Tissue-Specific Expression of Neurexin-1α Isoforms in Rat Organs

Russian Journal of Bioorganic Chemistry - Neurexins are a family of synaptic adhesion proteins that play a key role in synapse formation and maintenance. Neurexins undergo extensive alternative...     

Exercise Training Prevents TNF-α Induced Loss of Force in the Diaphragm of Mice

Rationale

Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are elevated in congestive heart failure and are known to induce the production of reactive oxygen species as well as to deteriorate respiratory muscle function.

Objectives

Given the antioxidative effects of exercise training, the aim of the present study was to investigate if exercise training is capable of preventing a TNF-α induced loss of diaphragmatic force in mice and, if so, to elucidate the potential underlying mechanisms.

Methods

Prior to intraperitoneal injection of TNF-α or saline, C57Bl6 mice were assigned to four weeks of exercise training or sedentary behavior. Diaphragmatic force and power generation were determined in vitro. Expression/activity of radical scavenger enzymes, enzymes producing reactive oxygen species and marker of oxidative stress were measured in the diaphragm.

Main Results

In sedentary animals, TNF-α reduced specific force development by 42% concomitant with a 2.6-fold increase in the amount of carbonylated α-actin and creatine kinase. Furthermore, TNF-α led to an increased NAD(P)H oxidase activity in both sedentary and exercised mice whereas xanthine oxidase activity and intramitochondrial ROS production was only enhanced in sedentary animals by TNF-α. Exercise training prevented the TNF-α induced force reduction and led to an enhanced mRNA expression and activity of glutathione peroxidase. Carbonylation of proteins, in particular of α-actin and creatine kinase, was diminished by exercise training.

Conclusion

TNF-α reduces the force development in the diaphragm of mice. This effect is almost abolished by exercise training. This may be a result of reduced carbonylation of proteins due to the antioxidative properties of exercise training.     

Proteomic Analysis of Signaling Network Regulation in Renal Cell Carcinomas with Differential Hypoxia-Inducible Factor-2α Expression

Background

The loss of von Hippel–Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α).

Methods and Principal Findings

We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography–mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335.

Conclusions and Significance

Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.     

Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in Vivo due to Impaired Signal Regulatory Protein α (SIRPα) Signaling

Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1α,25(OH)₂-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47^−/− mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)⁺ osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor κβ ligand) was reduced in CD47^−/− BMC, as compared with CD47^+/+ BMC. The stromal cell phenotype in CD47^−/− BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and α-1-collagen, and reduced mineral deposition, as compared with that in CD47^+/+ BMC. CD47 is a ligand for SIRPα (signal regulatory protein α), which showed strongly reduced tyrosine phosphorylation in CD47^−/− bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRPα cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47^−/− and non-signaling SIRPα mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47^−/− mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts.     

The Influence of Fluoride on the Expression of Inhibitors of Wnt/β-Catenin Signaling Pathway in Rat Skin Fibroblast Cells

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/β-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST),were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls(0 mg F(?)/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/β-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.     

Soluble extracellular domains of human SIRPα and CD47 expressed in Escherichia coli enhances the phagocytosis of leukemia cells by macrophages in vitro

Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Pioglitazone Normalizes Insulin Signaling in the Diabetic Rat Retina through Reduction in Tumor Necrosis Factor α and Suppressor of Cytokine Signaling 3

Dysfunctional insulin signaling is a key component of type 2 diabetes. Little is understood of the effects of systemic diabetes on retinal insulin signaling. A number of agents are used to treat patients with type 2 diabetes to normalize glucose levels and improve insulin signaling; however, little has been done to investigate the effects of these agents on retinal insulin signal transduction. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, would normalize retinal insulin signal transduction through reduced tumor necrosis factor α (TNFα) and suppressor of cytokine signaling 3 (SOCS3) activities in whole retina and retinal endothelial cells (REC) and Müller cells. To test this hypothesis, we used the BBZDR/Wor type 2 diabetic rat model, as well as REC and Müller cells cultured in normoglycemia and hyperglycemic conditions, to investigate the effects of pioglitazone on TNFα, SOCS3, and downstream insulin signal transduction proteins. We also evaluated pioglitazone''s effects on retinal function using electroretinogram and markers of apoptosis. Data demonstrate that 2 months of pioglitazone significantly increased electroretinogram amplitudes in type 2 diabetic obese rats, which was associated with improved insulin receptor activation. These changes occurred in both REC and Müller cells treated with pioglitazone, suggesting that these two cell types are key to insulin resistance in the retina. Taken together, these data provide evidence of impaired insulin signaling in type 2 diabetes rats, which was improved by increasing PPARγ activity. Further investigations of PPARγ actions in the retina may provide improved treatment options.     

The Suppressive Effect of Resveratrol on HIF-1α and VEGF Expression after Warm Ischemia and Reperfusion in Rat Liver

Background

Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in many human tumors and their metastases, and is closely associated with a more aggressive tumor phenotype. The aim of the present study was to investigate the effect of resveratrol (RES) on the expression of ischemic-induced HIF-1α and vascular endothelial growth factor (VEGF) in rat liver.

Methods

Twenty-four rats were randomized into Sham, ischemia/reperfusion (I/R), and RES preconditioning groups. I/R was induced by portal pedicle clamping for 60 minutes followed by reperfusion for 60 minutes. The rats in RES group underwent the same surgical procedure as I/R group, and received 20 mg/kg resveratrol intravenously 30 min prior to ischemia. Blood and liver tissue samples were collected and subjected to biochemical assays, RT-PCR, and Western blot assays.

Results

I/R resulted in a significant (P<0.05) increase in liver HIF-1α and VEGF at both mRNA and protein levels 60 minutes after reperfusion. The mRNA and protein expressions of HIF-1α and VEGF decreased significantly in RES group when compared to I/R group (P<0.05).

Conclusion

The inhibiting effect of RES on the expressions of HIF-1α and VEGF induced by I/R in rat liver suggested that HIF-1α/VEGF could be a promising drug target for RES in the development of an effective anticancer therapy for the prevention of hepatic tumor growth and metastasis.     

GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.     

Reproductive Senescence Blunts Response of Estrogen Receptor-α Expression to Estrogen Treatment in Rat Post-Ischemic Cerebral Microvessels

Background

Several studies demonstrate that estrogen treatment improves cerebral blood flow in ischemic brain regions of young ovariectomized (OVX) rats. Estrogen receptor-α (ER-α) may mediate estrogen’s beneficial actions via its effects on the cerebral microvasculature. However, estrogen-derived benefit may be attenuated in aged, reproductively senescent (RS) rats. Our goal was to determine the effects of aging, estrogen deprivation and estrogen repletion with oral conjugated estrogens (CE) on postischemic cerebral microvascular protein expression of ER-α and ER-β.

Methods

Fisher-344 (n = 37) female rats were randomly divided into the following groups: OVX, OVX CE-treated, RS untreated, and RS CE-treated. After 30 days pretreatment with CE (0.01 mg/kg) rats were subjected to15 min. transient global cerebral ischemia. Non-ischemic naïve, OVX and RS rats were used as controls. Expression of ER-α and ER-β in isolated cortical cerebral microvessels (20 to 100 µm in diameter) was assessed using Western blot and immunohistochemistry techniques.

Results

Age and reproductive status blunted nonischemic ER-α expression in microvessels of OVX rats (0.31±0.05) and RS rats (0.33±0.06) compared to naïve rats (0.45±0.02). Postischemic microvascular expression of ER-α in OVX rats (0.01±0.0) was increased by CE treatment (0.04±0.01). Expression of ER-α in microvessels of RS rats (0.03±0.02) was unaffected by CE treatment (0.01±0.02). Western blot data are presented as a ratio of ER-α or ER-β proteins to β-actin and. Oral CE treatment had no effect on ER-β expression in postischemic microvessels of OVX and RS rats. Statistical analysis was performed by One-Way ANOVA and a Newman-Keuls or Student’s post-hoc test.

Conclusion

Chronic treatment with CE increases ER-α but not ER-β expression in cerebral microvessels of OVX rats. Aging appears to reduce the normal ability of estrogen to increase ER-α expression in postischemic cerebral microvessels.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.     

Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E₂) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER⁻ and ER⁺ breast cancer cell lines, we showed that E₂ attenuated HLA-DR in two ER⁺ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER⁻ lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα⁺ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα⁻ VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E₂ and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E₂ treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E₂ inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E₂ in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα⁻ breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα⁺ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.