Induction of apoptosis in Eμ-myc lymphoma cells in vitro and in vivo through calpain inhibition

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eμ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eμ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eμ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eμ-myc lymphoma is calpain 1. Treatment of Eμ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eμ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eμ-myc lymphoma and calpain inhibition has therapeutic potential.     

Progestin signaling through mPRα in Atlantic croaker granulosa/theca cell cocultures and its involvement in progestin inhibition of apoptosis

Although there is substantial evidence that membrane progestin receptors (mPRs) perform a critical physiological role in meiotic maturation of fish oocytes, it is unknown whether they are also intermediaries in progestin signaling in the surrounding follicular cells. Here, we show that mPRα protein is located on the plasma membranes of both granulosa and theca cells (G/T cells) isolated from Atlantic croaker ovaries and is associated with the presence of a single high affinity, limited capacity, pertussis toxin-sensitive, specific progestin [17,20β,21-trihydroxy-4-pregnen-3-one (20β-S)] membrane binding site with the characteristics of mPRα. Treatment of G/T cells with 20β-S caused rapid G protein activation and a transient, pertussis toxin-sensitive, decrease in cAMP levels, whereas the selective nuclear progesterone receptor agonist, R5020, did not cause G protein activation, consistent with previous reports on mPRα signaling. 20β-S treatment decreased serum starvation-induced cell death in both G/T cells and in seatrout mPRα-transfected MDA-MB-231 cells, whereas R5020 was ineffective. Moreover, a selective mPRα agonist, 10-ethenyl-19-norprogesterone, mimicked the protective action of 20β-S against cell death, which was lost upon knockdown of mPRα protein but not after progesterone receptor knockdown, further demonstrating an involvement of mPRα. Signaling molecules involved in inhibition of apoptosis, Erk and serine-threonine kinase, were activated in G/T cells by 20β-S, which suggests a potential mechanism for mPRα inhibition of apoptosis. This is the first study to demonstrate endogenous mPR signaling in the ovarian follicle and to suggest a novel physiological role for mPRα in mediating the antiapoptotic actions of progestins in ovarian follicle cells.     

PKCα negatively regulates in vitro proplatelet formation and in vivo platelet production in mice

Proplatelet formation is a part of the intricate process by which platelets are generated by their precursor cell, the megakaryocyte. The processes that drive megakaryocyte maturation and platelet production are however still not well understood. The protein kinase C (PKC) family of serine/threonine kinases has been demonstrated as an important regulator of megakaryocyte maturation and proplatelet formation, but little investigation has been made on the individual isoforms. We have previously shown, in mouse models, that PKCα plays a vital role in regulating platelet function, so in this study we aimed to investigate the role of PKCα in megakaryocyte function using the same Prkca^?/? mice. We assessed the role of global PKC and specifically PKCα in proplatelet formation in vitro, analyzed polyploidy in Prkca^?/?-derived megakaryocytes and followed platelet recovery in platelet-depleted Prkca^?/? mice. We show reduced proplatelet formation in the presence of global PKC blockade. However, in the presence of a selective classical PKC isoform inhibitor, Go6976, proplatelet formation was conversely enhanced. PKCα null megakaryocytes also showed enhanced proplatelet formation, as well as a shift to greater polyploidy. In vivo, platelet production was enhanced in response to experimentally induced immune thrombocytopenia. In conclusion, our data indicate that classical PKC isoforms, and more specifically PKCα, are negative regulators of proplatelet formation. PKCα appears to negatively regulate endomitosis, with the enhanced polyploidy observed in Prkca^?/?-derived megakaryocytes. In vivo, these observations may culminate in the observed ability of Prkca^?/? mice to recover more rapidly from a thrombocytopenic insult.     

TNF-α production and apoptosis in hepatocytes after Listeria monocytogenes and Salmonella Typhimurium invasion

Invasion of hepatocytes by Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) can stimulate tumor necrosis factor alpha (TNF-α) release and induce apoptosis. In this study, we compared the behavior of hepatocytes invaded by three L. monocytogenes serotypes (LM-4a, LM-4b and LM-1/2a) and by ST to understand which bacterium is more effective in the infectious process. We quantified TNF-α release by ELISA, apoptosis rates by annexin V (early apoptosis) and TUNEL (late apoptosis) techniques. The cell morphology was studied too. TNF-α release rate was highest in ST-invaded hepatocytes. ST and LM-1/2a induced the highest apoptosis production rates evaluated by TUNEL. LM-4b produced the highest apoptosis rate measured by annexin. Invaded hepatocytes presented various morphological alterations. Overall, LM-4b and LM-1/2a proved to be the most efficient at cell invasion, although ST adapted faster to the environment and induced earlier hepatocyte TNF-α release.     

Inhibitory role of oxytocin on TNFα expression assessed in vitro and in vivo

Aim

Oxytocin administration to diet-induced obese (DIO) rodents, monkeys and humans decreases body weight and fat mass with concomitant improvements in glucose metabolism. Moreover, several studies show an immunomodulatory role of oxytocin in a number of settings (such as atherosclerosis, injury, sepsis). This study aims to shed some light on the effects of oxytocin on macrophage polarization and cytokine production, as well as its possible impact on these parameters in adipose tissue in DIO mice with impaired glucose metabolism.

Polo-like kinase 2 regulates selective autophagic α-synuclein clearance and suppresses its toxicity in vivo

An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.Parkinson disease (PD) is a neurodegenerative disorder characterized by the progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) (1, 2). Pathologically, PD is characterized by the presence, in surviving DA neurons, of intracellular inclusions called Lewy Bodies (LBs) and Lewy Neurites (LNs) (3). These fibrillar aggregates are mainly composed of the presynaptic protein α-synuclein (α-syn) (4). Converging evidence from pathologic, genetic, biochemical, and biophysical studies supports the hypothesis that α-syn accumulation and misfolding play a central role in the pathogenesis of PD and related disorders, which are altogether referred to as synucleinopathies (5).Although α-syn is constitutively phosphorylated at low levels (<4%) at S129 (pS129) in normal brains (6–8), a clear accumulation of pS129 (>90%) is found in LBs (8, 9) and in the brain of animal models of synucleinopathies (7, 10–12). The roles of this major posttranslational modification in regulating α-syn physiology and LB formation and/or neurodegeneration in PD remain elusive.Recently, our group and others (13–15) demonstrated that Polo-like kinase 2 (PLK2), a serine/threonine kinase playing a central role in cell division, oncogenesis, and synaptic regulation in the adult brain (16–19), efficiently phosphorylates α-syn, with an exclusive preference for the S129 site. Unlike other kinases that were reported to partially phosphorylate α-syn at S129, PLK2 phosphorylates α-syn with quantitative conversion in vitro (>95%) and in mammalian cell lines (13–15).Interestingly, PLK2 expression levels are significantly up-regulated in the midbrain of aged monkeys and correlate with increased pS129 levels in dopaminergic neurons (20). Furthermore, neuropathological analysis of diseased brains and tissues from α-syn transgenic animal models of synucleinopathies also showed a significant up-regulation of PLK2 protein levels, and colocalization with pS129 in neuronal cell bodies and terminals (13). Together, these observations suggest that PLK2 might play an important role in the modulation of α-syn physiology in normal and diseased conditions. However, whether this increase in PLK2 contributes to α-syn aggregation and neurodegeneration in PD or reflects an active cellular response to prevent these processes remains unknown. Motivated by these findings and the desire to learn more about the interplay between α-syn phosphorylation, PLK2, and neurodegeneration, we sought to investigate the effect of PLK2 expression and PLK2-induced phosphorylation on α-syn physiology and toxicity in vitro and in vivo.In the present study, we provide evidence that PLK2 binds directly to α-syn in an ATP-dependent manner and regulates α-syn selective clearance via the lysosome–autophagic degradation pathway. PLK2-mediated α-syn degradation is dependent on both PLK2 kinase activity and PLK2/α-syn protein–protein interaction. Moreover, we show that PLK2 WT, but not the kinase dead mutant, decreases α-syn protein levels in a rat genetic model of PD, reverses α-syn–induced toxicity, and prevents the motor deficits caused by the overexpression of human α-syn. Consistent with the role of PLK2 on α-syn degradation found in vitro, this effect depends on S129 phosphorylation. Collectively, our data support the neuroprotective role of PLK2 against PD pathology, which seems to be mediated by selective autophagy clearance of human α-syn, and suggest that modulating PLK2 activity is a viable therapeutic strategy for the treatment of PD and related disorders.     

Knockdown of RhoGDIα induces apoptosis and increases lung cancer cell chemosensitivity to paclitaxel

This study aimed to investigate the effects of RhoGDIα knockdown on apoptosis and the chemosensitivity of lung cancer cells to paclitaxel. The signaling proteins involved were also assessed. RhoGDIα expression was assessed by RT-PCR, Western blotting and immunohistochemistry. Apoptosis was determined by flow cytometric assessment, and cell viability was measured with the MTT assay. Phosphorylation levels of signaling proteins, ERK, JNK, Akt, Bad and IκBα were tested by Western blotting and immunohistochemistry. Positivity for RhoGDIα in lung cancer tissues was significantly higher than in paracancerous tissues. Downregulation of RhoGDIα was associated with significantly increased apoptosis and repressed cell viability. This effect could be due to the consequent upregulation of p-JNK, as well as decreased levels of p-ERK, p-Bad and p-IκBα. Knockdown of RhoGDIα strengthened the effect on apoptosis and inhibition of cell viability induced by paclitaxel treatment. This chemosensitization effect could be a result of the intensification of pro-apoptotic JNK activation, and repression of anti-apoptotic p-ERK, p-Bad and p-IκBα expression stimulated by paclitaxel. In summary, our study indicated that RhoGDIα could be a promising therapeutic target, and the combination of RhoGDIα siRNA and paclitaxel might be a valuable potential therapy for lung cancer treatment.     

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-β1

Purpose In the liver, transforming growth factor beta-1 (TGF-1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-1 in stages of chemically induced mouse liver carcinogenesis.Methods Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-1 (56 g or 200 g TGF-1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections.Results In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ß1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six g TGF-1/kg had no detectable effect on apoptosis.Conclusion These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.     

α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic β-cells

α1-antitrypsin (AAT) is a serine protease inhibitor, which recently has been shown to prevent type 1 diabetes (T1D) development, to prolong islet allograft survival and to inhibit β-cell apoptosis in vivo. It has also been reported that T1D patients have significantly lower plasma concentrations of AAT suggesting the potential role of AAT in the pathogenesis of T1D. We have investigated whether plasma-purified AAT can affect β-cell function in vitro. INS-1E cells or primary rat pancreatic islets were used to study the effect of AAT on insulin secretion after glucose, glucagon-like peptide-1 (GLP-1) and forskolin stimulation and on cytokine-mediated apoptosis. The secreted insulin and total cyclic AMP (cAMP) were determined using radioimmunoassay and apoptosis was evaluated by propidium iodide staining followed by FACS analysis. We found that AAT increases insulin secretion in a glucose-dependent manner, potentiates the effect of GLP-1 and forskolin and neutralizes the inhibitory effect of clonidine on insulin secretion. The effect of AAT on insulin secretion was accompanied by an increase in cAMP levels. In addition, AAT protected INS-1E cells from cytokine-induced apoptosis. Our findings show that AAT stimulates insulin secretion and protects β-cells against cytokine-induced apoptosis, and these effects of AAT seem to be mediated through the cAMP pathway. In view of these novel findings we suggest that AAT may represent a novel anti-inflammatory compound to protect β-cells under the immunological attack in T1D but also therapeutic strategy to potentiate insulin secretion in type 2 diabetes (T2D).     

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.     

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation

Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.Inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with IP₃ (IRBIT) was originally identified as a molecule that interacts with the intracellular calcium channel, IP₃R. IRBIT binds to and suppresses IP₃R activity in the resting state by blocking IP₃ access to IP₃R (1, 2). Our group and others have reported that IRBIT contributes to electrolyte transport, mRNA processing, and the maintenance of genomic integrity (3–9) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain, where it is most highly expressed (1).Calcium calmodulin (CaM) dependent kinase II alpha (CaMKIIα) is a Ser/Thr kinase that is abundant in the central nervous system and is activated by the binding of Ca²⁺–CaM. CaMKIIα phosphorylates various target proteins and is involved in the regulation of synaptic transmission and plasticity (10, 11). CaMKIIα is expressed in the hippocampus, neocortex, thalamus, hypothalamus, olfactory bulb, cerebellum, and basal ganglia (12, 13). Many studies involving mutant mice and also pharmacological studies have indicated that CaMKIIα activity is essential for the acquisition of memory and learning (14, 15). In addition, the appropriate regulation of CaMKIIα is required for cognitive function and mood control (16–18). Thus, aberrant CaMKIIα activity is associated with several neuronal disorders such as schizophrenia, autism spectrum disorder, attention-deficit hyperactivity disorder (ADHD), and drug addiction, in which hyperactivity and social abnormalities are frequently observed (19–23). However, the precise mechanism linking CaMKIIα dysregulation and mental disorders is poorly understood.Recent behavioral studies using knockout (KO) mouse models or pharmacological approaches have revealed that the dysregulation of dopamine (DA) systems is correlated with a hyperactive phenotype and social abnormalities (24–26). The catecholamines, DA and norepinephrine (NE) are biosynthesized from the amino acids phenylalanine and tyrosine. The sequence of steps starts with the enzyme, tyrosine hydroxylase (TH). Thus, TH is the rate-limiting enzyme for both DA and NE synthesis. The appropriate regulation of TH activity is important for the maintenance of normal brain function and mental state (27).In this study, we identified CaMKIIα as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses the kinase activity of CaMKIIα by inhibiting the binding of CaM to CaMKIIα. In addition, we found that mice deficient in IRBIT present with hyperactivity and social abnormalities. In IRBIT KO mice, we observed increased catecholamine levels and hyperphosphorylation of Ser19 on TH, which is known to enhance TH activity and increase the biosynthesis of DA and NE (27, 28). Thus, we have concluded that IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.     

RORα suppresses proliferation of vascular smooth muscle cells through activation of AMP-activated protein kinase

Background

Retinoic acid-related orphan receptor α (RORα) has been implicated in the progression of atherosclerosis, but its role in the proliferation of vascular smooth muscle cells (vSMCs) has not been fully examined. We previously reported that RORα activates AMP-activated protein kinase (AMPK), which is associated with the suppression of vSMC proliferation. Therefore, we investigated the suppressive function of RORα on the proliferation of vSMCs and the molecular mechanisms involved.

Results

First, RORα and its activator, cholesterol sulfate (CS), induced the activation of AMPK in both human aortic SMCs and rat A7r5 cells, which was accompanied by the suppression of mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1. Second, RORα and CS modulated the expression of cell-cycle-regulating factors, such as p53, p27, and cyclin D in vSMCs. Consistent with this, the overexpression of RORα or CS treatment suppressed the proliferation of human aortic SMCs and rat A7r5 cells, possibly through G₁ arrest. RORα and CS also inhibited the migration of A7r5 cells in two-dimensional and three-dimensional cell migration assays. Finally, we demonstrated that the infusion of adenovirus encoding RORα into arteries suppressed neointima formation after balloon injury in rats.

Conclusion

These results demonstrate that RORα inhibits vSMC proliferation through AMPK-induced mTOR suppression, and suggest that RORα is a therapeutic target for the cardiovascular diseases associated with vSMC proliferation.     

Roles of PLC-γ2 and PKCα in TPA-induced apoptosis of gastric cancer cells

AIM: To investigate the roles of PLCγ2 and PKCα in TPA-induced apoptosis of gastric cancer cells. METHODS: Human gastric cancer cell line MGCS0-3 was used. Protein expression levels of PLCγ2 and PKCα were detected by Western blot. Protein localization of PLCγ2 and PKCα was shown by immunofluoscence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among I 000 cells randomly. RESULTS: Treatment of gastric cancer cells MGCS0-3 with TPA not only up-regulated expression of PLC-γ2 protein, but also induced PLC-γ2 translocation from the cytoplasm to the nucleus. However, this process was not directly associated with apoptosis induction. Further investigation showed that PKCa translocation from the cytoplasm to the nucleus was correlated with initiation of apoptosis. To explore the inevitable linkage between PLC-γ2 and PKCα during apoptosis induction, PLC inhibitor U73122 was used to block PLC-γ2 translocation,in which neither stimulating PKCα translocation nor inducing apoptosis occurred in MGC80-3 cells. However, when U73122-treated cells were exposed to TPA, not only PLC-γ2, but also PKCα was redistributed. On the other hand, when cells were treated with PKC inhibitor alone, PLC-γ2 protein was still located in the cytoplasm. However, redistribution of PLC-γ2 protein occurred in the presence of TPA, no matter whether PKC inhibitor existed or not. CONCLUSION: PLC-γ2 translocation is critical in transmittingTPA signal to its downstream molecule PKCα. As an effector, PKCα directly promotes apoptosis of MGC80-3 cells. Therefore, protein translocation of PLCγ2 and PKCα is critical event in the process of apoptosis induction.