GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation.

ABSTRACT: BACKGROUND: After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl4) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl4 alone or CCl4 co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ. RESULTS: We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl4/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway. CONCLUSIONS: This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.

The role of PKCzeta in amikacin-induced apoptosis in the cochlea: prevention by aspirin.

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-kappaB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCzeta, a key regulator of NF-kappaB activation, in response to aminoglycoside treatment. Amikacin induced PKCzeta cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-kappaB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCzeta cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCzeta acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells.

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Chemoradiotherapy in malignant glioma: standard of care and future directions.

Glioma has been considered resistant to chemotherapy and radiation. Recently, concomitant and adjuvant chemoradiotherapy with temozolomide has become the standard treatment for newly diagnosed glioblastoma. Conversely (neo-)adjuvant PCV (procarbazine, lomustine, vincristine) failed to improve survival in the more chemoresponsive tumor entities of anaplastic oligoastrocytoma and oligodendroglioma. Preclinical investigations suggest synergism or additivity of radiotherapy and temozolomide in glioma cell lines. Although the relative contribution of the concomitant and the adjuvant chemotherapy, respectively, cannot be assessed, the early introduction of chemotherapy and the simultaneous administration with radiotherapy appear to be key for the improvement of outcome. Epigenetic inactivation of the DNA repair enzyme methylguanine methyltransferase (MGMT) seems to be the strongest predictive marker for outcome in patients treated with alkylating agent chemotherapy. Patients whose tumors do not have MGMT promoter methylation are less likely to benefit from the addition of temozolomide chemotherapy and require alternative treatment strategies. The predictive value of MGMT gene promoter methylation is being validated in ongoing trials aiming at overcoming this resistance by a dose-dense continuous temozolomide administration or in combination with MGMT inhibitors. Understanding of molecular mechanisms allows for rational targeting of specific pathways of repair, signaling, and angiogenesis. The addition of tyrosine kinase inhibitors vatalanib (PTK787) and vandetinib (ZD6474), the integrin inhibitor cilengitide, the monoclonal antibodies bevacizumab and cetuximab, the mammalian target of rapamycin inhibitors temsirolimus and everolimus, and the protein kinase C inhibitor enzastaurin, among other agents, are in clinical investigation, building on the established chemoradiotherapy regimen for newly diagnosed glioblastoma.

PPAR beta/delta Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKC alpha-Brief Report

Objectives-Peroxisome proliferator-activated receptor beta/delta (PPAR beta/delta) is a nuclear receptor found in platelets. PPAR beta/delta agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPAR beta/delta agonists on platelets must be nongenomic. Currently, the particular role of PPAR beta/delta receptors and their intracellular signaling pathways in platelets are not known. Methods and Results-We have used mice lacking PPAR beta/delta (PPAR beta/delta(-/-)) to show the effects of the PPAR beta/delta agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPAR beta/delta, however GW501516 had no PPAR beta/delta-specific effect on platelet aggregation. Studies in human platelets showed that PKC alpha, which can mediate platelet activation, was bound and repressed by PPAR beta/delta after platelets were treated with GW501516. Conclusions-These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk. (Arterioscler Thromb Vasc Biol. 2009; 29: 1871-1873.)

Neuropeptide Y expression and regulation in a differentiated rat insulin-secreting cell line.

Neuropeptide-Y (NPY) is a 36-amino acid peptide known to inhibit glucose-stimulated insulin secretion in various animal models in vitro and in vivo. NPY is thought to be one of the mediators of sympathetic action in the pancreas through nerve endings surrounding the islets, and it has recently been shown to be synthesized within the islets of Langerhans. To elucidate the potential role of NPY in the endocrine pancreas, we studied the expression and regulation of NPY secretion in a rat insulinoma cell line (INS-1). NPY mRNA and peptide are highly expressed and secreted by INS-1 cells. NPY levels were determined by a sensitive and specific two-site amplified enzyme-linked immunosorbent assay. Incubation of INS-1 cells with various glucose concentrations did not modify NPY secretion; however, stimulation of adenylate cyclase by forskolin induced a dose- and time-dependent increase in NPY release in the medium. The glucagon-like peptide-I-(7-36) amide (GLP-1), a known gluco-incretin in humans, induced at low concentration (10(-9) M) a similar expression of NPY mRNA and peptide secretion in INS-1 cells. On the other hand, the inhibition of cAMP accumulation by the alpha 2-adrenergic agonist clonidine decreased NPY secretion. In conclusion, 1) high levels of gene expression and secretion of NPY are found in a rat insulinoma cell line (INS-1). 2) Accumulation of cAMP induced by forskolin or a gluco-incretin (GLP-1) induces a further increase in NPY gene expression and release. 3) NPY secretion is not modulated by low or high glucose concentrations in the medium. 4) Induction of NPY, a known inhibitor of insulin secretion, may represent a novel counterregulatory mechanism of insulin secretion, limiting the stimulatory effect of GLP-1 on insulin secretion.

Adenosine A1 receptor activation is arrhythmogenic in the developing heart through NADPH oxidase/ERK- and PLC/PKC-dependent mechanisms.

Whether adenosine, a crucial regulator of the developing cardiovascular system, can provoke arrhythmias in the embryonic/fetal heart remains controversial. Here, we aimed to establish a mechanistic basis of how an adenosinergic stimulation alters function of the developing heart. Spontaneously beating hearts or dissected atria and ventricle obtained from 4-day-old chick embryos were exposed to adenosine or specific agonists of the receptors A(1)AR (CCPA), A(2A)AR (CGS-21680) and A(3)AR (IB-MECA). Expression of the receptors was determined by quantitative PCR. The functional consequences of blockade of NADPH oxidase, extracellular signal-regulated kinase (ERK), phospholipase C (PLC), protein kinase C (PKC) and L-type calcium channel (LCC) in combination with adenosine or CCPA, were investigated in vitro by electrocardiography. Furthermore, the time-course of ERK phosphorylation was determined by western blotting. Expression of A(1)AR, A(2A)AR and A(2B)AR was higher in atria than in ventricle while A(3)AR was equally expressed. Adenosine (100μM) triggered transient atrial ectopy and second degree atrio-ventricular blocks (AVB) whereas CCPA induced mainly Mobitz type I AVB. Atrial rhythm and atrio-ventricular propagation fully recovered after 60min. These arrhythmias were prevented by the specific A(1)AR antagonist DPCPX. Adenosine and CCPA transiently increased ERK phosphorylation and induced arrhythmias in isolated atria but not in ventricle. By contrast, A(2A)AR and A(3)AR agonists had no effect. Interestingly, the proarrhythmic effect of A(1)AR stimulation was markedly reduced by inhibition of NADPH oxidase, ERK, PLC, PKC or LCC. Moreover, NADPH oxidase inhibition or antioxidant MPG prevented both A(1)AR-mediated arrhythmias and ERK phosphorylation. These results suggest that pacemaking and conduction disturbances are induced via A(1)AR through concomitant stimulation of NADPH oxidase and PLC, followed by downstream activation of ERK and PKC with LCC as possible target.

Interplay of oxidative, nitrosative/nitrative stress, inflammation, cell death and autophagy in diabetic cardiomyopathy.

Diabetes is a recognized risk factor for cardiovascular diseases and heart failure. Diabetic cardiovascular dysfunction also underscores the development of diabetic retinopathy, nephropathy and neuropathy. Despite the broad availability of antidiabetic therapy, glycemic control still remains a major challenge in the management of diabetic patients. Hyperglycemia triggers formation of advanced glycosylation end products (AGEs), activates protein kinase C, enhances polyol pathway, glucose autoxidation, which coupled with elevated levels of free fatty acids, and leptin have been implicated in increased generation of superoxide anion by mitochondria, NADPH oxidases and xanthine oxidoreductase in diabetic vasculature and myocardium. Superoxide anion interacts with nitric oxide forming the potent toxin peroxynitrite via diffusion limited reaction, which in concert with other oxidants triggers activation of stress kinases, endoplasmic reticulum stress, mitochondrial and poly(ADP-ribose) polymerase 1-dependent cell death, dysregulates autophagy/mitophagy, inactivates key proteins involved in myocardial calcium handling/contractility and antioxidant defense, activates matrix metalloproteinases and redox-dependent pro-inflammatory transcription factors (e.g. nuclear factor kappaB) promoting inflammation, AGEs formation, eventually culminating in myocardial dysfunction, remodeling and heart failure. Understanding the complex interplay of oxidative/nitrosative stress with pro-inflammatory, metabolic and cell death pathways is critical to devise novel targeted therapies for diabetic cardiomyopathy, which will be overviewed in this brief synopsis. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Phosphorylation of CARMA1: the link(er) to NF-kappaB activation.

Antigen receptor-induced NF-kappaB activation depends on receptor-proximal and -distal signaling events. Two papers in this issue of Immunity demonstrate that PKC-dependent phosphorylation of CARMA1 is the critical molecular link that controls the activation of the IKK signalosome and NF-kappaB.

The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

The activation of the atypical PKC zeta in light-induced retinal degeneration and its involvement in L-DNase II control.

Light-induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase-independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI-derived DNase II (LEI/L-DNase II), a caspase-independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro-survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L-DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase-independent pathways.