Lale Ozcan

Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways.

ER stress-induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress-mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress-induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIgamma (CaMKIIgamma) and JNK. Remarkably, CaMKIIgamma was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress-induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIgamma-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress-induced apoptosis.

Role of Endoplasmic Reticulum Stress in Metabolic Disease and Other Disorders

Perturbations in the normal functions of the endoplasmic reticulum (ER) trigger a signaling network that coordinates adaptive and apoptotic responses. There is accumulating evidence implicating prolonged ER stress in the development and progression of many diseases, including neurodegeneration, atherosclerosis, type 2 diabetes, liver disease, and cancer. With the improved understanding of the underlying molecular mechanisms, therapeutic interventions that target the ER stress response would be potential strategies to treat various diseases driven by prolonged ER stress.

Calcium Signaling through CaMKII Regulates Hepatic Glucose Production in Fasting and Obesity

Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here, we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatocytes and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis.

Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B4 synthesis by inhibiting a calcium-activated kinase pathway

Significance Specialized proresolving mediators, such as resolvin D1 (RvD1), are endogenous molecules that both dampen inflammation without compromising host defense and promote tissue resolution. A prime example is RvD1’s ability to decrease the ratio of proinflammatory leukotriene B4 (LTB4) to proresolving lipoxin A4 (LXA4), but the mechanism is not known. We have discovered a new calcium kinase signaling pathway through which RvD1 lowers the nuclear:cytoplasmic ratio of 5-lipoxygenase (5-LOX), the common enzyme for LTB4 and LXA4 biosynthesis This shift in 5-LOX localization dampens LTB4 production and enhances LXA4 production. By providing a new mechanistic understanding of how RvD1 tempers inflammation to facilitate resolution, these findings can help devise new therapeutic strategies for diseases driven by nonresolving inflammation. Imbalances between proinflammatory and proresolving mediators can lead to chronic inflammatory diseases. The balance of arachidonic acid-derived mediators in leukocytes is thought to be achieved through intracellular localization of 5-lipoxygenase (5-LOX): nuclear 5-LOX favors the biosynthesis of proinflammatory leukotriene B4 (LTB4), whereas, in theory, cytoplasmic 5-LOX could favor the biosynthesis of proresolving lipoxin A4 (LXA4). This balance is shifted in favor of LXA4 by resolvin D1 (RvD1), a specialized proresolving mediator derived from docosahexaenoic acid, but the mechanism is not known. Here we report a new pathway through which RvD1 promotes nuclear exclusion of 5-LOX and thereby suppresses LTB4 and enhances LXA4 in macrophages. RvD1, by activating its receptor formyl peptide receptor2/lipoxin A4 receptor, suppresses cytosolic calcium and decreases activation of the calcium-sensitive kinase calcium-calmodulin-dependent protein kinase II (CaMKII). CaMKII inhibition suppresses activation P38 and mitogen-activated protein kinase-activated protein kinase 2 kinases, which reduces Ser271 phosphorylation of 5-LOX and shifts 5-LOX from the nucleus to the cytoplasm. As such, RvD1’s ability to decrease nuclear 5-LOX and the LTB4:LXA4 ratio in vitro and in vivo was mimicked by macrophages lacking CaMKII or expressing S271A-5-LOX. These findings provide mechanistic insight into how a specialized proresolving mediator from the docosahexaenoic acid pathway shifts the balance toward resolution in the arachidonic acid pathway. Knowledge of this mechanism may provide new strategies for promoting inflammation resolution in chronic inflammatory diseases.

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 ... 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.

Treatment of Obese Insulin-Resistant Mice with an Allosteric MAPKAPK2/3 Inhibitor Lowers Blood Glucose and Improves Insulin Sensitivity

The prevalence of obesity-induced type 2 diabetes (T2D) is increasing worldwide, and new treatment strategies are needed. We recently discovered that obesity activates a previously unknown pathway that promotes both excessive hepatic glucose production (HGP) and defective insulin signaling in hepatocytes, leading to exacerbation of hyperglycemia and insulin resistance in obesity. At the hub of this new pathway is a kinase cascade involving calcium/calmodulin-dependent protein kinase II (CaMKII), p38α mitogen-activated protein kinase (MAPK), and MAPKAPK2/3 (MK2/3). Genetic-based inhibition of these kinases improves metabolism in obese mice. Here, we report that treatment of obese insulin-resistant mice with an allosteric MK2/3 inhibitor, compound (cmpd) 28, ameliorates glucose homeostasis by suppressing excessive HGP and enhancing insulin signaling. The metabolic improvement seen with cmpd 28 is additive with the leading T2D drug, metformin, but it is not additive with dominant-negative MK2, suggesting an on-target mechanism of action. Allosteric MK2/3 inhibitors represent a potentially new approach to T2D that is highly mechanism based, has links to human T2D, and is predicted to avoid certain adverse effects seen with current T2D drugs.

Common Therapeutic Targets in Cardiometabolic Disease

Cardiovascular disease and insulin resistance syndrome interactions provide joint therapeutic targets. The interactions between cardiovascular disease (CVD) and insulin resistance syndromes suggest the possibility of joint targets for cardiometabolic research. The best drugs would go beyond minimizing adverse effects and have protective actions against both metabolic disease and CVD. In this perspective, we will outline a few examples in which a deep mechanistic understanding of the many cellular pathways that contribute to type 2 diabetes and CVD, regardless of cell type, have resulted in common upstream pathogenic pathways that can be therapeutically targeted.

Suppression of Adaptive Immune Cell Activation Does Not Alter Innate Immune Adipose Inflammation or Insulin Resistance in Obesity.

Obesity-induced inflammation in visceral adipose tissue (VAT) is a major contributor to insulin resistance and type 2 diabetes. Whereas innate immune cells, notably macrophages, contribute to visceral adipose tissue (VAT) inflammation and insulin resistance, the role of adaptive immunity is less well defined. To address this critical gap, we used a model in which endogenous activation of T cells was suppressed in obese mice by blocking MyD88-mediated maturation of CD11c+ antigen-presenting cells. VAT CD11c+ cells from Cd11cCre + Myd88 fl/fl vs. control Myd88 fl/fl mice were defective in activating T cells in vitro, and VAT T and B cell activation was markedly reduced in Cd11cCre + Myd88 fl/fl obese mice. However, neither macrophage-mediated VAT inflammation nor systemic inflammation were altered in Cd11cCre + Myd88 fl/fl mice, thereby enabling a focused analysis on adaptive immunity. Unexpectedly, fasting blood glucose, plasma insulin, and the glucose response to glucose and insulin were completely unaltered in Cd11cCre + Myd88 fl/fl vs. control obese mice. Thus, CD11c+ cells activate VAT T and B cells in obese mice, but suppression of this process does not have a discernible effect on macrophage-mediated VAT inflammation or systemic glucose homeostasis.

Hepatocyte-secreted DPP4 in obesity promotes adipose inflammation and insulin resistance

Obesity-induced metabolic disease involves functional integration among several organs via circulating factors, but little is known about crosstalk between liver and visceral adipose tissue (VAT). In obesity, VAT becomes populated with inflammatory adipose tissue macrophages (ATMs). In obese humans, there is a close correlation between adipose tissue inflammation and insulin resistance, and in obese mice, blocking systemic or ATM inflammation improves insulin sensitivity. However, processes that promote pathological adipose tissue inflammation in obesity are incompletely understood. Here we show that obesity in mice stimulates hepatocytes to synthesize and secrete dipeptidyl peptidase 4 (DPP4), which acts with plasma factor Xa to inflame ATMs. Silencing expression of DPP4 in hepatocytes suppresses inflammation of VAT and insulin resistance; however, a similar effect is not seen with the orally administered DPP4 inhibitor sitagliptin. Inflammation and insulin resistance are also suppressed by silencing expression of caveolin-1 or PAR2 in ATMs; these proteins mediate the actions of DPP4 and factor Xa, respectively. Thus, hepatocyte DPP4 promotes VAT inflammation and insulin resistance in obesity, and targeting this pathway may have metabolic benefits that are distinct from those observed with oral DPP4 inhibitors.

Endoplasmic Reticulum Stress in Cardiometabolic Disorders

When endoplasmic reticulum (ER) homeostasis is disrupted, an adaptive signaling pathway, called the unfolded protein response (UPR) is activated to help ER cope with the stress. The UPR is an important signal transduction pathway, crucial for the survival and function of all cells. Recently, there has been a substantial progress made in understanding the molecular mechanisms of physiological UPR regulation and its role in the pathogenesis of many diseases including metabolic diseases. Studies using mouse models lacking or overexpressing the factors involved in ER stress signaling as well as work performed on humans have revealed the contribution of UPR to disease progression. This review focuses on the regulation of UPR signaling and its relevance in pathogenesis of metabolic diseases.