CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2: Characterizing CD4+ T cells in Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Differential expression of TOX by skin-infiltrating T cells in Sézary syndrome and erythrodermic dermatitis.

BACKGROUND: The histopathologic differentiation between Sézary syndrome (SS) and erythrodermic dermatitis may be extremely difficult. In this immunohistochemical study, it was investigated if thymocyte selection-associated high mobility group box protein (TOX) and C-MYC can be used as additional diagnostic markers to differentiate between SS and erythrodermic dermatitis. METHOD: Paraffin-embedded skin biopsies from 15 SS patients and 17 erythrodermic dermatitis patients were stained and scored for TOX or C-MYC expression. RESULTS: Strong nuclear staining for TOX in more than 50% of skin-infiltrating T cells was observed in 13 of 15 (87%) SS cases, whereas erythrodermic dermatitis cases showed weak nuclear staining in 11-50% (median: 25%) of the T cells; strong nuclear staining as found in SS was never observed in erythrodermic dermatitis. No significant differences in C-MYC expression between SS and erythrodermic dermatitis were found. In most patients of both groups, percentages of C-MYC positive-cells varied between less than 10 and 25% of skin-infiltrating T cells. CONCLUSION: Our results suggest that strong expression of TOX in more than 50% of skin-infiltrating T cells in erythrodermic skin is a useful marker in the differentiation between SS and erythrodermic dermatitis, whereas staining for C-MYC does not contribute to differential diagnosis.

The expression of the tumour suppressor HBP1 is down-regulated by growth factors via the PI3K/PKB/FOXO pathway.

Growth factors inactivate the FOXO (forkhead box O) transcription factors through PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B). By comparing microarray data from multiple model systems, we identified HBP1 (high-mobility group-box protein 1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), PI3K and PKB, whereas it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukaemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and ChIP, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by shRNA increased the proliferation of human fibroblasts in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analysing a transcriptomics dataset from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumours that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K/FOXO pathway and controls cell proliferation in response to growth factors.

Activation of alternative NF-κB signaling during recovery of disuse-induced loss of muscle oxidative phenotype.

Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified, since the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-κB, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. Markers and regulators (including alternative NF-κB signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hindlimb suspension/reloading (HLS/RL) protocol. Expression levels of oxidative phosphorylation subunits and slow myosin heavy chain isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC) molecules PGC-1α and PGC-1ß and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α increased upon RL. PPAR-δ, nuclear respiratory factor 1 (NRF-1), NRF-2α, and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased, and phosphorylation of P100 and subsequent processing to P52 were elevated, reflecting alternative NF-κB activation. We conclude that RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-κB signaling.

miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1.

MicroRNAs have been implicated in regulating diverse cellular pathways. Emerging evidence indicate that the miR-17-92 cluster may have a causal role in breast cancer tumorigenesis as a novel class of oncogenes, but the role of these miRNAs in breast cancer invasion and migration remains unexplored. The aims of this study were to verify the effect of miR-17-5p (an important member of the miR-17-92 cluster) on the invasive and migratory ability of breast cancer cells. The matching of miR-17-5p and HMG box-containing protein 1 (HBP1) was predicted by TargetScan and confirmed by DNA constructs and luciferase target assay. The expression levels of miR-17-5p and its candidate target-HBP1 in MCF7 and MDA-MB-231 breast cancer cells were measured by real-time PCR and western blotting. Effects of miR-17-5p in cell cycle progression, proliferation, invasion and migration were evaluated by flow cytometry assay, 3-(4,-dimethy -lthiazol-2-yl)-2,-diphenyl -tetrazoliumbromide assay, soft-agar colony formation assay, and transwell invasive and migratory assay, respectively. The results showed that miR-17-5p was highly expressed in high-invasive MDA-MB-231 breast cancer cells but not in low-invasive MCF-7 breast cancer cells. Over-expression of miR-17-5p in MCF-7 cells rendered them the invasive and migratory abilities by targeting HBP1/ß-catenin pathway. On the other hand, down-regulation of endogenous miR-17-5p suppressed the migration and invasion of MDA-MB-231 cells in vitro. These findings suggest that miR-17-5p plays an important role in breast cancer cell invasion and migration by suppressing HBP1 and subsequent activation of Wnt/ß-catenin.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

BACKGROUND: Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. METHODS: We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. RESULTS: Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. CONCLUSIONS: We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

Sox10: a pan-schwannian and melanocytic marker.

S100 protein is a sensitive marker for melanomas and peripheral nerve sheath tumors. It is, however, expressed by other mesenchymal and epithelial tumors. Despite its low specificity, S100 protein is valuable for the diagnosis of desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific marker is available. Sox10 is a neural crest transcription factor crucial for specification, maturation, and maintenance of Schwann cells and melanocytes. Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors, mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear expression was found in 76 of 78 melanomas (97%) and 38 of 77 malignant peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71 melanomas (91%) and 23 malignant peripheral nerve sheath tumors (30%). Sox10 was diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen only in sustentacular cells of pheochromocytomas/paragangliomas, and occasionally carcinoid tumors from various organs, but it was not seen in the tumor cells. In normal tissues, Sox10 was expressed in Schwann cells, melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands. Sox10 reaction was not identified in any other mesenchymal and epithelial tumors except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not by other lymph node components such as dendritic cells. Our results indicate that Sox10 will serve as a more sensitive and specific marker for the diagnosis of melanocytic and schwannian tumors than S100 protein.

Differential expression and prognostic significance of SOX genes in pediatric medulloblastoma and ependymoma identified by microarray analysis.

The objective of this study was to identify differentially expressed and prognostically important genes in pediatric medulloblastoma and pediatric ependymoma by Affymetrix microarray analysis. Among the most discriminative genes, three members of the SOX transcription factor family were differentially expressed. Both SOX4 and SOX11 were significantly overexpressed in medulloblastoma (median, 11-fold and 5-fold, respectively) compared with ependymoma and normal cerebellum. SOX9 had greater expression in ependymoma (median, 16-fold) compared with normal cerebellum and medulloblastoma (p<0.001 for all comparisons). The differential expression of the SOX genes was confirmed at the protein level by immunohistochemical analysis. Survival analysis of the most discriminative probe sets for each subgroup showed that 35 and 13 probe sets were predictive of survival in patients with medulloblastoma and ependymoma, respectively. There was a trend toward better survival with increasing SOX4 expression in medulloblastoma. SOX9 expression was predictive for favorable outcome in ependymoma. The mRNA levels of BCAT1, a mediator of amino acid breakdown, were higher (median, 15-fold) in medulloblastoma patients with metastases compared with those without metastasized disease (p<0.01). However, the correlation between BCAT1 expression and metastatic medulloblastoma could not be confirmed at the protein level. The potential prognostic effect of the genes associated with outcome should be evaluated in ongoing studies using larger groups of patients. Furthermore, our findings support further analysis of the functional properties of the selected genes, especially SOX4 and BCAT1 for medulloblastoma and SOX9 for ependymoma, to evaluate the use of these genes as potential tumor markers, prognostic markers, and drug targets in pediatric brain tumors.

SOX9 is expressed in human fetal prostate epithelium and enhances prostate cancer invasion.

SOX9 is a transcription factor that plays a critical role in the development of multiple tissues. We previously reported that SOX9 in normal human adult prostate was restricted to basal epithelium. SOX9 was also expressed in a subset of prostate cancer (PCa) cells and was increased in relapsed hormone-refractory PCa. Moreover, SOX9 expression in PCa cell lines enhanced tumor cell proliferation and was beta-catenin regulated. Here we report additional in vivo results showing that SOX9 is highly expressed during fetal prostate development by epithelial cells expanding into the mesenchyme, suggesting it may contribute to invasive growth in PCa. Indeed, SOX9 overexpression in LNCaP PCa xenografts enhanced growth, angiogenesis, and invasion. Conversely, short hairpin RNA-mediated SOX9 suppression inhibited the growth of CWR22Rv1 PCa xenografts. These results support important functions of SOX9 in both the development and maintenance of normal prostate, and indicate that these functions contribute to PCa tumor growth and invasion.

Overexpression of HmgD causes the failure of pupariation in Drosophila by affecting ecdysone receptor pathway.

HmgD encodes Drosophila homologue of high mobility group proteins (HMGD), which are thought to have an architectural function in chromatin organization. However, current opinions about the function of HMGD in Drosophila development are controversial. Our previous studies have shown that ubiquitous overexpression of HmgD caused the formation of melanotic tumors in the Drosophila larvae by prematurely activating the Ras-MAPK pathway. Here we report that under maternal control, the viability of flies links with overexpression of HmgD, while under ubiquitous control, ActGal4, overexpressing HmgD animals, which display prolonged larval stages around day 13, developmentally stagnate in the larva-white pupa transition. Ecdysone feeding did not rescue overexpressing HmgD animals. RT-PCR analyses show that overexpression of HmgD does not affect the temporal expression pattern of ecdysone receptor gene EcR, whereas transcriptional patterns of some key regulatory genes, such as E74A, E74B, E75A, E75B, betaFTZ-F1, are changed greatly. These results suggest that ubiquitous overexpression of HmgD results in the failure of pupariation neither by affecting the process of ecdysone synthesis and release nor by abnormal EcR transcription, but by causing expression of EcR regulatory nuclear receptors out of schedule. The results led us to postulate that overexpression of HMGD likely changes the signaling cascade of Drosophila metamorphosis by an interaction between HMGD and DNA strands, and subsequently by an error of DNA binding abilities and transcriptional activities of some nuclear receptor genes. Arch. Insect Biochem. Physiol. 2008.

Ectopic SOX9 mediates extracellular matrix deposition characteristic of organ fibrosis.

Appropriate temporospatial expression of the transcription factor SOX9 is important for normal development of a wide range of organs. Here, we show that when SOX9 is expressed ectopically, target genes become expressed that are associated with disease. Histone deacetylase inhibitors in clinical trials for cancer therapy induced SOX9 expression via enhanced recruitment of nuclear factor Y (NF-Y) to CCAAT elements in the SOX9 proximal promoter. The effect of histone deacetylase inhibitors could be elicited in cells that normally lack SOX9, such as hepatocytes. In human fetal hepatocytes, this aberrant induction of SOX9 protein caused ectopic expression of COL2A1 and COMP1 that encode extracellular matrix (ECM) components normally associated with chondrogenesis. Previously, ectopic expression of this "chondrogenic" profile has been implicated in vascular calcification. More broadly, inappropriate ECM deposition is a hallmark of fibrosis. We demonstrated that induction of SOX9 expression also occurred during activation of fibrogenic cells from the adult liver when the transcription factor was responsible for expression of the major component of fibrotic ECM, type 1 collagen. These combined data identify new aspects in the regulation of SOX9 expression. They support a role for SOX9 beyond normal development as a transcriptional regulator in the pathology of fibrosis.

Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma.

Mantle cell lymphoma (MCL) is defined pathologically by the detection of CD20, CD5, and most importantly cyclin D1 (CCND1). Its distinction from other lymphomas is important for prognosis and appropriate therapy, but occasional cases may fail to express CCND1 and morphologic simulators may express CD20 and CD5 but not CD23. In this study, we show that the transcription factor Sox11 is specifically expressed in the nucleus of MCL compared with other lymphomas and benign lymphoid tissue. Although the role of Sox11 presently is not known in lymphocyte ontogeny, it is normally expressed in the developing central nervous system in the embryo and shows sequence homology with Sox4, a transcription factor crucial for B lymphopoiesis. Sox11 mRNA is increased in gliomas compared with healthy brain tissue, suggesting a role in malignant transformation and/or cell survival. Our novel finding of specific overexpression of Sox11 mRNA and nuclear protein in both cyclin D1-positive and - negative MCL may be useful for the diagnosis of MCL as a complement to cyclin D1 and also suggests a functional role for Sox11 in MCL.

SOX9 transduction of a human chondrocytic cell line identifies novel genes regulated in primary human chondrocytes and in osteoarthritis.

The transcription factor SOX9 is important in maintaining the chondrocyte phenotype. To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis.

Effects of IL-6 and soluble IL-6 receptor on the expression of cartilage matrix proteins in human chondrocytes.

Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.

Transcriptional dysregulation mediated by RUNX1-RUNX1T1 in normal human progenitor cells and in acute myeloid leukaemia.

The t(8;21)(q22;q22) occurs frequently in acute myelogenous leukaemia and gives rise to the transcription factor fusion protein, RUNX1-RUNX1T1 (also known as AML1-ETO). To identify the genes dysregulated by the aberrant transcriptional activity of RUNX1-RUNX1T1, we used microarrays to determine the effect of this mutation on gene expression in human progenitor cells and during subsequent development. Gene signatures of these developmental subsets were very dissimilar indicating that effects of RUNX1-RUNX1T1 are highly context dependent. We focused on gene changes associated with the granulocytic lineage and identified a clinically relevant subset of these by comparison with 235 leukaemia patient transcriptional signatures. We confirmed the overexpression of a number of significant genes (Sox4, IL-17BR, CD200 and gamma-catenin). Further, we show that overexpression of CD200 and gamma-catenin is also associated with the inv(16) abnormality which like RUNX1-RUNX1T1 disrupts core binding factor activity. We investigated the functional significance of CD200 and gamma-catenin overexpression in normal human progenitor cells. The effect of IL17 on growth was also assessed. Individually, none of these changes were sufficient to recapitulate the effects of RUNX1-RUNX1T1 on normal development. These data provide the most comprehensive and pertinent assessment of the effect of RUNX1-RUNX1T1 on gene expression and demonstrate the highly context-dependent effects of this fusion gene.

Alterations of the HBP1 transcriptional repressor are associated with invasive breast cancer.

Invasive breast cancer has a high risk of recurrence to incurable disease and needs improved prognostic and therapeutic tools. Our work combines clinical and molecular analyses to show that the transcriptional repressor HBP1 may be a new target for invasive breast cancer. Previous work indicated that HBP1 regulated proliferation and senescence and inhibited Wnt signaling. Two of these functions have been associated with invasive breast cancer. In 76 breast tumors, we identified 10 HBP1 mutations/variants that were associated with fully invasive breast cancer. In a separate analysis, we found that a subset of invasive breast cancer specimens also had reduced HBP1 mRNA levels. These clinical correlations suggested that mutation or reduction of HBP1 occurs in invasive breast cancer and that HBP1 might regulate the proliferation and invasiveness of this breast cancer type. Analysis of the HBP1 mutants showed they were functionally defective for suppressing Wnt signaling. To test the consequences of reduced HBP1 levels, we used RNA interference to knock down HBP1 and observed increased Wnt signaling, tumorigenic proliferation, and invasiveness in cell and animal breast cancer models. Lastly, statistical analysis of a breast cancer patient database linked reduced HBP1 expression to breast cancer recurrence. In considering two-gene criteria for relapse potential, reduced expression of HBP1 and SFRP1, which is another Wnt inhibitor that was recently linked to invasive breast cancer, strikingly correlated with recurrence. Together, these data indicate that HBP1 may be a molecularly and clinically relevant regulator of breast cancer transitions that eventually lead to poor prognosis.

Morphological characterization of intra-articular HMGB1 expression during the course of collagen-induced arthritis.

High-mobility group chromosomal box protein 1 (HMGB1) is a structural nuclear protein that promotes inflammation when present extracellularly. Aberrant, extracellular HMGB1 expression has been demonstrated in human and experimental synovitis. The aim of the present study was to elucidate the temporal and spatial expression of HMGB1 compared to that of the central mediators tumor necrosis factor (TNF) and interleukin-1-beta (IL-1beta) during the course of collagen-induced arthritis. Thus, Dark Agouti rats were immunized with homologous type II collagen and synovial tissue specimens were obtained at various time points prior to and during the course of clinical arthritis. Local cytokine responses were assessed by immunohistochemistry and by in situ hybridization. We demonstrate a distinct nuclear expression of HMGB1 at early disease-preceding time points. Preceding clinical onset by a few days, cytoplasmic HMGB1 expression was evident in synoviocytes within the non-proliferative lining layer. Pronounced cytoplasmic and additional extracellular HMGB1 expression coincided with the progression of clinical disease. In advanced arthritis, the number of cells with cytoplasmic HMGB1 expression was quantitatively comparable to that of cells expressing TNF and IL-1beta. Interestingly, although HMGB1 was abundantly expressed throughout the inflamed synovium at a protein level, upregulation of HMGB1 mRNA was restricted mainly to areas of cartilage and bone destruction. In conclusion, these new findings implicate a role for HMGB1 in both inducing and perpetuating inflammatory events of significant importance in the destructive processes in chronic arthritis.

Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation.

Alternative splicing produces functionally distinct proteins participating in cellular processes including differentiation and development. CoAA is a coactivator that regulates transcription-coupled splicing and its own pre-mRNA transcript is alternatively spliced. We show here that the CoAA gene is embryonically expressed and alternatively spliced in multiple tissues to three splice variants, CoAA, CoAM and CoAR. During retinoic-acid-induced P19 stem cell differentiation, the expression of CoAA undergoes a rapid switch to its dominant negative splice variant CoAM in the cavity of the embryoid body. CoAM functionally inhibits CoAA, and their switched expression up-regulates differentiation marker Sox6. Using a CoAA minigene cassette, we find that the switched alternative splicing of CoAA and CoAM is regulated by the cis-regulating sequence upstream of the CoAA basal promoter. Consistent to this, we show that p54(nrb) and PSF induce CoAM splice variant through the cis-regulating sequence. We have previously shown that the CoAA gene is amplified in human cancers with a recurrent loss of this cis-regulating sequence. These results together suggest that the upstream regulatory sequence contributes to alternative splicing of the CoAA gene during stem cell differentiation, and its selective loss in human cancers potentially deregulates CoAA alternative splicing and alters stem cell differentiation.

Overexpression of suppressor of cytokine signaling-3 in T cells exacerbates acetaminophen-induced hepatotoxicity.

Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4(+) T cells into T and B cell-deficient RAG-2(-/-) mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-gamma and TNF-alpha in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-gamma and TNF-alpha were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-gamma and TNF-alpha.