Cytokine Receptor Binding Research Articles

130. CONDITIONAL TARGETED DELETIONS OF STAT3 TO IDENTIFY ITS ROLE IN OOCYTESAND GRANULOSA CELLS

Signal transducers and activators of transcription-3 (STAT3) is a transcription factor activated by JAK kinases after cytokine-receptor binding. We have identified active phospho-STAT3 in granulosa cells of ovarian follicles from the very early activated stage. Abundant STAT3 has also been identified by us and others in oocytes, cumulus and granulosa cells of mature preovulatory follicles. Further, we have found the STAT responsive gene product SOCS4 is present in granulosa cells of early activated and growing follicles (1). To determine the role of STAT3 in follicle growth and ovulation we have generated three unique lines of conditional STAT3 null mice with STAT3 deletion in granulosa cells or oocytes. Mice with STAT3 gene sequenced flanked by LoxP elements (STAT3fl/fl) were crossed with lines expressing Cre-recombinase in granulosa cells (AmhR2-Cre, FSHR-Cre) or oocytes (ZP3-Cre). Fertility analysis of STAT3fl/fl;Zp3-Cre females crossed with wildtype males showed no effect on fertility from STAT3 deletion in the oocyte. In contrast, both FSHR-Cre and AmhR2-Cre mediated granulosa deficient STAT3 lines had significantly reduced litter sizes; 1.4-fold and 1.9-fold lower respectively compared to control littermates (STAT3fl/+;Cre+ or STAT3fl/fl;Cre-). Furthermore AmhR2-Cre mediated STAT3 deletion resulted in a significant 1.9-fold reduction in litters per month and an increased time to first litter. Surprisingly we did not find any difference in ovulation rate after eCG+hCG stimulated superovulation, nor in naturally cylcing mice. Zygotes flushed the morning after mating and placed into culture showed no deficit in cleavage of 2-cell embryos or development to blastocyst. A potential uterine deficiency was investigated and gross morphological observations indicate a defect in the uteri which is consistent with the recent report of Cre-recombinase expression in uterine cells derived from the Mullerian duct mesenchyme (2). Thus STAT3 deficiency in oocytes does not lead to infertility while AmhR2-Cre and FSHR-Cre mediated STAT3 deficiency leads to a sub-fertile phenotype. (1) Keightley RA, et al. 2009 Reprod, Fertil Dev 21(Suppl.): 509.(2) Gonzalez G, Behringer RR. 2009 Mol Reprod Dev 76: 678–688.

Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q.

To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor-related genes, using whole-genome array-CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high-grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1-q32.1 (64.5%), 1q32.1-q44 (59.2%), 8q11.21-q24.3 (48.7%) and a loss in 17p13.3-p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2-p21.1 and gain in 8q11.21-q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade-related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC.

Gene and protein expressions in human cord blood cells after exposure to acrylonitrile

Acrylonitrile is a very high volume industrial chemical used primarily in the manufacture of plastics and rubber, which displays a pronounced acute toxicity and may be carcinogenic. The damage to the hematopoietic function by acrylonitrile may result from interference with cytokine production and cytokine receptor binding. Our present data show that acrylonitrile modulates the expression of some genes implicated in cell differentiation, cell-cycle progression, and clonogenic potential of human cord blood cells. A macroarray hybridization analysis showed that expression of the CXCR4, MCP-1, and MRP8 genes was modified by acrylonitrile exposure. Moreover, the acrylonitrile cell target seems to be the myeloid compartment, as assessed by a CFU-GM assay. In particular, the downregulation of CXCR4, MCP1, and MRP8 can be responsible for the observed reduction of cell proliferation and clonogenic capability of CFU-GM precursors. A Western blot assay showed an acrylonitrile-dependent induction of Bax, while Bcl-2 expression changed only after 48 h of chemical exposure. Bax was overexpressed in respect to Bcl-2, and this fact can be responsible for the induction in cell death after 24 h of treatment. C-fos and c-jun were also downregulated after 24 h and 6 h of treatment, respectively.

The Jak1 SH2 Domain Does Not Fulfill a Classical SH2 Function in Jak/STATSignaling but Plays a Structural Role for Receptor Interaction andUp-regulation of Receptor SurfaceExpression

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP)-tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR.

Protein-based Strategies of Molecular Therapy in Allergy

The increasing knowledge concerning pathomechanism of hypersensitivity reactions enabled development of new strategies of allergy treatment. Since the molecular background of allergy includes some, still poorly defined, abnormalities in immunoregulatory cytokines network, the successful treatment should restore proper cytokine balance. Therefore, numerous studies concerning administration of missing cytokines or inhibition of excessive one have been undertaken. The possibilities to use IgE- and cytokine-neutralizing antibodies as well as soluble cytokine receptors are discussed in this review. Moreover, attempts to create various mutant cytokines that competitively block cytokine-receptor binding have been detaily described. Although majority of clinical trials analyzing those novel treatment approaches are not finished yet, the evaluation of their preliminary results suggests potential clinical usefulness of such treatment. Obviously, these molecular methods still require further studies, nevertheless, they could be considered as novel standard in allergy treatment pretty soon. Keywords: allergy, blocking antibodies, mutant cytokines, soluble cytokine receptors

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-alpha R beta L, IFN-gamma R alpha, IL-10R alpha, IL-2R beta, and IL-4R alpha. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the first 19 aa of JH6, and, second, a C-terminal region (JH6-3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7-6 impaired the association of IL-2R beta and IL-4R alpha chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-alpha R beta L or IL-10R alpha. Interestingly, regardless of the effect on receptor binding, removal of JH7-6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors.

Cytokine-directed therapies for asthma

Increasing knowledge of the pathophysiologic roles of various cytokines in atopic diseases has provided the basis for the development of novel therapies. Strategic approaches for cytokine inhibition include the blocking of transcription factors that lead to their expression, blockade after their release, cytokine receptor antagonism, and the inhibition of signaling pathways that are activated after cytokine-receptor binding. The proinflammatory cytokines IL-5, IL-4, IL-13, and TNF-α are among the therapeutic targets. Results with a humanized anti-IL-5 have been disappointing. Although successful in markedly reducing circulating eosinophils and in preventing eosinophil accumulation in airways, the humanized anti-IL-5 was unable to affect early or late responses to allergen or to reduce airway reactivity to methacholine challenge in patients with asthma. On the other hand, a soluble IL-4 receptor antagonist has shown clinical benefits for patients with moderate asthma who require daily inhaled corticosteroids. Agents that target IL-13 and TNF-α remain to be evaluated in asthmatic inflammation. The use of cytokines with anti-inflammatory effects may also have therapeutic value. The evaluation of such agents in human beings, including IL-10, IL-12, and IFN-γ, is at a preliminary stage, but so far results have not been encouraging. (J Allergy Clin Immunol 2001;108:S72-6.)

Expression and Ligand Binding Assays of Soluble Cytokine Receptor–Immunoglobulin Fusion Proteins

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)–mouse IgG1Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1sequence to encode a receptor ECD–IgG1fusion construct. The receptor–IgG1fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD–IgG1fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R–IgG1fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR–IgG1fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.

CONSTITUTE ACTIVATION OF CYTOKINE JAK/STAT SIGNALING PATHWAY PROTEINS IN EBV-TRANSFORMED B CELL LINES FROM PATIENTS WITH PTLD

127 B cell lymphomas are a major complication of the drug induced immunosuppression required for solid organ transplantation. Epstein-Barr virus (EBV) is the etiologic agent of this post-transplant lymphoproliferative disorder (PTLD). The molecular mechanisms involved in the cellular proliferation of these lymphomas are not yet clearly defined. From PTLD patients, we previously generated EBV infected spontaneously lymphoblastoid cell lines (SLCL) with morphologic and phenotypic similarities to PTLD tumors. SLCL treatment with IL-10 or IL-6 neutralizing antibodies, or with soluble IL-10 receptor inhibits cellular proliferation, indicating that SLCL utilize IL-10 and IL-6 as autocrine growth factors that are required for sustained proliferation. Cytokine receptor binding leads to the autophosphorylation of proteins of the Janus kinase (Jak) family, which then recruit STAT (signal transducers and activators of transcription) cytoplasmic proteins. Jak activated STAT proteins dimerize and translocate to the nucleus to control various cytokine activated genes. To understand the mechanisms by which IL-10 and IL-6 stimulate SLCL we analyzed the expression levels and tyrosine phosphorylation states of the Jak/STAT signal transduction pathway proteins. Three different SLCL, termed JB7, MF4 and VB4 were examined by Western blot and immunoprecipitation. All three SLCL express Jak1, with high levels of this protein particularly evident in the JB7 cell line. Tyk2 was found to be expressed by each of the SLCL at equivalent levels. STAT proteins expressed by these cells include STAT1, STAT2, STAT3, STAT5, and STAT6. STAT4 expression was not detected. To determine which of these proteins may be actively utilized by the SLCL, the tyrosine phosphorylation states of Jak proteins were examined by western blotting with the 4G10 anti-phosphotyrosine antibody. The JB7 line expresses a high level of constitutively tyrosine phosphorylated Jak1. Additionally, all three SLCL, but not the Daudi Burkitt's lymphoma B cell line express a high level of constitutively tyrosine phosphorylated Tyk2. Thus, the cytokine mediated constitutive activation of Jak1 and Tyk2 may be critical for the autonomous growth of SLCL. An understanding of the signal transduction molecules that participate in the cytokine dependent growth of EBV B cell lymphomas may aid in the treatment of PTLD.

CONSTITUTIVE ACTIVATION OF CYTOKINE JAK/STAT SIGNALING PATHWAY PROTEINS IN EBV-TRANSFORMED B CELL LINES FROM PATIENTS WITH PTLD

127 B cell lymphomas are a major complication of the drug induced immunosuppression required for solid organ transplantation. Epstein-Barr virus(EBV) is the etiologic agent of this post-transplant lymphoproliferative disorder (PTLD). The molecular mechanisms involved in the cellular proliferation of these lymphomas are not yet clearly defined. From PTLD patients, we previously generated EBV infected spontaneous lymphoblastoid cell lines (SLCL) with morphologic and phenotypic similarities to PTLD tumors. SLCL treatment with IL-10 or IL-6 neutralizing antibodies, or with soluble IL-10 receptor inhibits cellular proliferation, indicating that SLCL utilize IL-10 and IL-6 as autocrine growth factors that are required for sustained proliferation. Cytokine receptor binding leads to the autophosphorylation of proteins of the Janus kinase (Jak) family, which then recruit STAT (signal transducers and activators of transcription) cytoplasmic proteins. Jak activated STAT proteins dimerize and translocate to the nucleus to control various cytokine activated genes. To understand the mechanisms by which IL-10 and IL-6 stimulate SLCL we analyzed the expression levels and tyrosine phosphorylation states of the Jak/STAT signal transduction pathway proteins. Three different SLCL, termed JB7, MF4 and VB4 were examined by Western blot and immunoprecipitation. All three SLCL express Jak1, with high levels of this protein particularly evident in the JB7 cell line. Tyk2 was found to be expressed by each of the SLCL at equivalent levels. STAT proteins expressed by these cells include STAT1, STAT2, STAT3, STAT5, and STAT6. STAT4 expression was not detected. To determine which of these proteins may be actively utilized by the SLCL, the tyrosine phosphorylation states of Jak proteins were examined by western blotting with the 4G10 anti-phosphotyrosine antibody. The JB7 line expresses a high level of constitutively tyrosine phosphorylated Jak1. Additionally, all three SLCL, but not the Daudi Burkitt's lymphoma B cell line express a high level of constitutively tyrosine phosphorylated Tyk2. Thus, the cytokine mediated constitutive activation of Jak1 and Tyk2 may be critical for the autonomous growth of SLCL. An understanding of the signal transduction molecules that participate in the cytokine dependent growth of EBV B cell lymphomas may aid in the treatment of PTLD.

A new cytokine-receptor binding mode revealed by the crystal structure of the IL-1 receptor with an antagonist.

Inflammation, regardless of whether it is provoked by infection or by tissue damage, starts with the activation of macrophages which initiate a cascade of inflammatory responses by producing the cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (ref. 1). Three naturally occurring ligands for the IL-1 receptor (IL1R) exist: the agonists IL-1alpha and IL-1beta and the IL-1-receptor antagonist IL1RA (ref. 2). IL-1 is the only cytokine for which a naturally occurring antagonist is known. Here we describe the crystal structure at 2.7 A resolution of the soluble extracellular part of type-I IL1R complexed with IL1RA. The receptor consists of three immunoglobulin-like domains. Domains 1 and 2 are tightly linked, but domain three is completely separate and connected by a flexible linker. Residues of all three domains contact the antagonist and include the five critical IL1RA residues which were identified by site-directed mutagenesis. A region that is important for biological function in IL-1beta, the 'receptor trigger site' is not in direct contact with the receptor in the IL1RA complex. Modelling studies suggest that this IL-1beta trigger site might induce a movement of domain 3.

Particle concentration fluorescence as an alternative to radioactivity for cytokine receptor binding assays

To detect inhibitors of the type-I interleukin-1 receptor in a safe and cost-effective nonisotopic environment, the use of particle concentration fluorescence (PCF) as a detection method for a high volume receptor binding assay (RBA) has been explored. A cell-free PCF-RBA has been developed using a soluble form of the type-I IL-1 receptor (sIL-1R), a nonneutralizing anti-sIL-1R monoclonal antibody and two fluorescein-labelled tracers: IL-1α ∗ and IL-1ra fused to the Escherichia coli maltose binding protein (MBP-IL-1ra ∗). Both ligands showed saturable specific binding ( K A values in the 10 9 M −1 range), although signal intensity was about 3-fold higher with MBP-IL-1ra ∗. The data presented here suggest that the combined use of recombinant DNA and PCF technologies can replace the traditional cell-based, radioactive RBA when screening for inhibitors of cytokine receptors.

The Effects of Amine‐Carboxyborane Related Derivatives on UMR‐106Bone Metabolism

The amine-carboxyboranes and related derivatives have been shown to be potent anti-inflammatory and anti-osteoporosis agents. Their action in part appears to be mediated by the modulation of cytokines, e.g. TNFα or IL-1. Previous studies have demonstrated that LPS induced macrophages release of TNFα maximally at 60 to 90 min. and IL-1 from 5 to 8 hr. The amine-carboxyboranes reduced significantly the release of these cytokines but also blocked TNFα high affinity binding to UMR-106 receptor at 90 min. at 10 μM, and IL-1 high affinity binding at 5 hr. at 12.5 μM. In addition, the agents suppressed IL-8 binding to CHO K1 high affinity receptor at 24 hr. at 50 μM and IL-2 binding to HuT-8 receptors at 25 μM at 90 min. and 5 hr. Correlation of metabolic events associated with osteoporosis showed that at 90 min., when TNFα receptor binding was reduced by the agents, calcium uptake into UMR-106 cells was reduced at 10 μM as well as the acid and alkaline phosphatases, and the prostaglandin cyclo-oxygenase activities and adhesion of leukocytes and macrophages to UMR-106 cell monolayers. At 5hr. when the agents reduced IL-1 binding to UMR-106 receptors, calcitonin and 1,25-dihydrovitamin D3 binding was reduced by the agents as was acid and alkaline phosphatase, and 5′-lipoxygenase activities and white blood cell adhesion. At this time calcium uptake and proline incorporation was increased significantly by the agents. At later times e.g. 18-48 hr. calcium uptake was still increased, and NAG activity was inhibited in the presence of the agents. These effects may be related more to the inhibition of other cytokine receptor binding, e.g. IL-8. Thus, many of the observed metabolic effects of amine-carboxyboranes as antiosteoporosis agents can be correlated with their inhibition of cytokine high affinity binding to target cell receptors.

Are cytokines involved in osteoarthritic pathophysiology?

The putative role and mechanism of action of cytokines in the progression of arthritic diseases such as osteoarthritis (OA) has received particular attention because of the important interaction between articular cartilage and synovium in the pathophysiology of the diseased state. Maintaining matrix homeostasis in the normal adult cartilage phenotype requires normal turnover of matrix components, principally collagen and proteoglycan. Chondrocytes and synovial fibroblasts are targeted, via specific cell-surface receptors, by cytokines like interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) to produce matrix proteases and to suppress the synthesis of collagen and proteoglycan. Thus, cytokines not only favor tissue destruction, but also inhibit tissue repair. A structurally heterogeneous group of factors capable of directly antagonizing cytokine action is described, which acts either by blocking cytokine-receptor binding, inhibiting local cytokine synthesis, or complexing the cytokine into an inactive form. Furthermore, many growth factors, such as transforming growth factor-β (TGF-β), can counteract the net effect of cytokines by stimulating the synthesis of matrix components or natural inhibitors of cartilage degrading enzymes.