IL-4-induced Stat6 activities affect apoptosis and gene expression in breast cancer cells.

IL-4-induced Stat6 signaling is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression. Using EMSA gel shift assay and an antibody to Stat6, we phenotyped two breast cancer cell lines, ZR-75-1 being active Stat6(high) phenotype and BT-20 being defective Stat6(null) phenotype, respectively. Breast cancer cells carrying Stat6(null) phenotype exhibited increased spontaneous apoptosis compared with those carrying Stat6(high) phenotype. Expression microarray analyses demonstrated that IL-4 upregulated CCL26, SOCS1, CISH, EGLN3, and SIDT1, and downregulated DUSP1, FOS, and FOSB, respectively, in these breast cancer cells. Among those genes, CCL26 and SOCS1 were known genes regulated by IL-4/Stat6 pathway, but CISH, EGLN3, SIDT1, DUSP1, FOS, and FOSB were novel genes demonstrated to be IL-4 responsive for the first time. IL-4 also upregulated 38 genes unique to Stat6(null) BT-20 cells and 23 genes unique to Stat6(high) ZR-75-1 cells, respectively. Furthermore, Stat6(high) and Stat6(null) cells showed very different profiles of constitutively expressed genes relevant to apoptosis and metastasis among others, which serve as a valuable expression database and warrant for detailed studies of IL-4/Stat6 pathway in breast cancer.

High-throughput non-heme iron assay for animal tissues.

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.

Human B lymphoblast cell lines defective of Stat6 signaling produce high levels of proinflammatory cytokines IL-12, TNFalpha and IFNgamma.

Based on Stat6 gene knockout animal models, the Stat6 signaling pathway has been suggested to play a role in carcinogenesis and Th1/Th2 cytokine balance. Using a semiquantitative EMSA assay and EBV-transformed human B lymphoblast cell lines, we have previously identified three Stat6 activational phenotypes, termed as Stat6high, Stat6low, and Stat6null. A genetic mechanism has been proposed which determines the IL-4-induced activation of the human Stat6 signaling. With respect to the contribution of variant phenotypes to human disease, we further hypothesize that the Stat6null phenotype may result from a partial defect in Stat6 signaling which resembles Stat6 knockout animals in several functional aspects. The characterization of the human Stat6null phenotype stably displayed by the EBV-B cell lines is easily assailable and possesses important implications with respect to Th1/Th2 cytokine imbalance in diseases such as cancer development/metastasis and inflammatory diseases. In this study, we have extended our investigation to the downstream regulatory consequences associated with these Stat6 phenotypes. Production of three important proinflammatory cytokines, IL-12, TNFalpha and IFNgamma was examined in spontaneous EBV-B cell culture using ELISA methodology. Individual cell lines defined as Stat6null produced significantly higher levels of IL-12, TNFalpha and IFNgamma on day 4 in spontaneous culture in comparison with cell lines characterized as Stat6high and Stat6low. These observations of the human Stat6null phenotype, together with those accruing from Stat6 knockout mouse model studies, suggest that the Stat6 signaling pathway may play a role in maintaining the Th1/Th2 cytokine balance by directly and indirectly down-regulating the production of proinflammatory cytokines, a regulatory process which appears to go awry in inflammatory diseases. Moreover, observations from signal transduction studies in our human B lymphocyte model may be compatible with those in the chosen mouse B lymphocyte for establishing signaling networks by the Alliance for Cellular Signaling (AfCS).

Phenotyping of IL4-induced nuclear Stat6 activity in humans: quantitation after gel shift assay using immortalized cell lines.

The IL4-induced Stat6 signaling pathway is active in a variety of cell types, including different cancer cells, and plays an important role in the regulation of gene expression, such as CD23. There are large quantitative differences in DNA-binding activity of IL4-induced Stat6, which are useful for phenotyping activated Stat6 in normal and disease status. However, quantitation of activated Stat6 is challenging and a standardized methodology is needed. Here we have developed a semi-quantitative methodology using gel shift assay in which IL4-induced nuclear Stat6 activities are measured in human EBV-transformed lymphoblastoid cell lines. Using a DNA probe with high affinity Stat6-binding N4 motif and a specific antibody to Stat6, autoradiographs of EMSA gels are recorded by a scan imager and OD readings of antibody super-shifted Stat6 complex bands are obtained. OD readings of all test cell lines are referenced to that of a standard cell line placed in every single experiment and an OD ratio is obtained for each test cell, which allows assignment of Stat6 activational phenotypes. Using this methodology, we have been able to define three Stat6 activational phenotypes termed as Stat6high (intense banding), Stat6low (medium intensity banding), and Stat6null (very low to no discernible banding). These Stat6 phenotypes correlate well with levels of CD23 expression, but not with those of HLA-DR. Pedigree analysis has revealed a Mendelian inheritance pattern for Stat6 phenotypes. The methodology is useful in association studies in human cancer and autoimmune diseases. The Stat6null phenotype may result from a defect in Stat6 signaling which has important implications with respect to the pathogenesis of cancer and Th1/Th2 cytokine imbalance in general. In addition, the defective Stat6null lines discovered here may serve as a natural human model for comprehensive study in the same way as a Stat6 knockout null animal model does.

CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells.

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid alpha-galactosylceramide (alpha-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection. Overexpression of CD1d in transgenic (Tg) mice results in reduced numbers of NKT cells, and the residual NKT cells in CD1d-Tg mice exhibit both an altered Vbeta usage and a reduced sensitivity to antigen. Furthermore, bone marrow (BM) chimeras between Tg and WT mice reveal that CD1d-expressing BM-derived dendritic cells, but not thymic epithelial cells, mediate the efficient negative selection of NKT cells. Thus, our data suggest that NKT cells developmentally undergo negative selection when engaged by high-avidity antigen or abundant self-antigen.

The enigmatic role of the hemochromatosis protein (HFE) in iron absorption.

The HFE gene, a member of the class-I transplantation antigen gene family, is responsible for hereditary hemochromatosis, one of the most common inherited diseases in individuals of European descent. Patients exhibit predictable changes in iron homeostasis, including elevations in both transferrin saturation and serum ferritin levels. A subset of patients progress to overt clinical sequelae, resulting from iron overload. A hallmark of the disease is increased absorption of iron by the intestine. Although the HFE protein appears to modulate the function of the transferrin receptor in vitro, its precise role in vivo remains obscure. With multiple cell types involved in iron metabolism, the function of HFE is likely to be complex.

Genetic control of interleukin-4-induced activation of the human signal transducer and activator of transcription 6 signaling pathway.

The interleukin (IL)-4-induced Stat6 signaling pathway is active in a variety of cell types, including immune cells and cancer cells, and plays an important role in the regulation of gene expression, such as CD23 and major histocompatibility complex class II. Using a semiquantitative gel shift assay in which nuclear Stat6 activities were scored, three Stat6 activation phenotypes were defined as Stat6(high) (intense banding), Stat6(low) (medium intensity banding), and Stat6(null) (very low to no discernible banding). These Stat6 phenotypes correlated well with levels of CD23 expression, but not with those of human leukocyte antigen-DR cell-surface display. Pedigree analyses revealed a Mendelian inheritance pattern that can be explained by two STAT6 Pathway (STAT6P) activation genotypes, which we term A and a, where STAT6P*A determines an active Stat6 signaling and STAT6P*a determines an inactive Stat6 signaling, with incomplete dominance. Total Stat6 protein levels failed to correlate with the above Stat6 phenotypes allowing us to propose that IL-4-induced Stat6 signaling is a polygenic quantitative trait regulated by a collection of several contributing genetic loci that functionally interact. The Stat6(null) phenotype may result from a defect in Stat6 signaling, which has important implications with respect to the pathogenesis of cancer and Th1/Th2 cytokine imbalance in autoimmune diseases in general.

Genetic differences in hepatic lipid peroxidation potential and iron levels in mice.

Oxidative damage to macromolecules, including lipids, has been hypothesized as a mechanism of aging. One end product of lipid peroxidation, malondialdehyde (MDA), is often quantified as a measure of oxidative damage to lipids. We used a commercial colorimetric assay for MDA (Bioxytech LPO-586, Oxis International, Portland, OR) to measure lipid peroxidation potential in liver tissue from young (2 month) male mice from recombinant inbred (RI) mouse strains from the C57BL/6J (B6)xDBA/2J (D2) series (BXD). The LPO-586 assay (LPO) reliably detected significant differences (P<0.0001) in lipid peroxidation potential between the B6 and D2 parental strains, and yielded a more than two-fold variation across the BXD RI strains. In both B6 and D2 mice, LPO results were greater in old (23 month) mice, with a larger age-related increase in the D2 strain. As the level of iron can influence lipid peroxidation, we also measured hepatic non-heme iron levels in the same strains. Although iron level exhibited a slightly negative overall correlation (r(2)=0.119) with LPO results among the entire group of BXD RI strains, a sub-group with lower LPO values were highly correlated (r(2)=0.704). LPO results were also positively correlated with iron levels from a group of 8 other inbred mouse strains (r(2)=0.563). The BXD RI LPO data were statistically analyzed to nominate quantitaive trait loci (QTL). A single marker, Zfp4, which maps to 55.2 cM on chromosome 8, achieved a significance level of P<0.0006. At least two potentially relevant candidate genes reside close to this chromosomal position. Hepatic lipid peroxidation potential appears to be a strain related trait in mice that is amenable to QTL analysis.

Hemochromatosis protein (HFE) and tumor necrosis factor receptor 2 (TNFR2) influence tissue iron levels: elements of a common gut pathway?

Quantitative genetic analysis of hepatic and splenic iron levels in recombinant inbred mice yielded a quantitative trait locus that was found to coincide with the genomic locale encompassing the tumor necrosis factor receptor 2 gene (Tnfr2). When fed an iron-enriched diet, mice nullizygous with respect to Tnfr2, but not the Tnfr1 gene, showed a significant increase in splenic non-heme iron levels. This result contrasted with mice deficient in the hemochromatosis protein, HFE, which demonstrated a significant increase in normally high hepatic iron levels, but no change in splenic iron, when fed an iron-enriched chow. Both Tnfr2 knockout and HFE knockout mice fed an iron-enriched diet failed to demonstrate intestinal epithelial cell iron following the application of the Perls' stain, as compared to both Tnfr1 knockout and normal control mice. Moreover, intestinal intraepithelial lymphocytes (IELs) isolated from HFE knockout mice did not show an increase in TNF expression following challenge with the iron-enriched diet, in contrast to normal controls. These results suggest that HFE and TNFR2 are both involved in regulating iron deposition in tissues and that the regulation occurs at the level of the intestine through IEL-orchestrated production of TNF following the binding to TNFR2. These data suggest that HFE and TNFR2 may contribute to a common pathway of the iron stores regulator insuring the controlled efflux of gut iron.