The Janus family of protein tyrosine kinases (JAKs) and STAT transcription factors regulate cellular processes involved in cell growth, differentiation, and transformation through their association with cytokine receptors. The CIS family of proteins (also referred to as the SOCS or SSI family) has been implicated in the regulation of signal transduction by a variety of cytokines. Most of them appear to be induced after stimulation with several different cytokines, and at least three of them (CIS1, CIS3/SOCS3, and JAB/SOCS1) negatively regulate cytokine signal transduction by various means: CIS1 inhibits STAT5 activation by binding to cytokine receptors that recruit STAT5, whereas JAB/SOCS-1 and CIS3/SOCS-3 directly bind to the kinase domain of JAKs, thereby inhibiting tyrosine-kinase activity. Therefore, these CIS family members seem to function in a classical negative feedback loop of cytokine signaling. Biochemical characterization as well as gene disruption studies indicate that JAB/SOCS1/SSI-1 is an important negative regulator of interferon gamma signaling. The mechanisms by which these inhibitors of cytokine signal transduction exert their effects have been extensively studied and will provide useful information for regulating tyrosine-kinase activity.

Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.

Various cytokines utilize Janus kinase (JAK) and the STAT (signal transducers and activators of transcription) family of transcription factors to carry out their biological functions. Among STATs, two highly related proteins, STAT5a and STAT5b, are activated by various cytokines, including prolactin, growth hormone, erythropoietin, interleukin 2 (IL-2), and IL-3. We have cloned a STAT5-dependent immediate-early cytokine-responsive gene, CIS1 (encoding cytokine-inducible SH2-containing protein 1). In this study, we created CIS1 transgenic mice under the control of a beta-actin promoter. The transgenic mice developed normally; however, their body weight was lower than that of the wild-type mice, suggesting a defect in growth hormone signaling. Female transgenic mice failed to lactate after parturition because of a failure in terminal differentiation of the mammary glands, suggesting a defect in prolactin signaling. The IL-2-dependent upregulation of the IL-2 receptor alpha chain and proliferation were partially suppressed in the T cells of transgenic mice. These phenotypes remarkably resembled those found in STAT5a and/or STAT5b knockout mice. Indeed, STAT5 tyrosine phosphorylation was suppressed in mammary glands and the liver. Furthermore, the IL-2-induced activation of STAT5 was markedly inhibited in T cells in transgenic mice, while leukemia inhibitory factor-induced STAT3 phosphorylation was not affected. We also found that the numbers of gamma delta T cells, as well as those of natural killer (NK) cells and NKT cells, were dramatically decreased and that Th1/Th2 differentiation was altered in transgenic mice. These data suggest that CIS1 functions as a specific negative regulator of STAT5 in vivo and plays an important regulatory role in the liver, mammary glands, and T cells.

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.

Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation.

We have recently shown that, in human neutrophils, interleukin-10 (IL-10) fails to induce specific DNA-binding activities to the gamma-interferon response region (GRR), a regulatory element located in the FcgammaRI gene promoter, which is required for transcriptional activation by IL-10 and interferon gamma (IFNgamma) in monocytic cells. In this study, we report that IL-10 is also unable to induce the binding of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite the fact that both proteins are expressed in neutrophils. Whereas IFNgamma and granulocyte colony-stimulating factor (G-CSF) are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in polymorphonuclear neutrophils (PMN), IL-10 fails to trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcgammaRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible SH2-containing protein (CIS) gene family (which also includes CIS1, CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part, under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth arrest, macrophage-like differentiation, and nitric oxide synthesis, but not IL-6 mRNA expression. Collectively, our data suggest that, in neutrophils, the activation of STAT1 and STAT3 phosphorylation is neither required for CIS3/SOCS3 induction by IL-10 nor involved in the regulatory effects of IL-10 on cytokine production.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

OBJECTIVE

Cytokines play a key pathogenic role in rheumatoid arthritis (RA). Several cytokines signal through the JAK-STAT pathway, which is negatively regulated by the suppressors of cytokine signalling (SOCS) proteins. Since SOCS protein levels can profoundly modulate cellular responses to cytokines, we have investigated their expression in chronic RA.

METHODS

The levels of SOCS1-3 and CIS1 mRNA in peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MCs), purified T cells and monocytes from RA patients and healthy volunteers were studied using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). SOCS mRNA and protein expression in synovial tissues were examined by RT-PCR and immunohistochemistry.

RESULTS

The levels of SOCS1 and SOCS3 were significantly increased in PBMCs from RA patients when compared with healthy volunteers. These differences were mainly due to up-regulation of SOCS1 in PB T cells and of SOCS3 in PB monocytes. In addition, SOCS2 was up-regulated in PB T cells. Interestingly, SF T cells expressed lower and SF macrophages higher levels of SOCS molecules than their PB counterparts. Similarly, while a significant portion of macrophages in synovial tissues expressed SOCS1 and SOCS3 proteins, the majority of T cells remained SOCS negative. Finally, SOCS1 was up-regulated in the synovial membranes from patients with RA when compared with osteoarthritis.

CONCLUSIONS

SOCS expression levels are profoundly altered in RA, and the profile of SOCS expression is dependent on both the cell type as well as the cellular compartment.

We have reported JAK-signaling modulators, CIS1 (cytokine-inducible SH2 protein-1), CIS3 and JAB (JAK2 binding protein), which are structurally related. In M1 myeloid leukemia cells, CIS3 was induced by neither interleukin 6 (IL6) nor interferon gamma (IFNgamma), while JAB was induced strongly by IFNgamma and slightly by IL6 and leukemia inhibitory factor (ILF). Forced expression of CIS3 and JAB in M1 cells prevented IL6- or LIF-induced growth arrest and differentiation, even when their expression levels were comparable to endogenous ones in several cell lines such as HEL, UT-7, IFNgamma-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNgamma but not IFNbeta resulted in suppression of LIF-induced STAT3 activation and differentiation, further supporting that physiological level of JAB is sufficient to inhibit LIF-signaling. However, unlike JAB, CIS3 did not inhibit IFNgamma-induced growth arrest, suggesting a difference in cytokine specificity between CIS3 and JAB. CIS3 inhibited STAT3 activation with slower kinetics than JAB and allowed rapid c-fos induction and partial FcgammaRI expression in response to IL6. In 293 cells, CIS3 as well as JAB bound to JAK2 tyrosine kinase domain (JH1), and inhibited its kinase activity, however, the effect of CIS3 on tyrosine kinase activity was weaker than that of JAB, indicating that CIS3 possesses lower affinity to JAK kinases than JAB. These findings suggest that CIS3 is a weaker inhibitor than JAB against JAK signaling, and JAB and CIS3 possess different regulatory roles in cytokine signaling.

Anticancer drugs kill susceptible cells through induction of apoptosis. Alterations of apoptotic pathways in drug-resistant tumor cells leading to apoptosis deficiency might represent a potent mechanism conferring drug resistance. We have assessed the effect of etoposide and cisplatin on the apoptotic pathways of the drug-sensitive human melanoma cell line MeWo as well as its etoposide- and cisplatin-resistant sublines (MeWo(Eto01), MeWo(Eto1), (and) MeWoCis01, MeWo(Cis1)). Etoposide and cisplatin induced apoptosis in drug-sensitive MeWo cells as indicated by dose-dependent (i) cytochrome c release, (ii) caspase activation, (iii) DNA fragmentation, and (iv) cleavage of poly(ADP-ribose)polymerase. In contrast, whereas low etoposide-resistant cells (MeWo(Eto01)) demonstrated reduced but detectable apoptotic activities, highly etoposide-resistant cells (MeWo(Eto1)) did not exhibit any of the apoptotic events observed in etoposide-induced cell death downstream of a strongly reduced cytochrome c release. Highly cisplatin-resistant cells (MeWo(Cis1)), however, demonstrated a reduced caspase 9 activity and cytochrome c release but the extent of effector caspase activation as well as DNA fragmentation was comparable to that of sensitive MeWo cells at equitoxic concentrations. In addition, poly(ADP-ribose)polymerase cleavage was strongly reduced in highly cisplatin-resistant sublines. Taken together, sensitive and drug-resistant MeWo cells utilized different apoptotic pathways upon drug exposure in a drug-dependent fashion and apoptosis deficiency was strongly associated with the drug-resistant phenotype.

Deoxynivalenol (DON), a trichothecene mycotoxin found in grains and cereal-based foods worldwide, impairs weight gain in experimental animals but the underlying mechanisms remain undetermined. Oral exposure to DON induces rapid and transient upregulation of proinflammatory cytokine expression in the mouse. The latter are known to induce several suppressors of cytokine signaling (SOCS), some of which impair growth hormone (GH) signaling. We hypothesized that oral exposure to DON will induce SOCS expression in the mouse. Real-time PCR and cytokine bead array revealed that oral gavage with DON rapidly (1 h) induced tumor necrosis factor-alpha and interleukin-6 mRNA and protein expression in several organs and plasma, respectively. Upregulation of mRNAs for four well-characterized SOCS (CIS [cytokine-inducible SH2 domain protein], SOCS1, SOCS2, and SOCS3) was either concurrent with (1 h) or subsequent to cytokine upregulation (2 h). Notably, DON-induced SOCS3 mRNAs in muscle, spleen and liver, with CIS1, SOCS1, and SOCS2 occurring to a lesser extent. Hepatic SOCS3 mRNA was a very sensitive indicator of DON exposure with SOCS3 protein being detectable in the liver well after the onset of cytokine decline (5 h). Furthermore, hepatic SOCS upregulation was associated with about 75% suppression of GH-inducible insulin-like growth factor acid labile subunit. Taken together, DON-induced cytokine upregulation corresponded to increased expression of several SOCS, and was associated with suppression of GH-inducible gene expression in the liver.

BACKGROUND

This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.

METHODS

The expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.

RESULTS

We found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.

CONCLUSIONS

These data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.

Neutrophils are the first cell type to migrate out of the vascular space and into the inflammatory site during an acute inflammation. However, in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), a lack of clearance of neutrophils, imbalance between inflammatory mediators produced by neutrophils and their natural inhibitors make these cells a potential cause of tissue destruction in lung disease. Neutrophilic inflammation is generally characterised by high levels of local expression of activating cytokines (e.g., GM-CSF). Only a few studies have been published so far that have investigated the expression of genes preferentially expressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identification of potential novel therapeutic targets or markers of the disease. We performed representational difference analysis of cDNA, a sensitive PCR-based subtractive enrichment procedure, and isolated 12 genes, 1 EST clone, and 3 sequences not represented in the public databases. Differential expression for 9 of these clones was confirmed by Northern hybridisation. Of the above nine transcripts three were chosen and shown to be up-regulated in neutrophils cocultured with stimulated primary human bronchial epithelial cells using a semiquantitative RT-PCR approach. Among the known genes identified were HM-74, CIS1, Cathepsin C, alpha-enolase, CD44, and the gene Translocation Three Four (TTF), most of them previously not known to be involved in GM-CSF induced neutrophil activation. Along with its tissue and cellular distribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initial characterisation of a novel gene, P1-89 that is primarily expressed in granulocytes and is up-regulated in activated cells. Our results identify several important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflammations.

To investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild type and mutant sta7 Chlamydomonas reinhardtii. Mutants of the STA7 locus substitute synthesis of insoluble granular starch by that of small amounts of glycogen-like material. The mutants were previously shown to lack an 88 kDa debranching enzyme. Two distinct debranching activities were detected in wild-type strains. The 88 kDa debranching enzyme subunit missing in glycogen-producing mutants (CIS1) is shown to be part of a multimeric enzyme complex. A monomeric 95 kDa debranching enzyme (CLD1) cleaved alpha-1,6 linkages separated by as few as three glucose residues while the multimeric complex was unable to do so. Both enzymes were able to debranch amylopectin while the alpha-1,6 linkages of glycogen were completely debranched by the multimeric complex only. Therefore CLD1 and the multimeric debranching enzyme display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of both CIS1 and of the multimeric isoamylase complex. In contrast to rice and maize mutants that accumulate phytoglycogen owing to mutation of an isoamylase-type DBE, isoamylase depletion in Chlamydomonas did not result in any qualitative or quantitative difference in pullulanase activity.

Mitochondrial functions are essential for cell viability and rely on protein import into the organelle. Various disease and stress conditions can lead to mitochondrial import defects. We found that inhibition of mitochondrial import in budding yeast activated a surveillance mechanism, mitoCPR, that improved mitochondrial import and protected mitochondria during import stress. mitoCPR induced expression of Cis1, which associated with the mitochondrial translocase to reduce the accumulation of mitochondrial precursor proteins at the mitochondrial translocase. Clearance of precursor proteins depended on the Cis1-interacting AAA+ adenosine triphosphatase Msp1 and the proteasome, suggesting that Cis1 facilitates degradation of unimported proteins. mitoCPR was required for maintaining mitochondrial functions when protein import was compromised, demonstrating the importance of mitoCPR in protecting the mitochondrial compartment.

Prolactin (PRL) interacts with a single-chain prolactin-specific receptor of the cytokine receptor superfamily. PRL triggers the activation of JAK2 kinase, which phosphorylates the PRL receptor itself, and of STAT5, a member of the family of signal transducers and activators of transcription (STAT). We have shown that the STAT5-dependent immediate early gene, CIS1 (Cytokine-Inducible SH2 domain-containing protein-1), suppresses PRL-induced STAT5 activation in vitro as well as in transgenic mice. To facilitate the study of the interactions between CIS1 and the PRL receptor, we have developed the yeast tri-hybrid system, a modification of the yeast two-hybrid system. We expressed CIS1 fused to the DNA-binding domain and PRL receptor cytoplasmic domain fused to the transcription activation domain in the presence or absence of the tyrosine kinase domain of JAK2 in yeast. CIS1 bound to the PRL receptor cytoplasmic domain in a JAK2-dependent manner. Moreover, we determined that the phosphorylated Y532 of the murine PRL receptor is the binding site for CIS1. Interestingly, Y532 has been shown to be unnecessary for STAT5 activation, although CIS1 overexpression suppressed PRL-induced STAT5 activation. These data suggest that the suppression of STAT5 activation by CIS1 is not due to a simple competition with STAT5 but rather to a modification of the receptor by CIS1 binding.

The ability of five members of the cytokine-inducible SH2 protein family (CIS1-4) and JAK2 binding (JAB) protein to affect prolactin receptor (PRLR)-mediated activity was tested in human 293 embryonic kidney fibroblasts transiently transfected with rat PRLR, five concentrations of CIS/JAB Myc-tagged cDNAs and a STAT5-responsive reporter gene encoding luciferase. The protein expressions of CIS1, CIS2, CIS3 and JAB were comparable, whereas the level of CIS4 was slightly lower. PRLR-mediated luciferase activity was abolished in a dose-dependent manner in cells transfected with cDNA of CIS3 or JAB, even at concentrations below the level of protein detection by anti-Myc antibody. In contrast, CIS1, CIS2 and CIS4 had little or no effect, despite similar levels of expression. CIS1 expression in postpartum mouse mammary glands was high and changed little in the course of 3 days. CIS2 and CIS3 expression was also high and increased further, whereas JAB expression was very low. These results hint that at least in mammary gland CIS3 is likely the main physiological negative regulator of the PRLR-mediated JAK2/STAT5 pathway.

The hypolipidemic mechanism of chitosan was investigated in male Sprague-Dawley rats. Animals were divided into 5 groups (n = 8): a normal fat control group, a high-fat control group (HF), a positive control group (CR), and 2 chitosan groups (CIS1 and CIS2). Chitosan was fed at the beginning (CIS1) and after 2 weeks (CIS2). A commercial diet with 5% (wt/wt) cellulose (HF), cholestyramine (CR), or chitosan (CIS1, CIS2) was fed for 6 weeks. Chitosan did not affect food intake but decreased body weight gain and significantly increased fecal fat and cholesterol excretion, reduced the lipid level in plasma and liver, increased liver hepatic and lipoprotein lipase activities compared with HF (P < .05), and tended to relieve the degenerated fatty liver tissue. No significant differences in all measurements were found between the CIS1 and CIS2 groups although the CIS1 rats exhibited lower lipid levels compared to those in the CIS2 group. The results suggest that chitosan reduced the absorption of dietary fat and cholesterol in vivo and could effectively improve hypercholesterolemia in rats.

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

A family of cytokine-inducible SH2 proteins (CISs) has recently been identified and the members are growing in number. In this family, the central SH2 domain and approximately 40 amino acids at the C-terminus (CIS homology domain; CH domain) are well conserved, while the N-terminal region shares little similarity and varies in length. Most CISs appear to be induced by several cytokines and at least three of them (CIS1, CIS3 and JAB) negatively regulate cytokine signal transduction. Forced expression of CIS1 inhibits STAT5 activation by binding of CIS1 to cytokine receptors, and CIS3 and JAB directly bind to the kinase domain of JAKs, thereby inhibiting kinase activity. Therefore, these CIS family members seem to be present in a classical negative feedback loop of cytokine signaling. They may also play a role in the mutual suppression of cytokine actions frequently found in immune and inflammatory responses. Precise molecular mechanisms of the signal inhibition and their physiological functions will be addressed in the near future. The CH domain is also found in several interesting genes containing WD-40 repeats, SPRY domains, ankyrin repeats, and GTPases. However, the function of the CH domain remains to be determined.

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.

T cells are a key element in effective cancer immunity, recognizing MHC-antigen peptide complexes on the surface of antigen presenting cells and translating these signals into cytotoxic effector T cell responses. In this study, we systematically investigated by DNA array analysis the expression profiles of 514 immunologically relevant genes in naïve and SP2/0 tumor-specific activated mouse T cell populations. Our data shows that naïve T cells expressed 37 (i.e., 7.6% of the 514) transcripts with expression level (EL) values of>> or =2.0, while the activated T cells expressed 101 such transcripts. The expression levels of 9 (1.75% of 514) of the shared transcripts were equivalent in the two populations of T cells. Ninety-six genes were differently expressed upon T cell activation, with 71 (13.81%) being up-regulated and 25 (4.86%) down-regulated. The list of significantly affected genes includes numerous cytokines and their receptors (e.g., IL-2Ralpha, IL-6Ralpha, IL-7Ralpha, IL-16, IL-17R, TGF-beta), chemokines and chemokine receptors (e.g., RANTES, CCR7, CXCR4), alternate surface proteins (e.g., 4-1BB, GITR, integrins-alphaL and -beta7, L-selectin, CD6, CD45 and EMMPRIN), cytoplasmic signaling intermediates (e,g., GATA-3, 14-3-3-eta, CIS1, SMAD4 and JAK1) and an array of other molecules (e.g., NFkappa-B inducing kinase, LTBP3 and persephin), several of which are associated with Th1 responses, and T cell self-regulation or migration. Taken together, our data contribute to our understanding of the generalized processes that accompany T cell activation and, more specifically, to our understanding of the processes associated with T cell activation during antitumor responses.

A family of cytokine-inducible SH2 proteins (CISs) has recently been identified and the number of family members is growing. In this family, the central SH2 domain and C-terminal (about 40 amino acids) (CIS homology domain; CH domain) are well conserved, while N-terminal region shares little similarity and varies in length. Most of them appear to be induced after stimulation with several different cytokines and at least three of them (CIS1, CIS3 and JAB) negatively regulate cytokine signal transduction by various means. Forced expression of CIS1 inhibits STAT5 activation by binding to cytokine receptors, whereas CIS3 and JAB directly binds to the kinase domain of JAKs, thereby inhibiting tyrosine kinase activity. Therefore, these CIS family members seem to function in a classical negative feedback loop of cytokine signaling. They may also be involved in suppression between cytokines frequently found in immune and inflammatory responses. JAB is found to inhibit interferon signaling, suggesting that elevated expression of JAB is involved in interferon-resistance. The mechanisms by which these inhibitors of cytokine signal transduction exert their effects and their physiological functions are crucial issues which need to be and will be addressed in the near future.

Cisplatin is one of the most potent chemotherapeutic antitumor drugs used in the treatment of a wide range of solid tumors. Its primary dose-limiting side effect is nephrotoxicity. The organic anion transporter 5 (Oat5) is exclusively localized in the kidney. Oat5 urinary excretion was recently proposed as a potential early biomarker of acute kidney injury (AKI). The aim of this study was to evaluate Oat5 renal expression and its urinary excretion in rats exposed to different doses of cisplatin, in comparison with traditional markers of renal injury, like renal histology, creatinine and urea plasma levels, creatinine clearance, protein and glucose urinary levels and urinary alkaline phosphatase (AP) activity. Male Wistar rats were treated with a single injection of cisplatin at different doses of 1, 2, 5 and 10 mg/kg b.w., i.p. (<em>Cis1</em>, Cis2, Cis5 and <em>Cis1</em>0, n = 4, respectively) and experiments were carried out 48 h after cisplatin administration. The renal expression of Oat5 was evaluated by immunohistochemistry and Western blotting. Oat5 abundance, AP activity, creatinine, glucose and proteins were assayed in urine. Creatinine clearance and creatinine and urea plasma levels were also evaluated. In this experimental model, plasma urea and creatinine levels, creatinine clearance, AP urinary activity and protein and glucose urinary levels were significantly modified only at the highest cisplatin dose of 10 mg/kg b.w., i.p., as compared to control rats. In contrast, Oat5 urinary abundance was increased in a dose-related manner after the administration of cisplatin. Oat5 urinary abundance was elevated at a dose as low as 1 mg/kg b.w., i.p., implying renal perturbation, when no modifications of traditional markers of renal injury are yet observed. Oat5 renal expression was decreased in a dose-related manner, both in homogenates and apical membranes from cisplatin-treated kidneys. The increase in urinary Oat5 excretion might explain the decrease in the amount of Oat5 molecules in the renal tubule cells. Hence, the preclinical animal results showed in this work propose that Oat5 urinary excretion might potentially serve as a non-invasive early biomarker of cisplatin-induced AKI.