Bethany B. Moore

p53-Mediated Activation of miRNA34 Candidate Tumor-Suppressor Genes

BACKGROUND In response to varied cell stress signals, the p53 tumor-suppressor protein activates a multitude of genes encoding proteins with functions in cell-cycle control, DNA repair, senescence, and apoptosis. The role of p53 in transcription of other types of RNAs, such as microRNAs (miRNAs) is essentially unknown. RESULTS Using gene-expression analyses, reporter gene assays, and chromatin-immunoprecipitation approaches, we present definitive evidence that the abundance of the three-member miRNA34 family is directly regulated by p53 in cell lines and tissues. Using array-based approaches and algorithm predictions, we define genes likely to be directly regulated by miRNA34, with cell-cycle regulatory genes being the most prominent class. In addition, we provide functional evidence, obtained via antisense oligonucleotide transfection and the use of mouse embryonic stem cells with loss of miRNA34a function, that the BCL2 protein is regulated directly by miRNA34. Finally, we demonstrate that the expression of two miRNA34s is dramatically reduced in 6 of 14 (43%) non-small cell lung cancers (NSCLCs) and that the restoration of miRNA34 expression inhibits growth of NSCLC cells. CONCLUSIONS Taken together, the data suggest the miRNA34s might be key effectors of p53 tumor-suppressor function, and their inactivation might contribute to certain cancers.

CCR2-Mediated Recruitment of Fibrocytes to the Alveolar Space after Fibrotic Injury

Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45(+), CD13(+), collagen 1(+), CD34(-)). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2(+/+) mice than from CCR2(-/-) mice. Transplant of CCR2(+/+) bone marrow into CCR2(-/-) recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2(+/+) lung fibrocytes also migrated to injured airspaces of CCR2(-/-) recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury.

Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability

The present study was designed to elucidate the effects of the chemokine monocyte chemoattractant protein (MCP-1) on blood–brain barrier (BBB) permeability. Experiments were conducted under in vitro conditions (coculture of brain endothelial cells and astrocytes) to study the cellular effects of MCP-1 and under in vivo conditions (intracerebral and intracerebroventricular administration of MCP-1) to study the potential contribution of MCP-1 to BBB disruption in vivo. Our results showed that MCP-1 induces a significant increase in the BBB permeability surface area product for fluorescein isothiocyanate (FITC)-albumin under in vivo conditions, particularly during prolonged (3 or 7 days) exposure (0.096±0.008 versus 0.031±0.005 μL/g min in controls at 3 days, P<0.001). Monocyte chemoattractant protein-1 also enhanced (17-fold compared with control) the permeability of the in vitro BBB (coculture) model. At the cellular level, MCP-1 causes alteration of tight junction (TJ) proteins in endothelial cells (redistribution of TJ proteins determined by Western blotting and loss of immunostaining for occludin, claudin-5, ZO-1, ZO-2). Monocyte chemoattractant protein-1-induced alterations in BBB permeability are mostly realized through the CCR2 receptor. Absence of CCR2 diminishes any effect of MCP-1 on BBB permeability in vitro and in vivo. The permeability surface area product for FITC-albumin after 3 days exposure to MCP-1 was 0.096±0.006 and 0.032±0.007 μL/g min, in CCR2+/+ and CCR2−/− mice, respectively (P<0.001). Monocytes/macrophages also participate in MCP-1-induced alterations in BBB permeability in vivo. Monocytes/macrophages depletion (by clodronate liposomes) reduced the effect of MCP-1 on BBB permeability in vivo ∼2 fold. Our results suggest that, besides its main function of recruiting leukocytes at sites of inflammation, MCP-1 also plays a role in ‘opening’ the BBB.

Protection from Pulmonary Fibrosis in the Absence of CCR2 Signaling

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2−/− mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5−/− mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2−/− mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2−/− mice have increased levels of GM-CSF and reduced levels of TNF-α compared with FITC-treated CCR2+/+ mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation

RATIONALE Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis. OBJECTIVES To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs. METHODS Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured. MEASUREMENTS AND MAIN RESULTS We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-β-independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses. CONCLUSIONS This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ- and disease-specific manner.

Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis

RATIONALE Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process. OBJECTIVES To determine if a targeted injury to type II cells would result in pulmonary fibrosis. METHODS A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement. MEASUREMENTS AND MAIN RESULTS Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction. CONCLUSIONS The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells.

Prostate cancer is the second leading cause of malignancy-related mortality in males in the United States. As a solid tumor, clinically significant tumor growth and metastasis are dependent on nutrients and oxygen supplied by tumor-associated neovasculature. As such, there is a selective tumorigenic advantage for those neoplasms that can produce angiogenic mediators. We show here that human prostate cancer cell lines can constitutively produce angiogenic CXC chemokines. Tumorigenesis of PC-3 prostate cancer cells was shown to be attributable, in part, to the production of the angiogenic CXC chemokine, interleukin (IL)-8. Neutralizing antisera to IL-8 inhibits PC-3 tumor growth in a human prostate cancer/SCID mouse model. Furthermore, angiogenic activity in PC-3 tumor homogenates was attributable to IL-8. In contrast, the Du145 prostate cancer cell line uses a different angiogenic CXC chemokine, GRO-alpha, to mediate tumorigenicity. Neutralizing antisera to GRO-alpha but not IL-8 reduced tumor growth in vivo and reduced the angiogenic activity in tumor homogenates. Thus, prostate cancer cell lines can use distinct CXC chemokines to mediate their tumorigenicity.

Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule α, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.

γδ-T Cells Are Critical for Survival and Early Proinflammatory Cytokine Gene Expression During Murine Klebsiella Pneumonia

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR β- and/or δ-chain were intratracheally inoculated with Klebsiella pneumoniae. γδ T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only αβ-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in γδ-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-γ and TNF-α gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from γδ-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking γδ-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that γδ-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-γ and TNF-α.