Plasma S-nitrosothiol status in neonatal calves: ontogenetic associations with tissue-specific S-nitrosylation and nitric oxide synthase

Neonatal cattle and in part neonates of other species have manyfold higher plasma concentrations of nitrite plus nitrate than mature cows and subjects of other species, suggesting an enhanced and needed activation of the nitric oxide (NO) axis at birth. While the biological half-life of NO is short (<1 sec), its functionality can be prolonged, and in many regards more discretely modulated, when it reacts with low-molecular-weight and protein-bound thiols to form S-nitrosothiols (RSNO), from which NO subsequently can be rereleased. We used the calf as a model to test the hypothesis that plasma concentrations of RSNO are elevated at birth in mammals, correlate with ascorbate and urate levels, are selectively generated in critical tissue beds, and are generated in a manner temporally coincident with changes in tissue levels of active NO synthases (NOS). Plasma concentrations of RSNO, ascorbate, and urate were highest immediately after birth (Day 0), dropped >50% on Day 1, and gradually decreased over time, reaching a nadir in mature cattle. Albumin and immunoglobulin G were identified as major plasma RSNO. The presence of S-nitrosocysteine (SNC, a validated marker for S-nitrosylated proteins), inducible NOS (iNOS), and activated endothelial NOS (eNOS phosphorylated at Ser1177) in different tissues was analyzed by immunohistochemistry in another group of similar-aged calves. SNC, iNOS, and phosphorylated eNOS were detected in liver and ileum at the earliest timepoint of sampling (4 hrs after birth), increased between 4 and 24 hrs, and then declined to near-nondetectable levels by 2 weeks of life. Our data show that the neonatal period in the bovine species is characterized by highly elevated and coordinated NO-generating and nitrosylation events, with the ontogenetic changes occurring in iNOS and eNOS contents in key tissues as well as RSNO products and associated antioxidant markers.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3)

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

FTY720 suppresses interleukin-1beta-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism

BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.

Orthotopic transplantation of v-src-expressing glioma cell lines into immunocompetent mice: establishment of a new transplantable in vivo model for malignant glioma

OBJECT: The aim of this study was to develop and characterize a new orthotopic, syngeneic, transplantable mouse brain tumor model by using the cell lines Tu-9648 and Tu-2449, which were previously isolated from tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src transgenic mice. METHODS: Striatal implantation of a 1-microl suspension of 5000 to 10,000 cells from either clone into syngeneic B6C3F1 mice resulted in tumors that were histologically identified as malignant gliomas. Prior subcutaneous inoculations with irradiated autologous cells inhibited the otherwise robust development of a microscopically infiltrating malignant glioma. Untreated mice with implanted tumor cells were killed 12 days later, when the resultant gliomas were several millimeters in diameter. Immunohistochemically, the gliomas displayed both the astroglial marker GFAP and the oncogenic form of signal transducer and activator of transcription-3 (Stat3). This form is called tyrosine-705 phosphorylated Stat3, and is found in many malignant entities, including human gliomas. Phosphorylated Stat3 was particularly prominent, not only in the nucleus but also in the plasma membrane of peripherally infiltrating glioma cells, reflecting persistent overactivation of the Janus kinase/Stat3 signal transduction pathway. The Tu-2449 cells exhibited three non-random structural chromosomal aberrations, including a deletion of the long arm of chromosome 2 and an apparently balanced translocation between chromosomes 1 and 3. The GFAP-v-src transgene was mapped to the pericentromeric region of chromosome 18. CONCLUSIONS: The high rate of engraftment, the similarity to the high-grade malignant glioma of origin, and the rapid, locally invasive growth of these tumors should make this murine model useful in testing novel therapies for human malignant gliomas.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Proteomic alterations in heat shock protein 27 and identification of phosphoproteins in ascending aortic aneurysm associated with bicuspid and tricuspid aortic valve

Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. We have performed 2-dimensional gel electrophoresis and mass spectrometry coupled with dephosphorylation and phosphostaining experiments to reveal and define protein alterations and the high abundant structural phosphoproteins in BAV compared to TAV aortic aneurysm samples. 2-D gel patterns showed a high correlation in protein expression between BAV and TAV specimens (n=10). Few proteins showed significant differences, among those a phosphorylated form of heat shock protein (HSP) 27 with significantly lower expression in BAV compared to TAV aortic samples (p=0.02). The phosphoprotein tracing revealed four different phosphoproteins including Rho GDP dissociation inhibitor 1, calponin 3, myosin regulatory light chain 2 and four differentially phosphorylated forms of HSP27. Levels of total HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed that differences in HSP27 occur in the cytoplasma of VSMC's and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated.

Dual induction of PKR with E2F-1 and IFN-alpha to enhance gene therapy against hepatocellular carcinoma

Overexpression of the transcription factor E2F-1 induces apoptosis in tumor cells. This apoptotic effect is partly mediated through the induction of the double-stranded RNA-activated protein kinase (PKR). Here, we investigate if agents that upregulate PKR could enhance the apoptotic effect of E2F-1 overexpression in liver tumors. In human hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), adenovirus-mediated overexpression of E2F-1 (AdCMV-E2F) transcriptionally increased PKR mRNA. The subsequent increase of total and phosphorylated PKR protein was followed by induction of apoptosis. When AdCMV-E2F was combined with the PKR modifier interferon alpha (IFNalpha), PKR was additionally upregulated and both PKR activation and apoptosis were increased. Subcutaneous xenograft tumors were selectively targeted using an adenoviral vector expressing E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter (AdhTERT-E2F). Weekly systemic administration of AdhTERT-E2F inhibited tumor growth. The tumor suppressive effect of AdhTERT-E2F therapy was further enhanced in combination with IFNalpha.Our results demonstrate that PKR activating agents enhance the anti-tumor effect of E2F-1 overexpression in HCC in-vitro and in-vivo. Hence, modulation of PKR is a potential strategy to increase the efficacy of PKR-dependent anti-tumor therapies.

Severe structural damage of the seemingly non-diseased infrarenal aortic aneurysm neck

OBJECTIVE: The success of open and endovascular repair of abdominal aortic aneurysms (AAA) is hampered by postoperative dilatation of the anatomical neck of the AAA, which is used for graft attachment. The purpose of this study was to determine whether the macroscopically non-diseased infrarenal aortic neck of AAA is histologically and biochemically altered at the time of operative repair. METHODS: We harvested full-thickness aortic wall samples as longitudinal stripes spanning from AAA neck to aneurysmal sac in 22 consecutive patients undergoing open surgical AAA repair. Control tissue was obtained from five organ donors and five deceased subjects undergoing autopsy without evidence of aneurysmal disease. We assessed aortic media thickness, number of intact elastic lamellar units, media destruction, and neovascularization grade and performed immunohistochemistry for matrix metalloproteinase (MMP)-9 and phosphorylated c-Jun N-terminal kinase (p-JNK). MMP-9 and p-JNK protein expressions were quantified using Western Blots. RESULTS: The median thickness of the aortic media was 1150 mum in control tissue (range, 1000-1300), 510 mum in aortic necks (250-900), and 200 mum in aortic sacs (50-500, P from nonparametric test for trend <.001). The number of intact elastic lamellar units was 33 in controls (range, 33-55), 12 in aortic necks (0-31) and three in aortic sacs (0-10, P < .001). The expression of MMP-9 and p-JNK as assessed by Western Blots (P = .007 and .061, respectively) and zymography (P for trend <.001) were up regulated in both the AAA neck and sac compared with controls. Except for p-JNK expression, differences between tissues were similar after the adjustment for age, gender, and type of sampling. CONCLUSION: The seemingly non-diseased infrarenal AAA neck in patients with AAA undergoing surgical repair shows histological signs of destruction and upregulation of potential drug targets.

New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis. S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P(1)-S1P(5), and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P(1) receptor subtype. This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Coupling of mutated Met variants to DNA repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC. METHODS: Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting. RESULTS: Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation. CONCLUSIONS: Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Serine residues 1177/78/82 of the insulin receptor are required for substrate phosphorylation but not autophosphorylation

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

MEK/ERK-mediated phosphorylation of Bim is required to ensure survival of T and B lymphocytes during mitogenic stimulation

Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.