Hepatocellular expression of a dominant-negative mutant TGF-β type II receptor accelerates chemically induced hepatocarcinogenesis

The potent growth-inhibitory activity of cytokines of the transforming growth factor-beta (TGF-beta) superfamily and their widespread expression in epithelia suggest that they may play an important role in the maintenance of epithelial homeostasis. To analyse TGF-beta mediated tumor suppressor activity in the liver, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in hepatocytes under control of the regulatory elements of the human C-reactive protein gene promoter. Transgenic animals exhibited constitutive and liver-specific transgene expression. The functional inactivation of the TGF-beta signaling pathway in transgenic hepatocytes was shown by redu…

A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model

Alzheimer disease (AD) is characterized by excessive deposition of amyloid beta-peptides (A beta peptides) in the brain. In the nonamyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by the alpha-secretase within the A beta peptide sequence. Proteinases of the ADAM family (adisintegrin and metalloproteinase) are the main candidates as physiologically relevant alpha-secretases, but early lethality of knockout animals prevented a detailed analysis in neuronal cells. To overcome this restriction, we have generated transgenic mice that overexpress either ADAM10 or a catalytically inactive ADAM10 mutant. In this report we show that a moderate neuronal overexpression of ADAM10 i…

Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome.

Background & Aims: The HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is a life-threatening complication during pregnancy. The associated liver disease may be severe, and maternal hepatic complications may progress to the point that transplantation becomes necessary. CD95 (APO-1, Fas)-mediated apoptosis of liver cells is one of the major pathogenic mechanisms during liver disease. The interaction of CD95 with its ligand, CD95L(FasL), induces apoptosis and thus the source of the death-inducing ligand is critical for understanding the pathomechanism of liver damage involving the CD95-system. Methods: Sera from HELLP patients were analyzed and used in cell culture experiment…

Chemical skin carcinogenesis is prevented in mice by the induced expression of a TGF-β related transgene

Skin papillomas and squamous cell carcinomas (SCCs) are induced in mice by tumor initiation with a carcinogen followed by tumor promotion with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These usually arise from preneoplastic lesions characterized by epidermal proliferation and hyperplasia, dermal edema, and inflammation. To evaluate the role of polypeptide growth factors in chemically induced skin carcinogenesis, we used transgenic mice carrying the cDNA for a TGF-β related molecule, bone morphogenetic protein-4 (BMP-4), under the control of the regulatory elements of the cytokeratin IV* gene in a skin carcinogenesis protocol. Control non-transgenic littermates and BMP-4 …

The designer cytokine hyper-IL-6 mediates growth inhibition and GM-CSF-dependent rejection of B16 melanoma cells.

The low immunogenic B16 melanoma cell line was transfected with a mammalian expression vector containing the complementary DNA for a sIL-6R/IL-6 fusion protein, termed Hyper-IL-6 (H-IL-6), which was shown to have biological activities at 100-1000-fold lower concentrations than IL-6 in combination with sIL-6R. The secreted p84 glycoprotein was detected in the supernatant of transfected cells and was fully active on BAF3/gp130 cells, which respond to IL-6/sIL-6R but not to IL-6 alone. Administration of recombinant H-IL-6 to C57BL/6 mice resulted in a prolonged acute phase protein gene expression indicating long systemic persistence of the fusion protein. Transfected B16 cells (B16/H-IL6 cells…

Keratinocyte-Derived Granulocyte-Macrophage Colony Stimulating Factor Accelerates Wound Healing: Stimulation of Keratinocyte Proliferation, Granulation Tissue Formation, and Vascularization

Chronic, nonhealing wounds represent a major clinical challenge to practically all disciplines in modern medicine including dermatology, oncology, surgery, and hematology. In skin wounds, granulocyte-macrophage colony stimulating factor (GM-CSF) is secreted by keratinocytes shortly after injury and mediates epidermal cell proliferation in an autocrine manner. Many other cells involved in wound healing including macrophages, lymphocytes, fibroblasts, endothelial cells, and dendritic cells synthesize GM-CSF and/or are targets of this cytokine. Therefore, GM-CSF is a pleiotropic cytokine evoking complex processes during wound repair. Despite this complexity and the scarcity of mechanistic unde…

Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.

Bone morphogenetic protein-6 (BMP-6) belongs to the family of TGF-beta-related growth factors. In the developing epidermis, expression of BMP-6 coincides with the onset of stratification. Expression persists perinatally but declines after day 6 postpartum, although it can still be detected in adult skin by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. We constitutively overexpressed BMP-6 in suprabasal layers of interfollicular epidermis in transgenic mice using a keratin 10 promoter. All mice expressing the transgene developed abnormalities in the skin, indicating an active transgene-derived factor. Depending on the pattern of transgene expression, the effects on proli…

Regulation of IL-12 p40 Promoter Activity in Primary Human Monocytes: Roles of NF-κB, CCAAT/Enhancer-Binding Protein β, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E2

Abstract Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14+ monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-κB, and ETS were only found upon stimulation with LPS and IFN-γ. In contrast, a footprint over a purine-rich sequence at −155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of t…

TGF-beta regulates airway responses via T cells.

Abstract Allergic asthma is characterized by airway hyperreactivity, inflammation, and a Th2-type cytokine profile favoring IgE production. Beneficial effects of TGF-β and conflicting results regarding the role of Th1 cytokines have been reported from murine asthma models. In this study, we examined the T cell as a target cell of TGF-β-mediated immune regulation in a mouse model of asthma. We demonstrate that impairment of TGF-β signaling in T cells of transgenic mice expressing a dominant-negative TGF-β type II receptor leads to a decrease in airway reactivity in a non-Ag-dependent model. Increased serum levels of IFN-γ can be detected in these animals. In contrast, after injection of OVA …

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor β (TGFβ) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFβ type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice than in wi…

TGF-beta1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis.

Transforming growth factor-beta1 (TGF-beta1) is a powerful stimulus for collagen formation in vitro. To determine the in vivo effects of TGF-beta1 on liver fibrogenesis, we generated transgenic mice overexpressing a fusion gene [C-reactive protein (CRP)/TGF-beta1] consisting of the cDNA coding for an activated form of TGF-beta1 under the control of the regulatory elements of the inducible human CRP gene promoter. Two transgenic lines were generated with liver-specific overexpression of mature TGF-beta1. After induction of the acute phase response (15 h) with lipopolysaccharide (100 microgram ip), plasma TGF-beta1 levels reached600 ng/ml in transgenic animals, which is100 times above normal …

Susceptibility to collagen-induced arthritis is modulated by TGFβ responsiveness of T cells

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor beta (TGFbeta) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFbeta type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice t…

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE(-/-) mice, animals with macrophage specific TGF-ß1 overe…

Cloning and Targeted Deletion of the Mouse Fetuin Gene

We proposed that the alpha2-Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heter…

Cutting Edge: TGF-β Signaling Is Required for the In Vivo Expansion and Immunosuppressive Capacity of Regulatory CD4+CD25+ T Cells

Abstract Data regarding the role of TGF-β for the in vivo function of regulatory CD4+CD25+ T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-β signaling specifically in T cells was used to assess the role of endogenous TGF-β for the in vivo function of CD4+CD25+ Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4+CD25+ Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4+CD25+ Treg we could demonstrate that endogenous TGF-β promotes the expansion of CD4+CD25+ Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4+CD25+ Treg and were …

TGF-β Suppresses Tumor Progression in Colon Cancer by Inhibition of IL-6 trans-Signaling

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-depende…

Coexpression of TGF-β1 and IL-10 Enables Regulatory T Cells to Completely Suppress Airway Hyperreactivity

Abstract In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-β1. Using mice with impaired TGF-β signaling in T cells, we could demonstrate that TGF-β signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-β1 but de…

Hepatocellular Hyperplasia, Plasmacytoma Formation, and Extramedullary Hematopoiesis in Interleukin (IL)-6/Soluble IL-6 Receptor Double-Transgenic Mice

Cytokines interact not only with membrane anchored receptors, but also with specific soluble receptors which circulate in the bloodstream. In general, soluble cytokine receptors such as soluble tumor necrosis factor receptor, soluble interleukin 1 receptor, and soluble interleukin 4 receptor compete with their membrane-bound counterparts for the ligands and therefore act as antagonists. In contrast, soluble receptors for cytokines of the interleukin-6 (IL-6) family complex with their ligands act agonistically. Interestingly, the complex of IL-6 and the soluble interleukin 6 receptor (sIL-6R) activates target cells that do not express the membrane-bound IL-6R and therefore cannot respond to …

TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo.

Factors influencing the development of CD4+CD25+ T-cells in vivo are poorly understood. In order to investigate the contribution of TGFbeta1 to the development and function of CD4+CD25+ T-cells, we generated a gain of function mutation resulting in the overexpression of an active form of TGFbeta1 in T-cells under control of the human CD2 promoter. In peripheral lymphoid organs and in the thymus, the frequency of CD4+CD25+ T-cells was increased in transgenic mice. This appeared to be due to an autocrine effect of TGFbeta on T-cells, since concomitant impairment of TGFbeta-signaling in double transgenic mice resulted in a phenotype similar to wild type. In contrast, in single transgenic mice …

TGF-β superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of …

MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocyutes.

The ability to activate CD4 T cells is restricted to antigen-presenting cells that express major histocompatibility complex (MHC) class II molecules. Parenchymal cells normally do not express MHC class II molecules; however, in clinical hepatitis, viral or autoimmune, hepatocytes often exhibit aberrant MHC class II expression. It is not known whether MHC class II-expressing hepatocytes can function as antigen-presenting cells, but it has been suggested that aberrant MHC class II expression by parenchymal cells may cause autoimmune disease. Therefore, we generated transgenic mice that specifically overexpress class II transactivator molecules in hepatocytes. Hepatocytes from these mice exhib…

Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A.

Platelet derived growth factor (PDGF) plays a central role in repair mechanisms after acute and chronic tissue damage. To further evaluate the role of PDGF-A in liver fibrogenesis in vivo, we generated transgenic mice with hepatocyte-specific overexpression of PDGF-A using the CRP-gene promoter. Transgenic but not wildtype mice showed expression of PDGF-A mRNA in the liver. Hepatic PDGF-A overexpression was accompanied by a significant increase in hepatic procollagen III mRNA expression as well as TGF-beta1 expression. Liver histology showed increased deposition of extracellular matrix in transgenic but not in wildtype mice. PDGF-A-transgenic mice showed positive sinusoidal staining for alp…

P38 MAP Kinase Signaling Is Required for the Conversion of CD4+CD25− T Cells into iTreg

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25− T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25− T cells into …

Overexpression of bone morphogenetic protein-6 (BMP-6) in murine epidermis suppresses skin tumor formation by induction of apoptosis and downregulation of fos/jun family members.

Bone morphogenetic protein-6 (BMP-6) is a member of the transforming growth factor-beta superfamily. In murine skin, BMP-6 is highly expressed in postmitotic keratinocytes from day 15.5 p.c. till day 6 p.p. Expression in adult skin remains at very low levels, but pathological conditions such as wounding induce the expression of BMP-6. We demonstrate that tumor promotion by TPA (12-O-tetradecanoylphorbol-13-acetate) also induces expression of BMP-6 in suprabasal keratinocytes. This induction is due to post-transcriptional regulation since the level of BMP-6 mRNA remained unchanged. We performed two-stage skin carcinogenesis experiments with transgenic mice epidermally overexpressing BMP-6. T…

Expression of a dominant negative type II TGF-β receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development

The role of Transforming growth factor beta (TGF-beta) in carcinogenesis is complex. There are reports on both tumor inhibition and tumor promotion by TGF-beta. To elucidate the complex role of TGF-beta in epithelial carcinogenesis, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in the basal cell compartment and in follicular cells of the skin. Despite the reduced responsiveness of transgenic keratinocytes to TGF-beta, both proliferation and differentiation were normal in non-irritated epidermis of these transgenic mice. Thus, interruption of signaling of all three isoforms of TGF-beta in basal and follicular cells does not disturb tissue homeostas…

Impairment of TGF-β signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice

In autoimmune hepatitis, strong TGF-beta1 expression is found in the inflamed liver. TGF-beta overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-beta signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-beta type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared wit…

A key pathogenic role for the STAT1/T-bet signaling pathway in T-cell-mediated liver inflammation.

TH1 cytokines have been suggested to contribute to the pathogenesis of T-cell-mediated liver injury and inflammation. However, the molecular signaling pathways involved in such injury are still poorly understood. In the present study, we investigated the role of the STAT1/T-bet signaling pathway in a murine model of T-cell-mediated liver inflammation induced by the application of concanavalin A (Con A) using newly created STAT1 transgenic mice as well as STAT1- and T-bet-deficient mice. Liver injury induced by Con A was associated with an increase of both pSTAT1 and T-bet levels in the liver. Furthermore, functional studies suggested a pathogenic role for STAT1 in Con A-induced liver injury…

Induction of bone morphogenetic protein-6 in skin wounds. Delayed reepitheliazation and scar formation in BMP-6 overexpressing transgenic mice.

Growth factors of the transforming growth factor-beta superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-beta related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cell…

Granulocyte–Macrophage Colony-Stimulating Factor Is Essential for Normal Wound Healing

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing…

T Cell-Specific Overexpression of TGFß1 Fails to Influence Atherosclerosis in ApoE-Deficient Mice

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of T…

Resistance of keratinocytes to TGFbeta-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds.

The pleiotropic growth factor TGFβ plays an important role in regulating responses to skin injury. TGFβ targets many different cell types and is involved in all aspects of wound healing entailing inflammation,re-epithelialization, matrix formation and remodeling. To elucidate the role of TGFβ signal transduction in keratinocytes during cutaneous wound healing, we have used transgenic mice expressing a dominant negative type II TGFβ receptor exclusively in keratinocytes. We could demonstrate that this loss of TGFβ signaling in keratinocytes led to an accelerated re-epithelialization of full thickness excisional wounds accompanied by an increased proliferation in keratinocytes at the wound ed…