Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis.

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Defects in microvillus crosslinking sensitize to colitis and inflammatory bowel disease.

Intestinal epithelial cells are covered by the brush border, which consists of densely packed microvilli. The Intermicrovillar Adhesion Complex (IMAC) links the microvilli and is required for proper brush border organization. Whether microvillus crosslinking is involved in the intestinal barrier function or colitis is currently unknown. We investigate the role of microvillus crosslinking in colitis in mice with deletion of the IMAC component CDHR5. Electron microscopy shows pronounced brush border defects in CDHR5-deficient mice. The defects result in severe mucosal damage after exposure to the colitis-inducing agent DSS. DSS increases the permeability of the mucus layer and brings bacteria in direct contact with the disorganized brush border of CDHR5-deficient mice. This correlates with bacterial invasion into the epithelial cell layer which precedes epithelial apoptosis and inflammation. Single-cell RNA sequencing data of patients with ulcerative colitis reveals downregulation of CDHR5 in enterocytes of diseased areas. Our results provide experimental evidence that a combination of microvillus crosslinking defects with increased permeability of the mucus layer sensitizes to inflammatory bowel disease.

Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development.

It is established that tumor cell-derived VEGF acts on endothelial cells to promote angiogenesis and tumor growth. Here, we demonstrate that in K5-SOS-dependent mouse skin tumors, autocrine VEGF is required for tumor cell proliferation in a cell-autonomous and angiogenesis-independent manner. VEGF is upregulated in SOS-expressing tumors, and its deletion in epidermal cells delays tumorigenesis by suppressing angiogenesis and tumor cell proliferation. Epidermis-specific Flt1 deletion also impairs tumorigenesis and proliferation. Surprisingly, complete tumor inhibition occurs in the absence of VEGF in EGFR mutant mice, demonstrating that VEGFR and EGFR synergize in neoplastic cells to promote tumor growth. Mechanistically, K5-SOS upregulates VEGF, Flt1, and Neuropilin-1 in an Erk-dependent manner, thereby activating an autocrine proliferation loop, whereas EGFR prevents tumor cells from apoptosis. Moreover, Flt1 is upregulated in human SCC, and its inhibition in SCC cells impairs proliferation. Thus, in addition to regulating angiogenesis, VEGF has to be considered as a potent growth factor for epidermal tumors.

RNA editing of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure.

Epitranscriptomic events such as adenosine-to-inosine (A-to-I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q-to-R transition in the interactive C-terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient-derived RNA-Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients.

Bone morphogenetic protein signaling regulates skin inflammation via modulating dendritic cell function.

BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-ß family that signal via the BMP receptor (BMPR) signaling cascade, distinct from canonical TGF-ß signaling. BMP downstream signaling is strongly induced within epidermal keratinocytes in cutaneous psoriatic lesions, and BMP7 instructs monocytic cells to acquire characteristics of psoriasis-associated Langerhans dendritic cells (DCs). Regulatory T (Treg)-cell numbers strongly increase during psoriatic skin inflammation and were recently shown to limit psoriatic skin inflammation. However, the factors mediating Treg-cell accumulation in psoriatic skin currently remain unknown. OBJECTIVE: We sought to investigate the role of BMP signaling in Treg-cell accumulation in psoriasis. METHODS: The following methods were used: immunohistology of patients and healthy controls; ex vivo models of Treg-cell generation in the presence or absence of Langerhans cells; analysis of BMP versus canonical TGF-ß signaling in DCs and Treg cells; and modeling of psoriatic skin inflammation in mice lacking the BMPR type 1a in CD11c+ cells. RESULTS: We here demonstrated a positive correlation between Treg-cell numbers and epidermal BMP7 expression in cutaneous psoriatic lesions and show that unlike Treg cells from healthy skin, a portion of inflammation-associated Treg cells exhibit constitutive-active BMP signaling. We further found that BMPR signaling licenses inflammation-associated Langerhans cell/DC to gain an enhanced capacity to promote Treg cells via BMPR-mediated CD25 induction and that this effect is associated with reduced skin inflammation. CONCLUSIONS: Psoriatic lesions are marked by constitutive high BMP7/BMPR signaling in keratinocytes, which instructs inflammatory DCs to gain enhanced Treg-cell-stimulatory activity. Locally secreted BMP7 can directly promote Treg-cell generation through the BMP signaling cascade.

BMP7 aberrantly induced in the psoriatic epidermis instructs inflammation-associated Langerhans cells.

BACKGROUND: Epidermal hyperplasia represents a morphologic hallmark of psoriatic skin lesions. Langerhans cells (LCs) in the psoriatic epidermis engage with keratinocytes (KCs) in tight physical interactions; moreover, they induce T-cell-mediated immune responses critical to psoriasis. OBJECTIVE: This study sought to improve the understanding of epidermal factors in psoriasis pathogenesis. METHODS: BMP7-LCs versus TGF-ß1-LCs were phenotypically characterized and their functional properties were analyzed using flow cytometry, cell kinetic studies, co-culture with CD4 T cells, and cytokine measurements. Furthermore, immunohistology of healthy and psoriatic skin was performed. Additionally, in vivo experiments with Junf/fJunBf/fK5cre-ERT mice were carried out to assess the role of bone morphogenetic protein (BMP) signaling in psoriatic skin inflammation. RESULTS: This study identified a KC-derived signal (ie, BMP signaling) to promote epidermal changes in psoriasis. Whereas BMP7 is strictly confined to the basal KC layer in the healthy skin, it is expressed at high levels throughout the lesional psoriatic epidermis. BMP7 instructs precursor cells to differentiate into LCs that phenotypically resemble psoriatic LCs. These BMP7-LCs exhibit proliferative activity and increased sensitivity to bacterial stimulation. Moreover, aberrant high BMP signaling in the lesional epidermis is mediated by a KC intrinsic mechanism, as suggested from murine data and clinical outcome after topical antipsoriatic treatment in human patients. CONCLUSIONS: These data indicate that available TGF-ß family members within the lesional psoriatic epidermis preferentially signal through the canonical BMP signaling cascade to instruct inflammatory-type LCs and to promote psoriatic epidermal changes. Targeting BMP signaling might allow to therapeutically interfere with cutaneous psoriatic manifestations.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Interruption of vascular endothelial growth factor receptor 2 signaling induces a proliferative pulmonary vasculopathy and pulmonary hypertension.

Pulmonary arterial hypertension is a severe and progressive disease characterized by a pulmonary vascular remodeling process with expansion of collateral endothelial cells and total vessel occlusion. Endothelial cells are believed to be at the forefront of the disease process. Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptor, VEGF receptor-2 (VEGFR-2), play a central role in angiogenesis, endothelial cell protection, but also in the destabilization of endothelial barrier function. Therefore, we investigated the consequences of altered VEGF signaling in an experimental model, and looked for translational correlates of this observation in patients. We performed an endothelial cell-specific conditional deletion of the kinase insert domain protein receptor (kdr) gene, coding for VEGFR-2, in C57/BL6 mice (Kdr∆end) and held them in an environmental chamber with 10% FiO2 or under normoxia for 6 weeks. Kdr knockout led to a mild PH phenotype under normoxia that worsened under hypoxia. Kdr∆end mice exhibited a significant increase in pulmonary arterial wall thickness, muscularization, and VEGFR-3+ endothelial cells obliterating the pulmonary artery vessel lumen. We observed the same proliferative vasculopathy in our rodent model as seen in patients receiving anti-angiogenic therapy. Serum VEGF-a levels were elevated both in the experimental model and in humans receiving bevacizumab. Interrupted VEGF signaling leads to a pulmonary proliferative arteriopathy in rodents after direct ablative gene manipulation of Kdr. Histologically, similar vascular lesions can be observed in patients receiving anti-VEGF treatment. Our findings illustrate the importance of VEGF signaling for maintenance of pulmonary vascular patency.

Transcription-independent Induction of ERBB1 through Hypoxia-inducible Factor 2A Provides Cardioprotection during Ischemia and Reperfusion.

BACKGROUND: During myocardial ischemia, hypoxia-inducible factors are stabilized and provide protection from ischemia and reperfusion injury. Recent studies show that myocyte-specific hypoxia-inducible factor 2A promotes myocardial ischemia tolerance through induction of epidermal growth factor, amphiregulin. Here, the authors hypothesized that hypoxia-inducible factor 2A may enhance epidermal growth factor receptor 1 (ERBB1) expression in the myocardium that could interface between growth factors and its effect on providing tolerance to ischemia and reperfusion injury. METHODS: Human myocardial tissues were obtained from ischemic heart disease patients and normal control patients to compare ERBB1 expression. Myocyte-specific Hif2a or ErbB1 knockout mice were generated to observe the effect of Hif2a knockdown in regulating ERBB1 expression and to examine the role of ERBB1 during myocardial ischemia and reperfusion injury. RESULTS: Initial studies of myocardial tissues from patients with ischemic heart disease showed increased ERBB1 protein (1.12 ± 0.24 vs. 13.01 ± 2.20, P < 0.001). In contrast, ERBB1 transcript was unchanged. Studies using short hairpin RNA repression of Hif2A or Hif2a Myosin Cre+ mice directly implicated hypoxia-inducible factor 2A in ERBB1 protein induction during hypoxia or after myocardial ischemia, respectively. Repression of RNA-binding protein 4 abolished hypoxia-inducible factor 2A-dependent induction of ERBB1 protein. Moreover, ErbB1 Myosin Cre+ mice experienced larger infarct sizes (22.46 ± 4.06 vs. 46.14 ± 1.81, P < 0.001) and could not be rescued via amphiregulin treatment. CONCLUSIONS: These findings suggest that hypoxia-inducible factor 2A promotes transcription-independent induction of ERBB1 protein and implicates epidermal growth factor signaling in protection from myocardial ischemia and reperfusion injury.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients.

BACKGROUND & AIMS: Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis. METHODS: We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients' median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses. RESULTS: We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice. CONCLUSIONS: Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.

Feasibility of personalized treatment concepts in gastrointestinal malignancies: Sub-group results of prospective clinical phase II trial EXACT.

OBJECTIVE: Advances in high-throughput genomic profiling and the development of new targeted therapies improve patient's survival. In gastrointestinal (GI) malignancies, the concept of personalized medicine (PM) was not investigated so far. The aim of this prospective study was to evaluate the efficacy of a personalized treatment in GI patients who failed standard treatment. METHODS: Out of the original prospective clinical phase II EXACT trial, 21 (38%) GI cancer patients who had no further treatment options were identified. A molecular profile (MP) via a 50 gene next generation sequencing (NGS) panel in combination with immunohistochemistry (IHC) was conducted using real-time biopsy tumor material. Results were discussed by a multidisciplinary team (MDT) to translate the individual MP in an experimental treatment. RESULTS: Of the 55 patients originally included in the EXACT trial, 21 (38%) suffered from GI malignancies. The final analysis showed that 15 (71%) patients had experienced a longer progression-free survival (PFS) upon experimental targeted treatment (124 d, quartiles 70/193 d), when compared with the PFS achieved by the previous conventional therapy (62 d, quartiles 55/83 d) (P=0.014). Thirteen (62%) patients receiving targeted treatment experienced a disease control according to Response Evaluation Criteria in Solid Tumors (RECIST). Median overall survival (OS) from the start of experimental therapy to time of censoring or death was 193 d (quartiles 115/374 d). CONCLUSIONS: PM was not investigated in GI malignancies so far in a prospective trial. This study shows that treatment based on real-time molecular tumor profiling led to a superior clinical benefit, and survival as well as response was significantly improved when compared with previous standard medications.

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis.

The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.

Hepatocyte-Specific Deletion of EGFR in Mice Reduces Hepatic Abcg2 Transport Activity Measured by [11C]erlotinib and Positron Emission Tomography.

The epidermal growth factor receptor (EGFR) regulates cellular expression levels of breast cancer resistance protein (humans: ABCG2, rodents: Abcg2) via its downstream signaling pathways. Drugs that inhibit EGFR signaling (e.g., tyrosine kinase inhibitors, antibodies) may lead to ABCG2-mediated drug-drug interactions (DDIs) by changing the disposition of concomitantly administered ABCG2 substrate drugs. In this study, we used positron emission tomography and magnetic resonance imaging to compare disposition of the model Abcg2 substrate [11C]erlotinib in a mouse model of hepatocyte-specific deletion of EGFR (EGFR∆hep mice, n = 5) with EGFRfl/fl control mice (n = 6), which have normal EGFR expression levels in all tissues. Integration plot analysis was used to estimate the rate constants for transfer of radioactivity from the liver into bile (kbile) and from the kidney into urine (kurine). EGFR∆hep mice showed significantly lower radioactivity concentrations in the intestine (1.6-fold) and higher radioactivity concentrations in the urinary bladder (3.2-fold) compared with EGFRfl/fl mice. Kbile was significantly decreased (3.0-fold) in EGFR∆hep mice, whereas kurine was by 2.2-fold increased. Western blot analysis of liver tissue confirmed deletion of EGFR and showed significant decreases in Abcg2 and increases in P-glycoprotein (Abcb1a/b) expression levels in EGFR∆hep versus EGFRfl/fl mice. Our data show that EGFR deletion in hepatocytes leads to a reduction in Abcg2-mediated hepatobiliary clearance of a probe substrate accompanied by a shift to renal excretion of the drug, which raises the possibility that EGFR-inhibiting drugs may cause ABCG2-mediated DDIs.

Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo.

OBJECTIVE: Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. APPROACH AND RESULTS: We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. CONCLUSIONS: We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.

TNFalpha shedding and epidermal inflammation are controlled by Jun proteins.

Inducible epidermal deletion of JunB and c-Jun in adult mice causes a psoriasis-like inflammatory skin disease. Increased levels of the proinflammatory cytokine TNFalpha play a major role in this phenotype. Here we define the underlying molecular mechanism using genetic mouse models. We show that Jun proteins control TNFalpha shedding in the epidermis by direct transcriptional activation of tissue inhibitor of metalloproteinase-3 (TIMP-3), an inhibitor of the TNFalpha-converting enzyme (TACE). TIMP-3 is down-regulated and TACE activity is specifically increased, leading to massive, cell-autonomous TNFalpha shedding upon loss of both JunB and c-Jun. Consequently, a prominent TNFalpha-dependent cytokine cascade is initiated in the epidermis, inducing severe skin inflammation and perinatal death of newborns from exhaustion of energy reservoirs such as glycogen and lipids. Importantly, this metabolic "cachectic" phenotype can be genetically rescued in a TNFR1-deficient background or by epidermis-specific re-expression of TIMP-3. These findings reveal that Jun proteins are essential physiological regulators of TNFalpha shedding by controlling the TIMP-3/TACE pathway. This novel mechanism describing how Jun proteins control skin inflammation offers potential targets for the treatment of skin pathologies associated with increased TNFalpha levels.