Calcium signaling induced by 15-deoxy-prostamide-J2 promotes cell death by activating PERK, IP3R, and the mitochondrial permeability transition pore.

Melanoma is the deadliest form of skin cancer in the US. Although immunotherapeutic checkpoint inhibitors and small-molecule kinase inhibitors have dramatically increased the survival of patients with melanoma, new or optimized therapeutic approaches are still needed to improve outcomes. 15-deoxy-Δ12,14-prostamide J2 (15d-PMJ2) is an investigational small-molecule that induces ER stress-mediated apoptosis selectively in tumor cells. Additionally, 15d-PMJ2 reduces melanoma growth in vivo. To assess the chemotherapeutic potential of 15d-PMJ2, the current study sought to uncover molecular pathways by which 15d-PMJ2 exerts its antitumor activity. B16F10 melanoma and JWF2 squamous cell carcinoma cell lines were cultured in the presence of pharmacological agents that prevent ER or oxidative stress as well as Ca2+ channel blockers to identify mechanisms of 15d-PMJ2 cell death. Our data demonstrated the ER stress protein, PERK, was required for 15d-PMJ2-induced death. PERK activation triggered the release of ER-resident Ca2+ through an IP3R sensitive pathway. Increased calcium mobilization led to mitochondrial Ca2+ overload followed by mitochondrial permeability transition pore (mPTP) opening and the deterioration of mitochondrial respiration. Finally, we show the electrophilic double bond located within the cyclopentenone ring of 15d-PMJ2 was required for its activity. The present study identifies PERK/IP3R/mPTP signaling as a mechanism of 15d-PMJ2 antitumor activity.

Kinin B1 Receptor Blockade Prevents Angiotensin II-induced Neuroinflammation and Oxidative Stress in Primary Hypothalamic Neurons.

Neuroinflammation has become an important underlying factor in many cardiovascular disorders, including hypertension. Previously we showed that elevated angiotensin II (Ang II) and angiotensin II type I receptor (AT1R) expression levels can increase neuroinflammation leading to hypertension. We also found that kinin B1 receptor (B1R) expression increased in the hypothalamic paraventricular neurons resulting in neuroinflammation and oxidative stress in neurogenic hypertension. However, whether there are any potential interactions between AT1R and B1R in neuroinflammation is not clear. In the present study, we aimed to determine whether Ang II-mediated effects on inflammation and oxidative stress are mediated by the activation of B1R in mouse neonatal primary hypothalamic neuronal cultures. Gene expression and immunostaining revealed that both B1R and AT1R are expressed on primary hypothalamic neurons. Ang II stimulation significantly increased the expression of B1R, decreased mitochondrial respiration, increased the expression of two NADPH oxidase subunits (Nox2 and Nox4), increased the oxidative potential, upregulated several proinflammatory genes (IL-1ß, IL-6, and TNFα), and increased NF-kB p65 DNA binding activity. These changes were prevented by pretreatment with the B1R-specific peptide antagonist, R715. In summary, our study demonstrates a causal relationship between B1R expression after Ang II stimulation, suggesting a possible cross talk between AT1R and B1R in neuroinflammation and oxidative stress.

Expression of macrophage genes within skeletal muscle correlates inversely with adiposity and insulin resistance in humans.

Local inflammation in obese adipose tissue has been shown to contribute to insulin resistance; however, the role of macrophage infiltration within skeletal muscle is still debatable. This study aimed to evaluate the association of skeletal muscle macrophage gene expression with adiposity levels and insulin sensitivity in obese patients. Twenty-two nondiabetic obese patients and 23 healthy lean controls were included. Obese patients underwent a 3-month weight loss intervention. Macrophage gene expression in skeletal muscle (quantitative real-time polymerase chain reaction), body composition (dual-energy X-ray absorptiometry), and insulin sensitivity (homeostatic model assessment (HOMA) and oral glucose tolerance test) were compared between groups and their associations were analyzed. To validate skeletal muscle findings, we repeated the analyses with macrophage gene expression in adipose tissue. Expression levels of macrophage genes (CD68, CD11b, CD206, CD16, CD40, and CD163) were lower in skeletal muscle tissue of obese versus lean participants. Macrophage gene expression was also found to be inversely associated with adiposity, fasting insulin, and HOMA (r = -0.4 ∼ -0.6, p < 0.05), as well as positively associated with insulin sensitivity (r = 0.4 ∼ 0.8, p < 0.05). On the other hand, adipose tissue macrophage gene expression showed higher levels in obese versus lean participants, presenting a positive association with adiposity levels. Macrophage gene expression, in both skeletal and adipose tissue samples, was only minimally affected by the weight loss intervention. In contrast with the established positive relationship between adiposity and macrophage gene expression, an unexpected inverse correlation between these 2 variables was observed in skeletal muscle tissue. Additionally, muscle macrophage gene expression was inversely correlated with insulin resistance.

A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels.

EGF receptor (ERBB1) abundance in adipose tissue is reduced in insulin-resistant and type 2 diabetic women.

CONTEXT: Indications of adipose tissue dysfunction correlate with systemic insulin resistance and type 2 diabetes. It has been suggested that a defect in adipose tissue turnover may be involved in the development of these disorders. Whether this dysfunction causes or exacerbates systemic insulin resistance is not fully understood. OBJECTIVES, PARTICIPANTS, AND MEASURES: We tested whether the expression of members of the mitogenic ErbB family was reduced in adipose tissue of insulin-resistant individuals and whether ErbB1 and ErbB2 were involved in adipogenesis. Thirty-two women covering a wide range of body mass index values and insulin sensitivity participated in the cross-sectional portion of this study. We also studied preadipocytes isolated from 12 insulin-sensitive individuals to evaluate the impact of ErbB1 or ErbB2 inhibition on adipogenesis in vitro. For this purpose, we measured phospho-ErbB1 and phospho-ErbB2 levels using ELISA and the expression of peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ-regulated genes by real-time PCR. RESULTS: Among the ErbB family members, only ErbB1 expression was correlated with insulin sensitivity. Additionally, ErbB1 levels correlated positively with PPARγ and several PPARγ-regulated genes including acyl-coenzyme A synthetase long-chain family member 1 (ACSL1), adiponectin, adipose tissue triacylglycerol lipase (ATGL), diacylglycerol acyl transferase 1 (DGAT1), glycerol-3-phosphate dehydrogenase 1 (GPD1), and lipoprotein lipase (LPL), but negatively with CD36 and fatty acid-binding protein 4 (FABP4). In preadipocyte culture, ErbB1, but not ErbB2, inhibition was associated with a reduction in the expression of all the above-mentioned genes. CONCLUSIONS: These findings demonstrate a key role for ErbB1 in adipogenesis and suggest that lower ErbB1 protein abundance may lead to adipose tissue dysfunction.

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice.

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.

Orphan nuclear receptor NOR-1 enhances 3',5'-cyclic adenosine 5'-monophosphate-dependent uncoupling protein-1 gene transcription.

Prolonged cold exposure induces nonshivering thermogenesis primarily through beta-adrenergic- and cAMP-mediated regulation of uncoupling protein-1 (UCP1) in brown adipose tissue. Molecular mechanisms involved in this induction of Ucp1 gene transcription consists of an intricate interplay between many nuclear receptors in coordination with coactivators/corepressors. Recently, it has been shown that members of the nuclear receptor-4A (NR4A) family of orphan nuclear receptors (Nur77, Nurr1, and NOR-1) are highly responsive to cAMP-second messenger pathways. Here we have identified a new regulatory motif in the Ucp1 promoter that binds NR4As to stimulate Ucp1 gene transcription. Upon cold exposure of mice, or beta-agonist treatment of mouse and human adipocytes, the expression of NR4A nuclear receptors is rapidly induced, with NOR-1 being the most robust, and this precedes increases in Ucp1 expression. A dominant-negative mutant Nur-77 receptor that prevents the transcriptional activity of NR4A receptors blocked beta-adrenergic receptor-stimulated Ucp1 gene transcription. By gel shift and chromatin immunoprecipitation assays, we defined the sequence (-5.64 kb) in the Ucp1 promoter to which NOR-1 binds. In transient reporter assays, this element significantly augments the activity of a 3.7-kb Ucp1 promoter. These results extend our understanding of the combinatorial complexity in the signaling pathways that control this tissue-specific gene.

Requirement of vimentin filament assembly for beta3-adrenergic receptor activation of ERK MAP kinase and lipolysis.

Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.

p38 mitogen-activated protein kinase plays an inhibitory role in hepatic lipogenesis.

Hepatic lipogenesis is the principal route to convert excess carbohydrates into fatty acids and is mainly regulated by two opposing hormones, insulin and glucagon. Although insulin stimulates hepatic lipogenesis, glucagon inhibits it. However, the mechanism by which glucagon suppresses lipogenesis remains poorly understood. In this study, we have observed that p38 mitogen-activated protein kinase plays an inhibitory role in hepatic lipogenesis. Levels of plasma triglyceride and triglyceride accumulation in the liver were both elevated when p38 activation was blocked. Expression levels of central lipogenic genes, including sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase, hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl pyrophosphate synthase, and cytochrome P-450-51, were decreased in liver by fasting and in primary hepatocytes by glucagon but increased by the inhibition of p38. In addition, we have shown that p38 can inhibit insulin-induced expression of key lipogenic genes in isolated hepatocytes. Our results in hepatoma cells demonstrate that p38 plays an inhibitory role in the activation of the SREBP-1c promoter. Finally, we have shown that transcription of the PGC-1beta gene, a key coactivator of SREBP-1c, was reduced in liver by fasting and in isolated hepatocytes by glucagon. This reduction was significantly reversed by the blockade of p38. Insulin-induced expression of the PGC-1beta gene was enhanced by the inhibition of p38 but suppressed by the activation of p38. Together, we have identified an inhibitory role for p38 in the transcription of central lipogenic genes, SREBPs, and PGC-1beta and hepatic lipogenesis.

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation.

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.

p38 Mitogen-activated protein kinase plays a stimulatory role in hepatic gluconeogenesis.

Hepatic gluconeogenesis is essential for maintaining blood glucose levels during fasting and is the major contributor to postprandial and fasting hyperglycemia in diabetes. Gluconeogenesis is a classic cAMP/protein kinase A-dependent process initiated by glucagon, which is elevated in the blood during fasting and in diabetes. In this study, we have shown that p38 mitogen-activated protein kinase (p38) was activated in liver by fasting and in primary hepatocytes by glucagon or forskolin. Fasting plasma glucose levels were reduced upon blockade of p38 with either a chemical inhibitor or small interference RNA in mice. In examining the mechanism, inhibition of p38 suppressed gluconeogenesis in liver, along with expression of key gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Peroxisome proliferator-activated receptor gamma coactivator 1alpha and cAMP-response element-binding protein have been shown to be important mediators of hepatic gluconeogenesis. We have shown that inhibition of p38 prevented transcription of the PPARgamma coactivator 1alpha gene as well as phosphorylation of cAMP-response element-binding protein. Together, our results from in vitro and in vivo studies define a model in which cAMP-dependent activation of genes involved in gluconeogenesis is dependent upon the p38 pathway, thus adding a new player to our evolving understanding of this physiology.

Selective activation of mitogen-activated protein (MAP) kinase kinase 3 and p38alpha MAP kinase is essential for cyclic AMP-dependent UCP1 expression in adipocytes.

The sympathetic nervous system regulates the activity and expression of uncoupling protein 1 (UCP1) through the three beta-adrenergic receptor subtypes and their ability to raise intracellular cyclic AMP (cAMP) levels. Unexpectedly, we recently discovered that the cAMP-dependent regulation of multiple genes in brown adipocytes, including Ucp1, occurred through the p38 mitogen-activated protein kinases (MAPK) (W. Cao, K. W. Daniel, J. Robidoux, P. Puigserver, A. V. Medvedev, X. Bai, L. M. Floering, B. M. Spiegelman, and S. Collins, Mol. Cell. Biol. 24:3057-3067, 2004). However, no well-defined pathway linking cAMP accumulation or cAMP-dependent protein kinase (PKA) to p38 MAPK has been described. Therefore, in the present study using both in vivo and in vitro models, we have initiated a retrograde approach to define the required components, beginning with the p38 MAPK isoforms themselves and the MAP kinase kinase(s) that regulates them. Our strategy included ectopic expression of wild-type and mutant kinases as well as targeted inhibition of gene expression using small interfering RNA. The results indicate that the beta-adrenergic receptors and PKA lead to a highly selective activation of the p38alpha isoform of MAPK, which in turn promotes Ucp1 gene transcription. In addition, this specific activation of p38alpha relies solely on the presence of MAP kinase kinase 3, despite the expression in brown fat of MKK3, -4, and -6. Finally, of the three scaffold proteins of the JIP family expressed in brown adipocytes, only JIP2 co-immunoprecipitates p38alpha MAPK and MKK3. Therefore, in the brown adipocyte the recently described scaffold protein JIP2 assembles the required factors MKK3 and p38alpha MAPK linking PKA to the control of thermogenic gene expression.

Persistent nuclear factor-kappa B activation in Ucp2-/- mice leads to enhanced nitric oxide and inflammatory cytokine production.

One of the phenotypes of mice with targeted disruption of the uncoupling protein-2 gene (Ucp2-/-) is greater macrophage phagocytic activity and free radical production, resulting in a striking resistance to infectious microorganisms. In this study, the molecular mechanisms of this enhanced immune response were investigated. We found that levels of nitric oxide measured in either plasma or isolated macrophages from Ucp2-/- mice are significantly elevated in response to bacterial lipopolysaccharide challenge compared with similarly treated Ucp2+/+ mice. Likewise, expression of inducible nitric-oxide synthase and inflammatory cytokines is higher in Ucp2-/- mice in vivo and in vitro. Key steps in the activation cascade of nuclear factor (NF)-kappa B, including I kappa B kinase and nuclear translocation of NF-kappa B subunits, are all remarkably enhanced in Ucp2-/- mice, most notably even under basal conditions. The elevated basal activity of I kappa B kinase in macrophages from Ucp2-/- mice can be blocked by cell-permeable inhibitors of superoxide and hydrogen peroxide generation, but not by a specific inhibitor for inducible nitric-oxide synthase. Isolated mitochondria from Ucp2-/- cells produced more superoxide/hydrogen peroxide. We conclude that mitochrondrially derived reactive oxygen from Ucp2-/- cells constitutively activates NF-kappa B, resulting in a "primed" state to both potentiate and amplify the inflammatory response upon subsequent stimulation.

p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent transcription of the brown fat uncoupling protein 1 gene.

It is well established that catecholamine-stimulated thermogenesis in brown fat requires beta-adrenergic elevations in cyclic AMP (cAMP) to increase expression of the uncoupling protein 1 (UCP1) gene. However, little is known about the downstream components of the signaling cascade or the relevant transcription factor targets thereof. Here we demonstrate that cAMP- and protein kinase A-dependent activation of p38 mitogen-activated protein kinase (MAPK) in brown adipocytes is an indispensable step in the transcription of the UCP1 gene in mice. By phosphorylating activating transcription factor 2 (ATF-2) and peroxisome proliferator-activated receptor gamma (PPARgamma) coativator 1alpha (PGC-1alpha), members of two distinct nuclear factor families, p38 MAPK controls the expression of the UCP1 gene through their respective interactions with a cAMP response element and a PPAR response element that both reside within a critical enhancer motif of the UCP1 gene. Activation of ATF-2 by p38 MAPK additionally serves as the cAMP sensor that increases expression of the PGC-1alpha gene itself in brown adipose tissue. In conclusion, our findings illustrate that by orchestrating the activity of multiple transcription factors, p38 MAPK is a central mediator of the cAMP signaling mechanism of brown fat that promotes thermogenesis.