated activation of OGT.

Our knowledge of the role(s) of O-GlcNAc on the regulation of the cardiovascular system is in its infancy, and although there has been a relatively rapid growth in reports of O-GlcNAc in cardiomyocytes over the past few years,^7–9 O-GlcNAcylation of vasculature proteins has been much less well studied. Early work showed that O-GlcNAc and OGT proteins were increased in vascular smooth cells with hyperglycaemia,¹⁰ and subsequent studies demonstrated that diabetes and hyperglycaemia both result in lower endothelial eNOS protein but with an increase in inactivated and O-GlcNAcylated eNOS.^11,12 In contrast to these earlier studies, more recent reports have demonstrated that acute increases in O-GlcNAc improved the inflammatory responses in balloon-injured rat carotid arteries and attenuated neointimal formation.¹³ Similarly, early studies in cardiomyocytes linked increased O-GlcNAc levels seen in diabetes with impaired contractile function; however, there is now considerable evidence to indicate that acute activation of cellular O-GlcNAc levels is cardioprotective.^7–9

Thus, O-GlcNAcylation can be both a positive and a negative effector of cellular function in the cardiovascular system (Figure 1B), and this notion should not be surprising since OGT gene deletion is embryonically lethal.¹⁴ However, the first definitive evidence that O-GlcNAc synthesis was a pro-survival response was reported in 2004;⁵ prior to that time, the majority of the published literature focused on the concept that increased cellular O-GlcNAcylation was a contributing factor to pathophysiology in a number of chronic disease states. This dichotomy between the positive and negative effects of increased O-GlcNAc is both fascinating and challenging. Although excess substrate availability and cellular metabolism undoubtedly increases O-GlcNAcylation, protein O-GlcNAc levels are also increased by glucose deprivation¹⁵ and a variety of stress stimuli such as heat shock and oxidative stress.⁵ Therefore, O-GlcNAc levels in vascular tissues are likely modulated by nutrient availability, stress, or a combination of the two.

In conclusion, although it is clear that O-GlcNAc is vital for signal transduction and cellular function, the notion that increased O-GlcNAcylation is necessarily detrimental is overly simplistic and the effects of changes in O-GlcNAc levels on vascular function are likely context dependent. Nevertheless, the identification of the RhoA/Rho-kinase pathway as a downstream mediator of O-GlcNAc signalling in vascular smooth muscle² is a novel and important finding. However, since more than 600 O-GlcNAc-modified proteins have been identified, ranging from transcription factors to cytoskeletal and contractile proteins, it is unlikely to be the only O-GlcNAc-mediated regulatory factor in vascular function. At this time, the move to more translational studies is limited by our marked lack of understanding of the basic mechanisms regulating O-GlcNAc synthesis and degradation combined with the fact that only two enzymes control O-GlcNAc turnover and these are ubiquitously expressed in all cells and tissues. Thus, future investigations into protein O-GlcNAcylation and cardiovascular signalling should focus on the characterization of specific O-GlcNAcylated proteins, including the identification of specific modification sites, as well as identify those factors involved in regulating OGT and OGA activity. Such studies will provide a greater understanding as to how O-GlcNAc exerts both positive and negative effects and potentially provide an avenue for targeted interventions and therapies.

Conflict of interest: none declared.

Funding

J.C.C. is supported by NIH grants: HL101192, HL079364, and HL67464; S.A.M. is supported by the WSU College of Pharmacy.