Pivotal role of calcium/calmodulin-dependent protein kinase II in ER stress-induced apoptosis

Conditions that interfere with proper functioning of the Endoplasmic Reticulum (ER), such as the accumulation of misfolded proteins, lead to activation of the Unfolded Protein Response (UPR). Initially the UPR is protective, serving to restore ER homeostasis by reducing protein load and increasing the expression of protein folding chaperones. However, in certain cases when stress is prolonged or severe, the UPR can alternatively trigger apoptosis. Accordingly, ER stress-induced apoptosis has been implicated in the pathogenesis of several diseases such as diabetes, cancer, neurodegenerative disease, and atherosclerosis (1).

In the case of advanced atherosclerosis, a number of recent studies in humans and experimental animals, indicate that the UPR plays a pivotal role in the progression of disease. Advanced atherosclerotic plaques that are prone to rupture are characterized by a large necrotic core and thinning of the plaque fibrous cap, which separates thrombogenic intraplaque matrix from the overlying circulation. Macrophage apoptosis is a prominent feature of advanced atherosclerotic plaques. In the absence of efficient apoptotic cell clearance by phagocytosis, increased apoptosis contributes to necrotic core expansion. Both mechanistic studies in vitro and advanced plaque causation studies in vivo have shown that ER stress plays a role in advanced lesional macrophage apoptosis (2). Indeed, Myoishi et al. have shown a close correlation among UPR-effector C/EBP-homologous protein (CHOP) expression, apoptosis, and plaque vulnerability in human coronary artery lesions (3). There are many potential causes of ER stress in atherosclerotic plaques, including oxysterols, particularly 7-ketocholesterol; oxidized phospholipids; unesterified cholesterol; oxidant stress; saturated fatty acids; and hypoxia. Moreover, relevant to the epidemic of insulin resistance-driven coronary artery disease, insulin resistance is a potent inducer of the UPR in insulin-resistant macrophages (4).

Previous studies from our group have shown that the key initiating event in the apoptosis cascade in ER-stressed macrophages is release of calcium from ER stores into the cytosol. Certain ER stressors like unesterifed cholesterol promotes this calcium release through inhibition of the ER calcium reuptake pump, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (5). Additionally, we found that UPR itself amplifies this calcium release by activating inositol-3-phosphate receptor (IP3R) via CHOP-induced ER oxidase-1α (ERO1α) (6). In our recent study, we have revealed a novel mechanism where a calcium responsive kinase called calcium/calmodulin-dependent protein kinase II (CaMKII) is activated through ER-released calcium and this in turn serves as a unifying link between ER stress and the downstream apoptotic pathways (7).

The physiological release of calcium from the ER serves important signaling roles in maintaining normal cellular function. Calcium transfer from the ER to mitochondria contributes to a number of physiological events, notably cellular bioenergetics. However, after prolonged ER stress conditions, excess calcium release from ER and consequent calcium increase in the cytosol and in the mitochondrial matrix has been shown be involved in mitochondrial pathways of apoptosis (8). In our recent study (7), we found that CaMKII is essential for excess calcium uptake by the mitochondria, which in turn leads to outer mitochondrial membrane permeabilization and release of cytochrome c. Moreover, induction of the cell-surface death receptor Fas by ER stress in macrophages involves activation of CaMKII and c-jun amino terminal kinase (JNK) (Fig. 1). Consistent with these in vitro studies, our in vivo experiments also showed that CaMKII is important in macrophage apoptosis and loss of mitochondrial membrane potential induced by systemic ER stress.

Figure 1.

Scheme of CaMKII-mediated events lending to ER stress induced macrophage apoptosis. ER stress doplotes the calcium stores within the ER lumon. Calcium subsequently accumulates in the cytoplasm and activates CuMKII. CuMKII enables apoptosis through JNK-mediated Fas induction and promotion of mitochondrial calcium uptake, followed by mitochondrial membrane permeabilization and release of cytochrome c.

The molecular mechanisms involved in increased calcium transfer from the ER to the mitochondria under ER stress is not completely understood. Growing evidence indicates that calcium uptake into mitochondria is controlled by specific proteins residing at specific contact points between the ER and mitochondria known as mitochondrial-associated membranes (MAMs) (9) and by a calcium uniporter that has been identified through physiologic and pharmacologic means (10) but has not yet been cloned. In this context, it will be important to determine weather CaMKII is affecting these two processes in order to facilitate calcium uptake into the mitochondria.

Given that CaMKII acts as an upstream molecule regulating multiple apoptosis pathways, targeting CaMKII inhibition may have critical implications for the diseases related to ER stress-induced cell death, including that occurring in advanced atherosclerosis.