Protein kinase C-zeta mediates retinal degeneration in response to TNF.

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) zeta is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCzeta specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCzeta inhibitor. PKCzeta and NF-kappaB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCzeta was observed in the control eyes, TNF induced intense expression of PKCzeta mostly in bipolar cells processes. PKCzeta staining became nuclear when TNF was coinjected with PKCzeta inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCzeta-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCzeta-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCzeta signaling pathway.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Hepatitis B Virus e Antigen Physically Associates With Receptor-Interacting Serine/Threonine Protein Kinase 2 and Regulates IL-6 Gene Expression.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-κB activation. NF-κB is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-κB activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components.

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9.Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS).Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome.

Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Insight in protein S deficiency from mouse models

Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/-) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros-/-) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface.To extend the analysis of coagulation defects beyond the Pros-/- phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001).To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis.It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/-, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration.In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation.We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6-/-Pros-/- mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros-/- embryos.In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration.Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling.

Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

Calmodulin-dependent protein kinase IV during T-cell development.

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca(2+)-dependent gene expression, is itself under stringent regulatory control during tissue development.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis.

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.