Tumor Necrosis Factor Receptor-2 Signals Clear-Cell Renal Carcinoma Proliferation via Phosphorylated 4E Binding Protein-1 and Mitochondrial Gene Translation.

Clear-cell renal cell carcinoma (ccRCC), a tubular epithelial malignancy, secretes tumor necrosis factor (TNF), which signals ccRCC cells in an autocrine manner via two cell surface receptors, TNFR1 and TNFR2, to activate shared and distinct signaling pathways. Selective ligation of TNFR2 drives cell cycle entry of malignant cells via a signaling pathway involving epithelial tyrosine kinase, vascular endothelial cell growth factor receptor type 2, phosphatidylinositol-3-kinase, Akt, pSer727-Stat3, and mammalian target of rapamycin. In this study, phosphorylated 4E binding protein-1 (4EBP1) serine 65 (pSer65-4EBP1) was identified as a downstream target of this TNFR2 signaling pathway. pSer65-4EBP1 expression was significantly elevated relative to total 4EBP1 in ccRCC tissue compared with that in normal kidneys, with signal intensity increasing with malignant grade. Selective ligation of TNFR2 with the TNFR2-specific mutein increased pSer65-4EBP1 expression in organ cultures that co-localized with internalized TNFR2 in mitochondria and increased expression of mitochondrially encoded COX (cytochrome c oxidase subunit) Cox1, as well as nuclear-encoded Cox4/5b subunits. Pharmacologic inhibition of mammalian target of rapamycin reduced both TNFR2-specific mutein-mediated phosphorylation of 4EBP1 and cell cycle activation in tumor cells while increasing cell death. These results signify the importance of pSer65-4EBP1 in mediating TNFR2-driven cell-cycle entry in tumor cells in ccRCC and implicate a novel relationship between the TNFR2/pSer65-4EBP1/COX axis and mitochondrial function.

TNF, acting through inducibly expressed TNFR2, drives activation and cell cycle entry of c-Kit+ cardiac stem cells in ischemic heart disease.

TNF, signaling through TNFR2, has been implicated in tissue repair, a process that in the heart may be mediated by activated resident cardiac stem cells (CSCs). The objective of our study is to determine whether ligation of TNFR2 can induce activation of resident CSCs in the setting of ischemic cardiac injury. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+)/CD45(-)/VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA. We conclude that hypoxia-induced c-kit(+) CSC activation is mediated by TNF/TNFR2/Lin-28 signaling. These observations suggest that TNFR2 signaling in resident c-kit(+) CSCs induces cardiac repair, findings which provide further understanding of the unanticipated harmful effects of TNF blockade in human IHD.

DR3 signaling protects against cisplatin nephrotoxicity mediated by tumor necrosis factor.

The expression of death receptor 3 (DR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is up-regulated in human tubular epithelial cells (TECs) during renal injury, but its function in this setting remains unknown. We used cisplatin to induce renal injury in wild-type (DR3(+/+)) or congenitally deficient DR3(-/-) mice to examine the in vivo role of DR3. Cisplatin induced the expression of DR3, its ligand, TNF-like ligand 1A (TL1A), and TNF in TECs, as observed in human renal injury. Cisplatin increased apoptotic death of DR3(-/-) TECs by twofold compared with DR3(+/+) TECs, whereas it reduced the number of tubules expressing phospho-NF-κBp65(Ser276) by 50% at 72 hours. Similar degrees of induction of DR3, TL1A, and TNF, and changes in apoptosis and phospho-NF-κBp65(Ser276), were obtained in mouse kidney organ cultures treated with cisplatin for 3 hours, suggesting a direct effect on TECs. TNF was implicated in mediating cisplatin-induced tubular damage given that the in vivo co-administration of GM6001, an inhibitor of TNF maturation and release, significantly reduced TNF production and tubular damage. Moreover, TNF exacerbated, whereas TL1A reduced, cisplatin-induced apoptosis in the DR3(+/+) mouse proximal tubule cell line, TKPTS. Our data demonstrate that cisplatin-induced nephrotoxicity is mitigated by DR3 signaling, suggesting that this occurs by antagonizing pro-apoptotic signals induced by TNF. Therefore, activating DR3 may be beneficial in reducing acute kidney injury.

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

TNFR2 ligation in human T regulatory cells enhances IL2-induced cell proliferation through the non-canonical NF-κB pathway.

Human T regulatory cells (T regs) express high levels of TNF receptor 2 (TNFR2). Ligation of TNFR2 with TNF, which can recognise both TNFR1 and TNFR2, or with a TNFR2-selective binding molecule, DARPin 18 (D18) activates canonical NF-κB signalling, assessed by IκBα degradation, and the magnitude of the response correlates with the level of TNFR2 expression. RNA-seq analysis of TNF- or D18-treated human T regs revealed that TNFR2 ligation induces transcription of NFKB2 and RELB, encoding proteins that form the non-canonical NF-κB transcription factor. In combination with IL2, D18 treatment is specific for T regs in (1) stabilising NF-κB-inducing kinase protein, the activator of non-canonical NF-κB signalling, (2) inducing translocation of RelB from cytosol to nucleus, (3) increasing cell cycle entry, and (4) increasing cell numbers. However, the regulatory function of the expanded T regs is unaltered. Inhibition of RelB nuclear translocation blocks the proliferative response. We conclude that ligation of TNFR2 by D18 enhances IL2-induced T regs proliferation and expansion in cell number through the non-canonical NF-κB pathway.

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia.

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 10(6) copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG(0) phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 µM and 5 µM) showed an increase in events within the subG(0) phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 µM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies.

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML.

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3' UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.

A rapid and sensitive method for measuring cell adhesion.

We have adapted the CyQuant(R) assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant(R) assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.

CCN3: a key growth regulator in Chronic Myeloid Leukaemia.

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. We have shown previously that the negative growth regulator, CCN3, is down-regulated as a result of Bcr-Abl kinase activity and that CCN3 has a reciprocal relationship of expression with BCR-ABL. We now show that CCN3 confers growth regulation in CML cells by causing growth inhibition and regaining sensitivity to the induction of apoptosis. The mode of CCN3 induced growth regulation was investigated in K562 CML cells using gene transfection and treatment with recombinant CCN3. Both strategies showed CCN3 regulated CML cell growth by reducing colony formation capacity, increasing apoptosis and reducing ERK phosphorylation. K562 cells stably transfected to express CCN3 showed enhanced apoptosis in response to treatment with the tyrosine kinase inhibitor, imatinib. Whilst CCN3 expression was low or undetectable in CML stem cells, primary CD34+ CML progenitors were responsive to treatment with recombinant CCN3. This study shows that CCN3 is an important growth regulator in haematopoiesis, abrogation of CCN3 expression enhances BCR-ABL dependent leukaemogenesis. CCN3 restores growth regulation, regains sensitivity to the induction of apoptosis and enhances imatinib cell kill in CML cells. CCN3 may provide an additional therapeutic strategy in the management of CML.

[Expression in Escherichia coli, purification and enzymatic properties of chicken aminopeptidase H].

Aminopeptidase H (APH) is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b(+). The recombined vector was transformed into E. coli Rosetta(DE3), and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same mo lecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94.7 mg/L till 6 hours, which contained 16.7% of the total protein. Moreover, this recombinant protein showed similar prop erties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties. Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity. These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.

Capillary electrophoresis of erythrocytes.

Capillary electrophoresis (CE) of erythrocytes from different sources under various conditions is reported in this paper. It was found that erythrocyte samples from sheep, duck, and human showed characteristic and reproducible elution peaks, and that the retention times of A-, B-, AB-, and O-type erythrocytes from human blood were distinctively different; even subtle differences, among individuals with the same blood type could be detected by CE. A strictly linear correlation was obtained between the peak area and the amount of human erythrocyte over a range of 4.8 x 10(2)-1.9 x 10(4) cells (r=0.999), indicating that CE could be used for rapid and accurate quantification of erythrocytes. Using this CE protocol, the decrease of the surface electrical charge of erythrocyte during storage was confirmed. Therefore, this work demonstrated that CE could be a useful alternative for characterizing and quantifying erythrocytes or other cells.

Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain alpha-neurotoxin from Naja atra venom.

Atratoxin-b, a short-chain alpha-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other alpha-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1).