Christopher J. Ong

Anti-IL-4 treatment prevents dermal collagen deposition in the tight-skin mouse model of scleroderma

The tight‐skin ( Tsk  /+) mutant mouse, a putative murine model of scleroderma, is characterized primarily by the excessive deposition of collagen and other extracellular matrix molecules in the dermis, and also by a developmentally acquired defect in pulmonary architecture. Passive transfer experiments have suggested an etiologic role for the immune system in Tsk  /+ dermal pathology. In addition, CD4+ T lymphocytes have been shown to be required for the excessive accumulation of dermal collagen in these mice. As IL‐4, a product of differentiated CD4+ T cells, is capable of regulating the synthesis of various matrix molecules (including type I collagen) by fibroblasts in vitro, we investigated the potential role of IL‐4 in mediating Tsk  /+ dermal fibrosis. Confirming that Tsk  /+ cells are capable of responding to IL‐4, we found receptors for this cytokine on Tsk  /+ embryonic fibroblasts and a dermal fibroblast cell line derived from these mice. Furthermore, IL‐4 receptors on Tsk  /+ fibroblasts were functional since IL‐4 stimulation in vitro increased type I collagen secretion from these cells. These results demonstrated the potential for IL‐4 to be directly involved in the excessive deposition of dermal collagen in Tsk  /+ mice. Critical insight into the role played by IL‐4 in mediating the dermal phenotype, however, was obtained following the administration of neutralizing anti‐IL‐4 antibodies to Tsk  /+ mice. This treatment prevented the development of dermal fibrosis, leading to normalization of dermal collagen content. Given the requirement for CD4+ T cells in Tsk  /+ dermal fibrosis, our results suggest that Th2 cells and/or factors elaborated by this T cell subset may play a key role in regulating dermal collagen content in this strain.

Divergent mechanisms utilized by SOCS3 to mediate interleukin-10 inhibition of tumor necrosis factor α and nitric oxide production by macrophages

The cytokine interleukin-10 (IL-10) potently inhibits macrophage function through activation of the transcription factor STAT3. The expression of SOCS3 (suppressor of cytokine signaling-3) has been shown to be induced by IL-10 in a STAT3-dependent manner. However, the relevance of SOCS3 expression to the anti-inflammatory effect of IL-10 on macrophages has been controversial. Through kinetic analysis of the requirement for SOCS3 in IL-10 inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNFα) transcription and translation, SOCS3 was found to be necessary for TNFα expression during the early phase, but not the late phase of IL-10 action. SOCS3 was essential for IL-10 inhibition of LPS-stimulated production of iNOS (inducible nitric-oxide synthase) protein and nitric oxide (NO). To determine the domains of SOCS3 protein important in mediating these effects, SOCS3–/– macrophages were reconstituted with SOCS3 mutated for the SH2, KIR, SOCS box domains, and tyrosines 204 (Tyr204) and 221 (Tyr221). The SH2 domain, SOCS box, and both Tyr204 and Tyr221 were required for IL-10 inhibition of TNFα mRNA and protein expression, but interestingly the KIR domain was necessary only for IL-10 inhibition of TNFα protein expression. In contrast, Tyr204 and Tyr221 were the only structural features of SOCS3 that were necessary in mediating IL-10 inhibition of iNOS protein expression and NO production. These data define SOCS3 as an important mediator of IL-10 inhibition of macrophage activation and that SOCS3 interferes with distinct LPS-stimulated signal transduction events through differing mechanisms.

PTEN Loss Promotes Mitochondrially Dependent Type II Fas-Induced Apoptosis via PEA-15

ABSTRACT Two distinct biochemical signals are delivered by the CD95/Fas death receptor. The molecular basis for the differential mitochondrially independent (type I) and mitochondrially dependent (type II) Fas apoptosis pathways is unknown. By analyzing 24 Fas-sensitive tumor lines, we now demonstrate that expression/activity of the PTEN tumor suppressor strongly correlates with the distinct Fas signals. PTEN loss-of-function and gain-of-function studies demonstrate the ability to interconvert between type I and type II Fas pathways. Importantly, from analyses of Bcl-2 transgenic Pten+/− mice, Pten haploinsufficiency converts Fas-induced apoptosis from a Bcl-2-independent to a Bcl-2-sensitive response in primary thymocytes and activated T lymphocytes. We further show that PTEN influences Fas signaling, at least in part, by regulating PEA-15 phosphorylation and activity that, in turn, regulate the ability of Bcl-2 to suppress Fas-induced apoptosis. Thus, PTEN is a key molecular rheostat that determines whether a cell dies by a mitochondrially independent type I versus a mitochondrially dependent type II apoptotic pathway upon Fas stimulation.

Oncolysis of Prostate Cancers Induced by Vesicular Stomatitis Virus in PTEN Knockout Mice

Vesicular stomatitis virus (VSV) is an oncolytic virus which selectively infects and kills cancer cells. The goal of the present study was to determine the safety and efficacy of VSV treatment of prostate tumors that arise in situ in immunocompetent, transgenic prostate-specific PTEN-null (PTEN(-/-)) mice. Interferon-sensitive VSV(AV3 strain), which expresses luciferase, was injected intraprostatically into tumor-bearing PTEN(-/-) and control mice and then monitored for tissue bioluminescence over 96 hours. Virus readily dispersed throughout the bodies of mice after only 3 hours; however, it persisted at high levels for >72 hours in PTEN(-/-) mice, but at relatively low levels and for only approximately 48 hours in controls. Plaque assays provided a similar pattern, with much higher concentrations of replicating virus in prostates of PTEN(-/-) mice than in controls. Transient, low levels of virus were detected in the spleens of both groups. Apoptotic analyses by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that VSV(AV3) is able to selectively infect and kill prostate cells in PTEN(-/-) mice, while sparing normal cells in control mice. The primary mechanism for cell kill is apparently apoptotic oncolysis as opposed to neutrophil invasion as has been reported using xenograft models. These results suggest that control of locally advanced human prostate cancer may be achievable through intraprostatic injection and amplification of a safe oncolytic virus, such as VSV(AV3).

Integrin-linked kinase regulates cell proliferation and tumour growth in murine colitis-associated carcinogenesis

Background: Integrins are transmembrane cell surface receptors that mediate cell–cell and cell–matrix contacts. Integrin-linked kinase (ILK) is the binding partner of β1 and β3 integrins, and has been ascribed essential roles in development, angiogenesis and tumourigenesis. However, in vivo evidence for the latter is currently lacking. Aim: The hypothesis that epithelial cell-specific deletion of ILK would impact on murine tumourigenesis was tested using a colitis-associated cancer model. Methods: To create intestinal epithelial cell ILK knockout animals, Fabp/Cre mice (Cre recombinase expressed under the control of a modified Fabp promoter) were used, and they were mated with mice carrying a loxP-flanked (floxed) ILK gene (ILKflox/flox). Results: ILK intestinal knockout mice exhibited a reduction in the size of the caecum, and reduced crypt height in the colon. Immunohistochemical analysis confirmed that there was diminished ILK expression, and bromodeoxyuridine (BrdU) staining was significantly reduced in the knockout animals as compared with the wild-type animals in both the caecum and colon (p<0.001 for both). Following azoxymethane and dextran sodium sulfate (DSS) treatment, fewer total tumours were observed in the ILK knockout animals, which were mosaic with respect to ILK expression. Cyclin D1, Snail, fibronectin and matrix metalloproteinase 9 (MMP9) were all reduced, and active caspase 3 increased, in tumours from ILK knockout mice, as compared with wild-type mice, on immunohistochemical analysis. Using small interfering RNA (siRNA) to knock down ILK in colonic cancer cell lines, it was confirmed that it is capable of regulating cyclin D1, Snail, MMP9 and fibronectin transcription. Conclusions: From these findings, it is concluded that ILK plays an important role in intestinal epithelial cell proliferation, and that it influences the development of colitis-associated cancer, through modulation of cyclin D1, the extracellular matrix and MMP9.

Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP) in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.

Role of EIF5A2, a downstream target of Akt, in promoting melanoma cell invasion

Background:Cutaneous melanoma is a life-threatening skin cancer because of its poorly understood invasive nature and high metastatic potential. This study examines the importance of eukaryotic translation initiation factor 5A2 (EIF5A2) in melanoma pathogenesis.Methods:We examined EIF5A2 expression in 459 melanocytic lesions using tissue microarray. In addition, melanoma cell lines were subjected to invasion and cell proliferation assays, zymography, FACS and real-time PCR to investigate the role of EIF5A2 in cancer progression.Results:Positive EIF5A2 staining increased from dysplastic naevi to primary melanomas (PMs; P=0.001), and further increased in metastatic melanomas (P=0.044). Eukaryotic translation initiation factor 5A2 expression was correlated with melanoma thickness (P<0.001) and was inversely correlated with the 5-year survival of PM patients especially those with tumour⩽2 mm thick. Strikingly, none of the latter died within 5 years in EIF5A2-negative staining group. Cox regression analysis revealed that EIF5A2 is an independent prognostic marker. Further, we found that EIF5A2 is a novel downstream target of phosphorylated Akt. Both melanoma cell invasion and MMP-2 activity increased and decreased with EIF5A2 overexpression and knockdown, respectively.Conclusion:We for the first time showed that EIF5A2, as a target of PI3K/Akt, promotes melanoma cell invasion and may serve as a promising prognostic marker and a potential therapeutic target for melanoma.

Prognostic significance of KAI1/CD82 in human melanoma and its role in cell migration and invasion through the regulation of ING4

KAI1/CD82 is a member of the transmembrane 4 superfamily, which was first identified as a metastasis suppressor for prostate cancer. The expression of KAI1 was found to be reduced in many types of cancers, including prostate, breast, ovarian, cervical and endometrial cancer. However, the role of KAI1 in melanoma pathogenesis is not known. In this study, we investigated the expression level of KAI1 in a large set of melanocytic lesions at different stages. We found that the expression of KAI1 is significantly decreased during melanoma progression. In fact, KAI1 expression is drastically reduced in primary melanoma compared with dysplastic nevi (P = 1.8×10(-4)) and further reduced in metastatic melanoma compared with primary melanoma (P = 9.4 × 10(-15)). Furthermore, decreased KAI1 staining is strongly correlated with a worse 5 year and 10 year patient survival. Multivariate Cox regression analysis showed that KAI1 is also an independent prognostic factor for both 5 year and 10 year survival. Moreover, we found that overexpression of KAI1 significantly inhibited melanoma cell migration through suppression of Rho-associated kinase-mediated formation of stress fiber. Our data also suggested that overexpression of KAI1 significantly inhibited melanoma cell invasion by reducing the activity of metalloproteinase-2. In addition, we found that suppression of melanoma cell migration by KAI1 is mediated by another tumor-suppressor protein called inhibitor of growth 4 through the regulation of p65. Taken together, our data suggest that KAI1 may be used as a promising prognostic marker and a possible therapeutic target for human melanoma.

High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO‐induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non‐derepressible‐2(GCN2) kinase stress‐responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO‐induced low tryptophan environment. IDO‐expressing human fibroblasts were co‐cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO‐expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO‐induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co‐cultured with IDO‐expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT‐expressing vector, was rescued significantly in tryptophan‐deficient but not IDO‐expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO‐mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO‐generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non‐immune cells such as skin cells acts as a protective mechanism against IDO‐induced GCN2 activation, therefore, makes them resistant to the amino acid‐deprived environment caused by IDO. J. Cell. Physiol. 225: 196–205, 2010.

Differentiation of Definitive Endoderm from Mouse Embryonic Stem Cells

Efficient production of definitive endoderm from embryonic stem (ES) cells opens doors to the possibilities of differentiation of endoderm-derived tissues such as the intestines, pancreas, and liver that could address the needs of people with chronic diseases involving these organs. The lessons learned from developmental biology have contributed significantly to in vitro differentiation of definitive endoderm. Gastrulation, a process that results in the production of all three embryonic germ cell layers, definitive endoderm, mesoderm, and ectoderm, is an important step in embryonic development. Gastrulation occurs as a result of the events that are orchestrated by the signaling pathways involving Nodal, FGF, Wnt, and BMP. Understanding these signaling pathways has led to the introduction of key ingredients such as Activin A, FGF, Wnt, and BMP to the differentiation protocols that have been able to produce definitive endoderm from ES cells. Efficient production of definitive endoderm needs to meet the specific criteria that include (a) increase in the production of markers of definitive endoderm such as Sox 17, FOXA2, GSC, and Mixl1; (b) decrease in the production of markers of primitive/visceral/parietal endoderm, Sox 7 and OCT4; and (c) decrease in the mesoderm markers (Brachyury, MEOX) and ectoderm markers (Sox1 and ZIC1).