COMMENTARIES

The Significance of PKC to Atherosclerosis and Diabetes Macrovascular Complications

Francesco Beguinot, Dipartimento di Biologia e Patologia Cellulare e Molecolare "Federico II" University of Naples & IEOS, CNR, Via S. Pansini 5, Naples 80131, Italy, Email: beguino@unina.it

Classification of PKCs

 

Protein kinases C (PKCs) are a family of kinase phosphorylating protein substrates on serine or threonine residues. At least ten PKC isoforms have been described and usually classified according to whether they contain domains that bind Ca²⁺ and/or diacylglycerol (DAG), both of which induce the kinase activity [1]. Conventional PKCs, including the α, β1, β2 and γ isoforms, bind both activators; novel PKCs, including the δ, ε, h, and q isoforms, bind DAG but not Ca²⁺, and atypical PKCs (ζ and ι isoforms) bind neither (the mouse and rat homologue of human PKCι is termed PKCλ). PKC is ubiquitously expressed, but different tissues feature specific isoform distribution, due to tissue-specific transcriptional regulation of distinct genes encoding each isoform. Homologies have been identified between protein kinase A, protein kinase B (Akt), and PKC. In addition, similarities exist in regulation of these enzyme activities by phosphorylation [2].

 

PKC Regulation

 

Induction of PKC often occurs as result of phospholipase C activation following ligand binding of G-protein-coupled receptors and hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP₂). DAG, the first product of this reaction, directly activates eight out of ten PKC isoforms by binding. Inositol 1,4,5-trisphosphate (IP₃), the PIP₂ head group, opens the IP₃-gated Ca²⁺ transport channels in the endoplasmic reticulum membrane and increases intracellular Ca²⁺ concentration, thereby indirectly activating Ca²⁺-dpendent PKCs. Induction of phospholipase D also may contribute to PKC activation by phosphatidylcholine hydrolysis and generation of DAG. Finally, high glucose concentrations in diabetes cause increased activity of PKC and increased DAG intracellular mass in several tissues and cultured cells including renal glomeruli [3]-cultured aortic endothelial cells and vascular smooth muscle cells [4]. There is evidence that the increased DAG production occurring under such circumstances is largely dependent on de novo synthesis rather than hydrolysis reactions [4,5].

 

PKC in Diabetes and Insulin Resistance

 

The PKC isoforms undergoing activation in diabetes vary depending on the cell type. In the aorta of murine models of diabetes, PKCβ2 activity is increased [6]. In aortic vascular smooth muscle cells cultured in media containing high glucose concentrations, PKCβ2 and PKCδ activation has been demonstrated [7], while induction of PKCα, β1, and ε have been reported in the retina [8] and renal glomeruli [9] of diabetic rats. Importantly, treatment with ruboxistaurin (LY333531), a β-specific PKC inhibitor, improved retinal circulation, albumin excretion rate, and glomerular filtration rate in diabetic rats, supporting the concept that activation of PKCβ1 or β2 is involved in diabetes vascular dysfunction in different districts [10]. Indeed, in diabetes and other conditions accompanied by insulin resistance, the activation of PKC in vascular cells is now thought to play a major role in mediating the consequences of elevated plasma and tissue concentrations of glucose and non-esterified fatty acids on deranged vascular cell signaling.

 

PKC and Diabetes Macrovascular Lesions

 

Initial studies on the significance of vascular PKC activation to diabetes and its long-term complications were primarily focused on microvascular dysfunction, but increasing evidence supports the concept that PKCs have a role in several mechanisms promoting atherosclerosis and/or inhibiting anti-atherogenic mechanisms. There is direct evidence that PKCβ/apolipoprotein E double knockout mice feature reduced aortic atherosclerosis compared with apolipoprotein E knockout mice [11], supporting the role of PKCβ in atherogenesis even in nondiabetic conditions. Much evidence now indicates that vascular DAG accumulation and ensuing PKC activation in diabetes causes endothelial dysfunction by inducing activation of vascular NAD(P)H oxidase, eNOS dysfunction, and induction of endothelin-1 (ET-1). Vascular remodeling by vascular smooth muscle cell proliferation and apoptosis is controlled by PKC, either via the induction of the DAG/PKC pathway or intermediary signaling mechanisms, e.g. upon angiotensin II stimulation. Also, leukocyte adhesion, monocyte transdifferentiation, and foam cell formation have been discovered to be regulated, in part, through PKC activation [12]. In all of these mechanisms, the precise identification of the responsible PKC isoform(s) is still preliminary and represents an important area of current investigation and industrial interest.

 

PKCβ May Affect Early Atherosclerosis Event

 

One of the earliest events leading to development of the atherosclerotic plaque is the accumulation of lipids in the intima, followed by modified LDL engulfment of monocyte-derived macrophage and foam cell formation [13]. The oxLDL induce ICAM-1 in isolated porcine coronary arteries, leukocyte adhesiveness to endothelium (14), and macrophage proliferation in culture [15]. At least in part, ICAM-1 induction and macrophage growth can be prevented by PKC inhibitors including calphostin C [14,15]. In cultured macrophages, oxLDL enhances expression of the CD36 scavenger receptor, which is prevented by calphostin C [16]. In addition, exposure of monocyte-derived macrophages to elevated glucose concentrations induces another scavenger receptor, LOX-1 [17]. Li and coworkers have shown that the induction of LOX-1 expression by glucose is accompanied by PKCβ2 membrane translocation and is blocked by calphostin C as well as the specific PKCβ inhibitor LY379196 [17]. In this same study, ROS production was identified as an event upstream to PKCβ2 activation and LOX-1 induction as both were inhibited by the antioxidant agent N-acetyl-1-cysteine. These studies provided evidence supporting the specific role of the PKC2 isoform in foam cell formation by transducing several different effects of oxLDL.

 

Reducing PKC Overactivation in Diabetes

 

PKCs are emerging as promising target molecules mediating diabetes-dependent long-term complications. The PKCβ inhibitor ruboxistaurine is presently being tested in ongoing clinical trials with diabetes microvascular end-points. Whether PKC inhibition can also be adopted to prevent atherosclerotic plaque formation deserves further investigation. However, strategies distinct from direct pharmacological inhibition of PKC may also be adopted to reduce PKC overactivation and signaling in diabetes. For instance, different PPARγ agonists have been reported to inhibit glucose activation of PKC in endothelial [18] and vascular smooth muscle cells [19] by DAG kinase induction and reduction of DAG intracellular mass [18]. Whether similar strategies may add to the diabetologist armamentarium represents an exciting new area of industrial research.

References

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