Cardiotrophin-like Cytokine Factor Research Articles

Novel potential biomarkers for ST-elevation myocardial infarction identified by proteomic analysis of plasma-derived microparticles

Purpose: Microparticles (MP) are cell-derived membrane vesicles of less than a micron known to be increased in the blood of patients with acute coronary syndromes. They derive from various cells, most notably platelets. Our objective was to compare the proteome of plasma-derived microparticles from ST-elevation myocardial infarction (STEMI) patients and stable coronary artery disease (SCAD) controls, and to detect MP proteins differentially regulated between both groups. Methods: Plasma MP were isolated from 10 STEMI patients and 10 SCAD matched controls. We followed a standardized procedure to guarantee the purity of the microparticle population. MPs were characterized by FACS and by electron microscopy. Proteome analysis was based on high-resolution two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Validations were by 1D- and 2D-western blotting in an independent cohort of patients. Results: For the proteomic analysis, samples were pooled for each condition (STEMI and SCAD), and four gels (technical replicates) were run. 1228 spots were detected per gel. The number of differentially regulated spots was of 223 (fold change ≥ 1.5 and p < 0.05). We focused on the analysis of 115 differentially regulated spots with a fold change ≥ 2 and p < 0.05. From those, 71 were up-regulated in MP from STEMI patients, whereas 44 were up-regulated in SCAD controls. Some of the differentially regulated proteins identified play a central role in the activation of the classical pathway of the complement system and are involved in inflammatory response (e.g. complement C3 and C4a). Others are important in the maintenance of hemostasis and participate in platelet activation (e.g. von Willebrand factor, fibrinogen, fibronectin). Another protein identified was alpha-2-macroglobulin, which plays an important role in thrombogenesis and has been recently found to be up-regulated in plasma MP from deep venous thrombosis patients. Interestingly, cardiotrophin-like cytokine factor 1 (CLCF1) was found to be up-regulated in STEMI samples. This is a cytokine with B-cell stimulating capability that we recently found to be present in platelet-derived MP. Conclusions: We performed the first high-resolution proteome analysis of plasma-derived MP from STEMI and SCAD patients. We provide a panel of proteins that vary between both conditions and that might constitute a promising source of biomarkers for STEMI.

Abstract 168: Cytokine signaling through the JAK2/STAT3 pathway is a viable therapeutic target in non-small cell lung cancer

Abstract The emergence of molecular methods for classifying solid tumors based upon specific “driver mutations” has made rationally-designed chemotherapy possible in a variety of cancers. Non-small cell lung carcinoma (NSCLC) is a prototypical example of these clinical advances, and is routinely treated with targeted kinase inhibitors including erlotinib and gefitinib. Despite the success of this approach, drug resistance continues to be a significant problem in patients with advanced NSCLC. Constitutive activation of STAT3 is a common feature in non-small cell lung carcinoma (NSCLC), and has been proposed to play an important role in tumor cell resistance to conventional and targeted small-molecule therapies. Previous studies have suggested that STAT3 can be activated in NSCLC by hyperactive growth factor receptors or cytokine signaling, though the interplay between STAT3 activation and other well-characterized oncogenic “driver” mutations in NSCLC has not been fully characterized. We analyzed the activation status of key growth factor signaling pathways in seven NSCLC lines with four distinct classes of driver mutations, including activating mutations to KRAS and several receptor tyrosine kinases (EGFR, MET, PDGFRA). Targeted pharmacologic inhibitors were used to evaluate the relative contribution of each oncogene to signaling pathway activation. While common downstream effectors such as ERK, AKT, mTOR were repressed by these drugs, we observed that STAT3 remained activated in all seven cell lines independent of mutational subtype. However, pharmacologic or genetic inhibition of the gp130/Janus kinase (JAK2) signaling pathway uniformly disrupted STAT3 activation. Interestingly, only 3/7 lines produced significant levels of the cytokine interleukin-6 (IL-6), which was previously suggested to be the key mediator of gp130/JAK2 signaling in NSCLC. Gene expression and ELISA analysis of other IL-6 family ligands suggests that cardiotrophin-like cytokine factor-1 (CLCF1) may be responsible for pathway activation in the other four cell lines lacking IL-6. At the functional level, treatment of cells with the JAK1/2 inhibitor ruxolitinib had little effect on proliferation and viability in two-dimensional culture, though it inhibited anchorage-independent growth of NSCLC cells in soft agar. Ruxolitinib also slowed growth of xenografted NSCLC cells, indicating that JAK2 activity is required for optimal growth of lung tumors in vivo. Together these results demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and may play a key role in growth and/or progression of lung tumors. We suggest that addition of a JAK2 inhibitor to therapies which target commonly altered oncogenes such as KRAS or EGFR will improve clinical outcomes for NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 168. doi:1538-7445.AM2012-168

Role of cytokine receptor-like factor 1 in hepatic stellate cells and fibrosis

To elucidate the role of cytokine receptor-like factor 1 (CRLF1) in hepatic stellate cells and liver fibrosis. Rat hepatic stellate cells (HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro. Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays. Injections of carbon tetrachloride (CCl(4)) for 4 wk were employed to induce liver fibrosis. The degree of fibrosis was assessed by Sirius red staining. Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver. The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro. Expression of CRLF1, CLCF1 and ciliary neurotrophic factor receptor (CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Expression of profibrotic cytokines and collagens was analyzed by the same method. CRLF1 is a secreted cytokine with unknown function. Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin (IL)-6. Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs. Likewise, expression of CRLF1 was very low in normal livers, but was highly upregulated in fibrotic livers, where its expression correlated with the degree of fibrosis. A cofactor of CLRF1, cardiotrophin-like cytokine factor 1 (CLCF1), and the receptor which binds CRLF1/CLCF1 dimer, the CNTFR, were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers, indicating a constitutive expression. Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines, suggesting that the factor itself is not pro-inflammatory. Ectopic expression in quiescent HSCs, however, retarded their activation into myofibroblasts and specifically decreased expression of type III collagen. Inhibition of type III collagen expression by CRLF1 was also seen in the whole liver. Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type III collagen in fibrosis. By regulating activation of HSCs and expression of type III collagen, CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.

Expression of Bcl2l1, Clcf1, IL-28ra and Pias1 in the mouse heart after single and repeated administration of chlorpromazine

Several chlorpromazine (CPZ)-related deaths have been suspected in forensic autopsies but these are difficult to identify precisely because only low concentrations of CPZ can usually be detected. Patients on CPZ therapy exhibit various cardiovascular diseases, such as arrhythmia and cardiomyopathy. As our previous study revealed that CPZ administration affects the expression of immediate early genes that are induced before any other genes, we expected that CPZ probably affects the heart and, in particular, the gene expression in heart. CPZ changes tumor necrosis factor (TNF) production. After stimulation of TNF, the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway is activated via TNF receptor (TNF-R), and the pathway participates in the regulation of cellular responses such as apoptosis [1]. We used semi-arrays to determine the JAK-STAT signaling pathway in a mouse cardiomyocyte cell line, HL-1, and real-time quantitative-PCR to determine whether the semi-array data applied in vivo in mouse heart after single and once-daily repeated (1–4 weeks) low-dose (0.75 mg/kg) or high-dose (7.5 mg/kg) CPZ treatment. We found that expression of B cell lymphoma 2 like 1 ( Bcl2l1), Cardiotrophin-like cytokine factor 1 (Clcf1), Interleukin-28 receptor alpha ( IL-28ra) and Protein inhibitor of activated STAT-1 ( Pias1) were significantly changed in vivo. All these genes are associated with apoptosis. The expression level of Bcl2l1 was elevated after a single high-dose CPZ treatment and after 1 week of repeated high doses, but returned to baseline from week 2 to week 4. Clcf1 and IL-28ra expression increased from week 2 or 3 after low-dose CPZ treatment. Pias1 also increased from week 2 after low-dose CPZ treatment. Our results indicate that different doses of CPZ can induce distinct patterns of gene expression for preventing the apoptotic progression in mouse cardiomyocytes, suggesting that CPZ can affect cardiomyocytes via the JAK-STAT signaling pathway and that this might lead to cardiomyopathy. In addition, our data may help to clarify the pathophysiology of cardiomyopathy induced by CPZ and to diagnose cardiac sudden death following CPZ treatment.

Mutations in Cytokine Receptor-Like Factor 1 ( CRLF1) Account for Both Crisponi and Cold-Induced Sweating Syndromes

Crisponi syndrome is a rare autosomal recessive disorder characterized by congenital muscular contractions of facial muscles, with trismus in response to stimuli, dysmorphic features, bilateral camptodactyly, major feeding and respiratory difficulties, and access of hyperthermia leading to death in the first months of life. The overlap with Stuve-Wiedemann syndrome (SWS) is striking, but the two conditions differ in that congenital lower limb bowing is absent in Crisponi syndrome, whereas it is a cardinal feature of SWS. We report here the exclusion of the leukemia inhibitory factor receptor gene in Crisponi syndrome and the identification of homozygote or compound heterozygote cytokine receptor-like factor 1 (CRLF1) mutations in four children from three unrelated families. The four mutations were located in the immunoglobulin-like and type III fibronectin domains, and three of them predicted premature termination of translation. Using real-time quantitative polymerase chain reaction, we found a significant decrease in CRLF1 mRNA expression in patient fibroblasts, which is suggestive of a mutation-mediated decay of the abnormal transcript. CRLF1 forms a heterodimer complex with cardiotrophin-like cytokine factor 1, and this heterodimer competes with ciliary neurotrophic factor for binding to the ciliary neurotrophic factor receptor (CNTFR) complex. The identification of CRLF1 mutations in Crisponi syndrome supports the key role of the CNTFR pathway in the function of the autonomic nervous system.

STAT3-induced WNT5A signaling loop in embryonic stem cells, adult normal tissues, chronic persistent inflammation, rheumatoid arthritis and cancer (Review)

Leukemia inhibitory factor (LIF), oncostatin M, leptin, ciliary neurotrophic factor, cardiotrophin 1, cardiotrophin-like cytokine factor 1, interleukin 6 (IL6), interleukin 11 and interleukin 27 activate the gp130-JAK-STAT3 signaling cascade. Here, WNT5A was characterized as the evolutionarily conserved target of the STAT3 signaling cascade based on 11-bp-spaced tandem STAT3-binding sites within intron 4 of human, chimpanzee, cow, mouse and rat WNT5A orthologs. Canonical WNT5A signaling through Frizzled and LRP5/LRP6 receptors activates FGF20, WISP1, MYC and CCND1 transcription for the maintenance of stem/progenitor cells, while non-canonical WNT5A signaling through Frizzled and ROR2/PTK7/RYK receptors activates the RHOA, JNK, NLK and NFAT signaling cascades for the control of tissue polarity, cell adhesion or movement. LIF-induced Wnt5a activates canonical Wnt signaling in mouse embryonic stem cells for self-renewal. STAT3-induced Wnt5a activates non-canonical Wnt signaling in rat cardiac myocytes for N-cadherin-dependent aggregation. IL6, secreted from epithelial cells or macrophages, induces WNT5A upregulation in mesenchymal cells. WNT5A then activates canonical WNT signaling in epithelial cells. IL6-induced WNT5A activates canonical WNT signaling for autocrine proliferation of human synovial fibroblasts in rheumatoid arthritis. IL-6 signaling is activated during human chronic atrophic gastritis with Helicobacter pylori infection, and aberrant Stat3 signaling activation gives rise to mouse gastric tumors. WNT5A is frequently upregulated in human primary gastric cancer due to tumor-stromal interaction. WNT5A might be downregulated in advanced cancer with poorer prognosis due to genetic alterations compensating WNT5A signaling. Oncogenic WNT5A activates canonical WNT signaling in cancer stem cells for self-renewal, and non-canonical WNT signaling at the tumor-stromal interface for invasion and metastasis. SNP of genes encoding components of the cytokine-induced WNT5A signaling loop is a predicted risk factor for RA and cancer, especially diffuse-type gastric and pancreatic cancer. Humanized anti-IL6 receptor antibody and WNT5A mimetic small-molecule antagonist could be applied to personalized medicine for RA and cancer driven by the IL6-induced WNT5A signaling loop.

Cardiotrophin-Like Cytokine/Cytokine-Like Factor 1 is an Essential Trophic Factor for Lumbar and Facial MotoneuronsIn Vivo

The ciliary neurotrophic factor alpha-receptor (CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf-/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.