Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells.

The POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.

The CUG-translated WT1, not AUG-WT1, is an oncogene.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

TRAF1 Is Critical for DMBA/Solar UVR-Induced Skin Carcinogenesis.

TRAF1 is a member of the TRAF protein family, which regulates the canonical and noncanonical NF-κB signaling cascades. Although aberrant TRAF1 expression in tumors has been reported, the role of TRAF1 remains elusive. Here, we report that TRAF1 is required for solar UV-induced skin carcinogenesis. Immunohistochemical analysis showed that TRAF1 expression is up-regulated in human actinic keratosis and squamous cell carcinoma. In vivo studies indicated that TRAF1 expression levels in mouse skin are induced by short-term solar UV irradiation, and a long-term skin carcinogenesis study showed that deletion of TRAF1 in mice results in a significant inhibition of skin tumor formation. Moreover, we show that TRAF1 is required for solar UV-induced extracellular signal-regulated kinase-5 (ERK5) phosphorylation and the expression of AP-1 family members (c-Fos/c-Jun). Mechanistic studies showed that TRAF1 expression enhances the ubiquitination of ERK5 on lysine 184, which is necessary for its kinase activity and AP-1 activation. Overall, our results suggest that TRAF1 mediates ERK5 activity by regulating the upstream effectors of ERK5 and also by modulating its ubiquitination status. Targeting TRAF1 function might lead to strategies for preventing and treating skin cancer.

Gestational loss and growth restriction by angiogenic defects in placental growth factor transgenic mice.

OBJECTIVE: Angiogenesis is an important biological process during development, reproduction, and in immune responses. Placental growth factor (PlGF) is a member of vascular endothelial growth factor that is critical for angiogenesis and vasculogenesis. We generated transgenic mice overexpressing PlGF in specifically T cells using the human CD2-promoter to investigate the effects of PlGF overexpression. APPROACH AND RESULTS: Transgenic mice were difficult to obtain owing to high lethality; for this reason, we investigated why gestational loss occurred in these transgenic mice. Here, we report that placenta detachment and inhibition of angiogenesis occurred in PlGF transgenic mice during the gestational period. Moreover, even when transgenic mice were born, their growth was restricted. CONCLUSIONS: Conclusively, PlGF overexpression prevents angiogenesis by inhibiting Braf, extracellular signal-regulated kinase activation, and downregulation of HIF-1α in the mouse placenta. Furthermore, it affected regulatory T cells, which are important for maintenance of pregnancy.

Kaempferol targets RSK2 and MSK1 to suppress UV radiation-induced skin cancer.

Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The MAPK cascades are activated by SUV irradiation. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. In this study, phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol, a natural flavonol, found in tea, broccoli, grapes, apples, and other plant sources, is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated group exhibited a substantial reduction in SUV-induced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1.

ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal.

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2(-/-) ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF- culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1(-/-) and jnk2(-/-) murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Enforced expression of roquin protein in T cells exacerbates the incidence and severity of experimental arthritis.

To investigate the role of Roquin, a RING-type ubiquitin ligase family member, we used transgenic mice with enforced Roquin expression in T cells, with collagen-induced arthritis (CIA). Wild-type (WT) and Roquin transgenic (Tg) mice were immunized with bovine type II collagen (CII). Arthritis severity was evaluated by clinical score; histopathologic CIA severity; proinflammatory and anti-inflammatory cytokine levels; anti-CII antibody levels; and populations of Th1, Th2, germinal center B cells, and follicular helper T cells in CIA. T cell proliferation in vitro and cytokine levels were determined to assess the response to CII. Roquin Tg mice developed more severe CIA and joint destruction compared with WT mice. Production of TNF-α, IFN-γ, IL-6, and pathogenic anti-collagen CII-specific IgG and IgG2a antibodies was increased in Roquin Tg mice. In addition, in vitro T cell assays showed increased proliferation and proinflammatory cytokine production in response to CII as a result of enforced Roquin expression in T cells. Furthermore, the Th1/Th2 balance was altered by an increased Th1 and decreased Th2 population. These findings suggest that overexpression of Roquin exacerbates the development of CIA and that enforced expression of Roquin in T cells may promote autoimmune diseases such as CIA.

Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B.

Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.

Over-expression of extracellular superoxide dismutase in mouse synovial tissue attenuates the inflammatory arthritis.

Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular- superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC- SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1ß, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.

ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4.

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins ßTrCP1 or ßTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

Regulation of inflammatory responses and fibroblast-like synoviocyte apoptosis by calcineurin-binding protein 1 in mice with collagen-induced arthritis.

OBJECTIVE: Calcineurin-binding protein 1 (CABIN-1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLS), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of CABIN-1 in FLS apoptosis is not clear. This study was undertaken to define the regulatory role of CABIN-1 in FLS from mice with collagen-induced arthritis (CIA). METHODS: Transgenic mice overexpressing human CABIN-1 in joint tissue under the control of a type II collagen promoter were generated. Expression of human CABIN-1 (hCABIN-1) in joints and FLS was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLS was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were stained with hematoxylin and eosin and with tartrate-resistant acid phosphatase for histologic analysis. RESULTS: Human CABIN-1-transgenic mice with CIA had less severe arthritis than wild-type mice with CIA, as assessed according to hind paw thickness and histologic features. The milder arthritis was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by a significantly greater number of TUNEL-positive cells in synovial tissue. Expression of inflammatory cytokines and MMPs in the transgenic mice with CIA was reduced, and they exhibited decreased Akt activation and increased expression of p53, caspase 3, caspase 9, and Bax. CONCLUSION: Our findings demonstrate that hCABIN-1 plays a critical role in promoting apoptosis of FLS and in attenuating inflammation and cartilage and bone destruction in RA. These results help elucidate the pathogenic mechanisms of RA and suggest that CABIN-1 is a potential target for treatment of this disease.

Overexpression of cathepsin S induces chronic atopic dermatitis in mice.

Atopic dermatitis (AD) is a chronically relapsing, non contagious pruritic skin disease with two phases: acute and chronic. Cysteine protease cathepsin S (CTSS) is involved in inflammatory processes, possibly leading to atherosclerosis and asthma. Recently, it has been reported that CTSS can arouse a predominant sensation of itch accompanied by classical ligand­receptor signaling [corrected]. Recently, CTSS was shown to be a ligand for proteinase-activated receptor-2 (PAR-2), which is associated with itching. In this study, we show that CTSS-overexpressing transgenic (TG) mice spontaneously develop a skin disorder similar to chronic AD. The results of this study suggest that CTSS overexpression triggers PAR-2 expression in dendritic cells (DCs), resulting in the promotion of CD4(+) differentiation, which is involved in major histocompatibility complex (MHC) class II expression. In addition, we investigated mast cells and macrophages and found significantly higher mean levels of T helper type 1 (Th1) cell-associated cytokines than T helper type 2 (Th2) cell-associated cytokines in CTSS-overexpressing TG mice. These results suggest that increased PAR-2 expression in DCs as a result of CTSS overexpression induces scratching behavior and Th1 cell-associated cytokine expression, and can trigger chronic AD symptoms.

The role of Roquin overexpression in the modulation of signaling during in vitro and ex vivo T-cell activation.

The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4(+) T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

Tissue-specific expression of human calcineurin-binding protein 1 in mouse synovial tissue can suppress inflammatory arthritis.

Calcineurin (CN) is a calcium- and calmodulin-dependent serine/threonine phosphatase. In immune cells, CN controls the activity of a wide range of transcription factors, including nuclear factor of activated T, nuclear factor-kappa B, c-fos, and Elk-1. CN plays an important role in synoviocyte activation and arthritis progression in vivo and this function is tightly linked to dysregulated intracellular Ca(2+) store and Ca(2+) response triggered by proinflammatory cytokines. In the present study, transgenic mice expressing human calcineurin-binding protein 1 (hCabin1) were generated, driven by type II collagen promoter, and the efficiency of these mice was investigated by experimental arthritis. These transgenic mice successfully expressed hCabin1 in joint tissue as well as other organs such as liver, heart, and brain. The overexpression of hCabin1 reduced the disease severity during collagen-induced arthritis. In fibroblast-like synoviocytes (FLSs) from hCabin1 transgenic mice, the productions of these cytokines, including interleukin (IL)-2, IL-4, and IFN-γ, were decreased and matrix metalloproteinases were also depressed in transgenic mice FLS. In addition, these effects were only found in the joint tissue, which is a major inflammation site. These findings will provide a better knowledge of the pathogenic mechanisms of rheumatoid arthritis and a potential animal model of the chronic inflammatory conditions, including atherosclerosis and transplantation.

Embryonic stem-cell-preconditioned microenvironment induces loss of cancer cell properties in human melanoma cells.

The cancer microenvironment affects cancer cell proliferation and growth. Embryonic stem (ES)-preconditioned 3-dimensional (3-D) culture of cancer cells induces cancer cell reprogramming and results in a change in cancer cell properties such as differentiation and migration in skin melanoma. However, the mechanism has not yet been clarified. Using the ES-preconditioned 3-D microenvironment model, we provide evidence showing that the ES microenvironment inhibits proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. We also found that the ES microenvironment suppresses telomerase activity and thereby induces senescence in SK-MEL-28 cells. Furthermore, we observed that gremlin, an antagonist of BMP4, is secreted from ES cells and plays an important role in cellular senescence. Knocking down gremlin in the ES microenvironment increases proliferation and anchorage-independent growth of SK-MEL-28 melanoma cells. Taken together, these results demonstrated that gremlin is a crucial factor responsible for abrogating melanoma properties in the ES-preconditioned 3-D microenvironment.

Overexpression of Jazf1 induces cardiac malformation through the upregulation of pro-apoptotic genes in mice.

The transcription factor Juxtaposed with another zinc finger gene 1 (JAZF1) is a zinc finger protein that binds to the nuclear orphan receptor TR4. Recent evidence indicates that TR4 receptor functions as both a positive and negative regulator of transcription, but the role of JAZF1 in transcriptional mechanisms has not been elucidated. Recently, the incidence rate of congenital heart malformations was reported to be significantly elevated in patients who had neurofibromatosis 1 (NF1) with chromosomal microdeletion syndrome. Furthermore, Joined to JAZF1 (SUZ12) is expressed at high levels in the hearts of adult patients with NF1 microdeletion syndrome. Therefore, we hypothesized that ectopic expression of JAZF1 may lead to cardiac malformations that deleteriously affect the survival of neonates and adults. We sought to elucidate the role of JAZF1 in cardiac development using a Jazf1-overexpressing (Jazf1-Tg) mouse model. In Jazf1-Tg mice, Jazf1 mRNA expression was significantly elevated in the heart. Jazf1-Tg mice also showed cardiac defects, such as high blood pressure, electrocardiogram abnormalities, apoptosis of cardiomyocytes, ventricular non-compaction, and mitochondrial defects. In addition, we found that the expression levels of pro-apoptotic genes were elevated in the hearts of Jazf1-Tg mice. These findings suggest that Jazf1 overexpression may induce heart failure symptoms through the upregulation of pro-apoptotic genes in cardiomyocytes.

Spatiotemporal expression of tmie in the inner ear of rats during postnatal development.

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.

Ectopic expression of tmie transgene induces various recovery levels of behavior and hearing ability in the circling mouse.

The circling (cir/cir) mouse is one of the murine models for human non-syndromic deafness DFNB6. The mice have abnormal circling behavior, suggesting a balanced disorder and profound deafness. The causative gene was transmembrane inner ear (tmie) gene of which the mutation is a 40-kb genomic deletion including tmie gene itself. In this study, tmie-overexpression trasngenic mice were established. Individuals with germline transmission have been mated with circling homozygous mutant mice (cir/cir) in order to produce the transgenic mutant mice (cir/cir-tg) as a gene therapy. After the genotyping, phenotypic analyses were performed so that the insertion of the new gene might compensate for the diseases such as hearing loss, circling behavior, or swimming inability. Some individuals exhibited complete recovery in their behavior and hearing but the others did not show any amelioration in behavior or hearing. Individual mice had very different levels of tmie transgene expression in the cochlea. These results clearly indicate that tmie protein plays an important role when the appropriate expression level of tmie was expressed in the inner ear. The protein levels were variable in each individual and these are thought to induce the differences in disease amelioration levels.

The therapeutic effect of extracellular superoxide dismutase (EC-SOD) mouse embryonic fibroblast (MEF) on collagen-induced arthritis (CIA) mice.

Rheumatoid arthritis is a chronic inflammatory disease. The generation of reactive oxygen species (ROS) within an inflamed joint has been suggested as playing a significant pathogenic role. Extracellular superoxide dismutase (EC-SOD) is a major scavenger enzyme of ROS, which has received growing attention for its therapeutic potential. To investigate the therapeutic effect of EC-SOD in mice with collagen-induced arthritis (CIA), we used mouse embryonic fibroblast (MEF) of transgenic mice that overexpresses EC-SOD on the skin by using hK14 promoter. DBA/1 mice that had been treated with bovine type II collagen were administrated subcutaneous injections of EC-SOD transgenic MEF (each at 1.4 x 10(60 cells) on days 28, 35, and 42 after primary immunization. To test EC-SOD activity, blood samples were collected in each group on day 49. The EC-SOD activity was nearly 1.5-fold higher in the transgenic MEF-treated group than in the nontransgenic MEF-treated group (p < 0.05). The severity of arthritis in mice was scored in a double-blind manner, with each paw being assigned a separate clinical score. The severity of arthritis in EC-SOD transgenic MEF-treated mice was significantly suppressed in the arthritic clinical score (p < 0.05). To investigate the alteration of cytokine levels, ELISA was used to measure blood samples. Levels of IL-1beta and TNF-alpha were reduced in the transgenic MEF-treated group (p < 0.05). Abnormalities of the joints were examined by H&E staining. There were no signs of inflammation except for mild hyperplasia of the synovium in the transgenic MEF-treated group. The proliferation of CII-specific T cells was lower in the transgenic MEF-treated mice than in those in the other groups. The transfer of EC-SOD transgenic MEF has shown a therapeutic effect in CIA mice and this approach may be a safer and more effective form of therapy for rheumatoid arthritis.