Ugra S. Singh

Cannabinoid-induced apoptosis in immune cells as a pathway to immunosuppression

Cannabinoids are a group of compounds present in Cannabis plant (Cannabis sativa L.). They mediate their physiological and behavioral effects by activating specific cannabinoid receptors. With the recent discovery of the cannabinoid receptors (CB1 and CB2) and the endocannabinoid system, research in this field has expanded exponentially. Cannabinoids have been shown to act as potent immunosuppressive and anti-inflammatory agents and have been shown to mediate beneficial effects in a wide range of immune-mediated diseases such as multiple sclerosis, diabetes, septic shock, rheumatoid arthritis, and allergic asthma. Cannabinoid receptor 1 (CB1) is mainly expressed on the cells of the central nervous system as well as in the periphery. In contrast, cannabinoid receptor 2 (CB2) is predominantly expressed on immune cells. The precise mechanisms through which cannabinoids mediate immunosuppression is only now beginning to be understood and can be broadly categorized into four pathways: apoptosis, inhibition of proliferation, suppression of cytokine and chemokine production and induction of T regulatory cells (T regs). Studies from our laboratory have focused on mechanisms of apoptosis induction by natural and synthetic cannabinoids through activation of CB2 receptors. In this review, we will focus on apoptotic mechanisms of immunosuppression mediated by cannabinoids on different immune cell populations and discuss how activation of CB2 provides a novel therapeutic modality against inflammatory and autoimmune diseases as well as malignancies of the immune system, without exerting the untoward psychotropic effects.

PKC mediates cyclic stretch‐induced cardiac hypertrophy through Rho family GTPases and mitogen‐activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch‐induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho‐guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12‐myristate 13‐acetate, indicating that phorbol ester‐sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral‐mediated gene transfer of wild‐type (WT) and dominant‐negative (DN) mutants of PKCα and δ, we found that stretch‐induced activation of Rho GTPases and phosphorylation of Rho‐GDI were mainly regulated by PKCα. PKCδ was involved in regulation of the activation of Rac1. Stretch‐induced increases in [3H]‐leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCα and δ, suggesting that PKCα and δ are both required in stretch‐induced hypertrophy, through Rho GTPases‐mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen‐activated protein (MAP) kinases. Stretch‐stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCα and δ, and that of MKK3/p38 inhibited by DN PKCδ. The phosphorylation of ERK and JNK induced by overexpression of WT PKCα, and the phosphorylation of p38 induced by WT PKCδ, were regulated by Rho GTPases. This study represents the first evidence that PKCα and δ are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch‐induced hypertrophic process.

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH‐SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH‐SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH‐SY5Y cells in a transamidation‐independent manner. RA‐induced activation of Rac1 is mediated by phosphatidylinositol 3‐kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over‐expression of constitutively active PI3K or Rac1‐V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over‐expression of dominant negative Rac1‐N17, blocks RA‐induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH‐SY5Y cells.

Role of transglutaminase II in retinoic acid‐induced activation of RhoA‐associated kinase‐2

Transamidation is a post‐translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP‐binding protein, participating in signal transduction pathways as a non‐conventional G‐protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGase. The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G‐protein, showing increased binding to its downstream target, RhoA‐associated kinase‐2 (ROCK‐2). Upon binding to RhoA, ROCK‐2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA‐stimulated interaction between RhoA and ROCK‐2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

Resveratrol Restores Nrf2 Level and Prevents Ethanol-Induced Toxic Effects in the Cerebellum of a Rodent Model of Fetal Alcohol Spectrum Disorders

In humans, ethanol exposure during pregnancy produces a wide range of abnormalities in infants collectively known as fetal alcohol spectrum disorders (FASD). Neuronal malformations in FASD manifest as postnatal behavioral and functional disturbances. The cerebellum is particularly sensitive to ethanol during development. In a rodent model of FASD, high doses of ethanol (blood ethanol concentration 80 mM) induces neuronal cell death in the cerebellum. However, information on potential agent(s) that may protect the cerebellum against the toxic effects of ethanol is lacking. Growing evidence suggests that a polyphenolic compound, resveratrol, has antioxidant and neuroprotective properties. Here we studied whether resveratrol (3,5,4′-trihydroxy-trans-stilbene), a phytoalexin found in red grapes and blueberries, protects the cerebellar granule neurons against ethanol-induced cell death. In the present study, we showed that administration of resveratrol (100 mg/kg) to postnatal day 7 rat pups prevents ethanol-induced apoptosis by scavenging reactive oxygen species in the external granule layer of the cerebellum and increases the survival of cerebellar granule cells. It restores ethanol-induced changes in the level of transcription factor nuclear factor-erythroid derived 2-like 2 (nfe2l2, also known as Nrf2) in the nucleus. This in turn retains the expression and activity of its downstream gene targets such as NADPH quinine oxidoreductase 1 and superoxide dismutase in cerebellum of ethanol-exposed pups. These studies indicate that resveratrol exhibits neuroprotective effects in cerebellum by acting at redox regulating proteins in a rodent model of FASD.

Resveratrol prevents embryonic oxidative stress and apoptosis associated with diabetic embryopathy, and improves glucose and lipid profile of diabetic dam

SCOPE Diabetic embryopathy, a consequence of diabetic pregnancy, is associated with increase in embryonic oxidative stress and apoptosis, which lead to severe embryonic damage at early stage of organogenesis. METHODS AND RESULTS This study investigated if resveratrol, found in red grapes and blue-berries, may prevent diabetes-induced oxidative stress and apoptosis in embryos and have beneficial effects in diabetic dams. A rodent model of diabetic embryopathy was used. Diabetes was associated with lowered reduced glutathione levels (26.98%), increased total thiol (100.47%) and lipid peroxidation (124.73%) in embryos, and increased blood sugar (384.03%), cholesterol (98.39%) and triglyceride (1025.35%) in diabetic dams. Increased apoptosis (272.20%) was also observed in the embryos of diabetic dams. Administration of resveratrol (100 mg/kg body weight (b.w.)) during pregnancy prevented both oxidative stress and apoptosis in embryos. Resveratrol reduced embryonic maldevelopment by improving embryo weight (41.23%), crown rump length (16.50%) and somite number (11.22%). It further improved the glucose (33.32%) and lipid (cholesterol 41.74%, triglyceride 60.64%) profile of the diabetic dams, which also represents the protective role of resveratrol in diabetes. CONCLUSION Resveratrol was found to prevent embryonic oxidative stress and apoptosis. It also improved glucose and lipid profile of diabetic dams, indicating the beneficial effects in diabetic pregnancy.

Ethanol Impairs Activation of Retinoic Acid Receptors in Cerebellar Granule Cells in a Rodent Model of Fetal Alcohol Spectrum Disorders

BACKGROUND Ethanol is the main addictive and neurotoxic constituent of alcohol. Ethanol exposure during embryonic development causes dysfunction of the central nervous system (CNS) and leads to fetal alcohol spectrum disorders. The cerebellum is one of the CNS regions that are particularly vulnerable to ethanol toxic effects. Retinoic acid (RA) is a physiologically active metabolite of vitamin A that is locally synthesized in the cerebellum. Studies have shown that RA is required for neuronal development, but it remains unknown if ethanol impairs RA signaling and thus induces neuronal malformations. In this study, we tested the hypothesis that ethanol impairs the expression and activation of RA receptors in cerebellum and in cerebellar granule cells. METHODS The cerebellum of ethanol unexposed and exposed pups was used to study the expression of retinoic acid receptors (RARs or RXRs) by immunohistochemistry and by Western blot analysis. We also studied the effect of ethanol on expression of RA receptors in the cerebellar granule cells. Activation of RA receptors (DNA-binding activities) in response to high-dose ethanol was determined by electrophoretic mobility shift and supershift assays. RESULTS Findings from these studies demonstrated that ethanol exposure reduced the expression of RARalpha/gamma while it increased the expression of RXRalpha/gamma in the cerebellum and in cerebellar granule neurons. Immuno-histological studies further strengthened the expression pattern of RA receptors in response to ethanol. The DNA-binding activity of RARs was reduced, while DNA-binding activity of RXRs was increased in response to ethanol exposure. CONCLUSION For the first time, our studies have demonstrated that high-dose ethanol affects the expression and activation of RA receptors, which could impair the signaling events and induce harmful effects on the survival and differentiation of cerebellar granule cells. Taken together, these findings could provide insight into the treatment options for brain defects caused by excessive ethanol exposure, such as in Fetal Alcohol Spectrum Disorders.

Hyperglycemia Inhibits Retinoic Acid–Induced Activation of Rac1, Prevents Differentiation of Cortical Neurons, and Causes Oxidative Stress in a Rat Model of Diabetic Pregnancy

Diabetes is a risk factor for neuronal dysfunction. Impairment in signaling mechanisms that regulate differentiation of neurons is hypothesized to be one of the main causes of neuronal dysfunction. Retinoic acid, a physiologically active retinoid synthesized from vitamin A, regulates neuronal differentiation during embryonic development and is required for maintenance of plasticity in differentiated neurons. To date, little is known about the molecular events underlying hyperglycemia-induced complications in the central nervous system (CNS). Here, we provide evidence, in a diabetes rat model, of hyperglycemia-induced oxidative stress along with apoptotic stress in developing cortical neurons isolated from 16-day-old rat embryos. We also demonstrate impaired retinoic acid signaling that is involved in neuronal differentiation. Retinoic acid–induced neurite outgrowth and expression of neuronal markers were reduced in this model. The activation of small–molecular weight G-protein, Rac1, that mediates these effects was also reduced. Retinoic acid applied at a physiological concentration significantly decreased hyperglycemia-induced oxidative stress and thus supported the antioxidant defense system. These results suggest that diabetes-induced neuronal complications during pregnancy might be due to impaired retinoic acid signaling, and exogenously administered retinoic acid may be useful against CNS complications associated with diabetes.

Role of AKT/PKB signaling in fibroblast growth factor-1 (FGF-1)-induced angiogenesis in the chicken chorioallantoic membrane (CAM)

Transfection of chicken chorioallantoic membranes (CAMs) with a chimeric secreted version of fibroblast growth factor‐1 (sp‐FGF‐1) gene construct leads to a significant increase in vascularization. Though FGF‐stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF‐1‐induced angiogenesis are poorly understood in vivo. This study was designed to investigate the role of the AKT (PKB) kinase signaling pathway in mediating sp‐FGF‐1‐induced angiogenesis in the chicken CAM. The involvement of the AKT pathway was demonstrated by up‐regulation of AKT1 mRNA expression in sp‐FGF‐1 compared to vector alone control transfected CAMs as demonstrated by real‐time RT‐PCR. Western analysis using an antibody specific to the activated AKT (phosphorylated AKT), demonstrated an increase in AKT activity in sp‐FGF‐1 compared to vector control transfected CAMs. More importantly, the AKT inhibitor ML‐9 significantly reduced sp‐FGF‐1‐induced angiogenesis in CAMs. These results indicate that AKT signaling plays a role in FGF‐1‐stimulated angiogenesis in vivo and the AKT pathway may serve as a therapeutic target for angiogenesis‐associated diseases.

Diabetic complications in pregnancy: is resveratrol a solution?

Diabetes is a metabolic disorder that, during pregnancy, may affect fetal development. Fetal outcome depends on the type of diabetes present, the concentration of blood glucose and the extent of fetal exposure to elevated or frequently fluctuating glucose concentrations. The result of some diabetic pregnancies will be embryonic developmental abnormalities, a condition referred to as diabetic embryopathy. Tight glycemic control in type 1 diabetes during pregnancy using insulin therapy together with folic acid supplementation are partially able to prevent diabetic embryopathy; however, the protection is not complete and additional interventions are needed. Resveratrol, a polyphenol found largely in the skins of red grapes, is known to have antidiabetic action and is in clinical trials for the treatment of diabetes, insulin resistance, obesity and metabolic syndrome. Studies of resveratrol in a rodent model of diabetic embryopathy reveal that it significantly improves the embryonic outcome in terms of diminishing developmental abnormalities. Improvements in maternal and embryonic outcomes observed in rodent models may arise from resveratrols antioxidative potential, antidiabetic action and antidyslipidemic nature. Whether resveratrol will have similar actions in human diabetic pregnancy is unknown. Here, we review the potential therapeutic use of resveratrol in diabetes and diabetic pregnancy.