David B. Williams

p53 Mediates TNF-Induced Epithelial Cell Apoptosis in IBD

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS(-/-) mice. IEC p53 levels and apoptosis were reduced in IL-10(-/-) colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF-treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF-treated CUC patients.

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Background: Mitochondrial Ogg1 prevents oxidant (H2O2 and asbestos)-induced Aco-2 degradation and apoptosis. Results: Oxidant stress caused preferential AEC mtDNA > nuclear DNA damage, mt-p53 translocation, and apoptosis; effects were blocked by mt-hOgg1 or Aco-2. Conclusion: mt-hOgg1 and Aco-2 preserve AEC mtDNA, preventing oxidant-induced p53 activation and apoptosis. Significance: mt-hOgg1/Aco-2 effects on mtDNA may be an innovative target for preventing degenerative diseases. Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.

Electrophysiological Effects of Burimamide and 16,16-Dimethyl Prostaglandin E2 on The Canine Gastric Mucosa

The electrophysiological effects of two potent inhibitors of gastric acid secretion, burimamide and 16,16-dimethyl prostaglandin E2 (dm-PGE2), were determined in an in vivo histamine-stimulated canine stomach preparation and an in vitro canine gastric mucosal preparation. In the in vivo stomach preparation, intravenous burimamide caused a decrease in acid secretion, an increase in transmucosal potential difference (PD) and the relative resistance (R) was essentially unchanged. Intravenous dm-PGE2 also inhibited acid secretion and increased PD but, in contrast to burimamide, increased R. In the in vitro preparation, the unidirectional flux of sodium from mucosa to serosa increased after dm-PGE2 but not after burimamide. Passive sodium fluxes and unidirectional chloride fluxes were not altered after either agent. These findings suggest that increased active transport of sodium from mucosa to serosa is at least partially responsible for the observed increase in transmural PD with dm-PGE2, an agent which also decreases hydrogen ion transport. With burimamide the increased PD was due primarily to inhibition of hydrogen ion secretion.

p53 and PUMA independently regulate apoptosis of intestinal epithelial cells in patients and mice with colitis.

BACKGROUND & AIMS Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs. METHODS Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53⁻/⁻, Bid⁻/⁻, Bim⁻/⁻, Bax3⁻/⁻, Bak⁻/⁻, PUMA⁻/⁻, and Noxa⁻/⁻ mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis. RESULTS Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53⁻/⁻ and PUMA⁻/⁻ mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53⁻/⁻ mice indicated that PUMA-mediated apoptosis was independent of p53. CONCLUSIONS In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis.

The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis.

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

Asbestos-Induced Pulmonary Fibrosis Is Augmented in 8-Oxoguanine DNA Glycosylase Knockout Mice

Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 μl) or titanium dioxide (100 μg/50 μl), crocidolite or Libby amphibole asbestos (100 μg/50 μl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target.

SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

Alveolar epithelial cell (AEC) mitochondrial dysfunction and apoptosis are important in idiopathic pulmonary fibrosis and asbestosis. Sirtuin 3 (SIRT3) detoxifies mitochondrial reactive oxygen species, in part, by deacetylating manganese superoxide dismutase (MnSOD) and mitochondrial 8‐oxoguanine DNA glycosylase. We reasoned that SIRT3 deficiency occurs in fibrotic lungs and thereby augments AEC mtDNA damage and apoptosis. Human lungs were assessed by using immunohistochemistry for SIRT3 activity via acetylated MnSODK68. Murine AEC SIRT3 and cleaved caspase‐9 (CC‐9) expression were assayed by immunoblotting with or without SIRT3 enforced expression or silencing. mtDNA damage was measured by using quantitative PCR and apoptosis via ELISA. Pulmonary fibrosis after asbestos or bleomycin exposure was evaluated in 129SJ/wild‐type and SIRT3‐knockout mice (Sirt3−/−) by using fibrosis scoring and lung collagen levels. Idiopathic pulmonary fibrosis lung alveolar type II cells have increased MnSODK68 acetylation compared with controls. Asbestos and H2O2 diminished AEC SIRT3 protein expression and increased mitochondrial protein acetylation, including MnSODK68. SIRT3 enforced expression reduced oxidant‐induced AEC OGG1K338/341 acetylation, mtDNA damage, and apoptosis, whereas SIRT3 silencing promoted these effects. Asbestos‐ or bleomycin‐induced lung fibrosis, AEC mtDNA damage, and apoptosis in wild‐type mice were amplified in Sirt3−/− animals. These data suggest a novel role for SIRT3 deficiency in mediating AEC mtDNA damage, apoptosis, and lung fibrosis.—Jablonski, R. P., Kim, S.‐J., Cheresh, P., Williams, D. B., Morales‐Nebreda, L., Cheng, Y., Yeldandi, A., Bhorade, S., Pardo, A., Selman, M., Ridge, K., Gius, D., Budinger, G. R. S., Kamp, D. W. SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis. FASEB J. 31, 2520–2532 (2017). www.fasebj.org

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn’s disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/β-catenin–mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/β-catenin signaling correlates with inflammation status. TNF-deficient (Tnf−/−) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM–derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2−/− BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/β-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/β-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced β-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/β-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Asbestos-Induced Gastrointestinal Cancer: An Update

Asbestos-related diseases, such as malignancies and asbestosis, remain a significant occupational and public health concern. Asbestos is still widely used in many developing countries despite being a recognized carcinogen that has been banned over 50 countries. The prevalence and mortality from asbestos-related diseases continue to pose challenges worldwide. Many countries are now experiencing an epidemic of asbestos-related disease that is the legacy of occupational exposure during the 20th century because of the long latency period (up to 40 years) between initial asbestos exposure and exhibition of disease. However, the gastrointestinal (GI) cancers resulting from asbestos exposure are not as clearly defined. In this review, we summarize some of the recent epidemiology of asbestos-related diseases and then focus on the evidence implicating asbestos in causing GI malignancies. We also briefly review the important new pathogenic information that has emerged over the past several years that may account for asbestos-related gastrointestinal cancers. All types of asbestos fibers have been implicated in the mortality and morbidity from GI malignancies but the collective evidence to date is mixed. Although the molecular basis of GI cancers arising from asbestos exposure is unclear, there have been significant advances in our understanding of mesothelioma and asbestosis that may contribute to the pathophysiology underlying asbestos-induced GI cancers. The emerging new evidence into the pathogenesis of asbestos toxicity is providing insights into the molecular basis for developing novel therapeutic strategies for asbestos-related diseases in future management.

ID: 117: SIRT3, THE MAJOR ANTI-AGING MITOCHONDRIAL DEACETYLASE, IS IMPORTANT FOR PREVENTING PULMONARY FIBROSIS

Objectives Alveolar epithelial cell (AEC) injury from ‘exaggerated’ lung aging and mitochondrial dysfunction play vital roles in the development of lung fibrosis. Our group, using the asbestos lung fibrosis paradigm, has shown that AEC mitochondrial reactive oxygen species (ROS) mediate asbestos-induced AEC mitochondrial DNA (mtDNA) damage and apoptosis by a mitochondria-regulated (intrinsic) death pathway. Sirtuin 3 (SIRT3), the anti-aging major mitochondrial deacetylase governing mitochondrial function, mitigates oxidative stress and fibrosis in non-lung models though deacetylation of diverse substrates including MnSOD (Chen et al EMBO Rep 2011) and OGG1 (Cheng et al Cell Death Dis 2013). We previously reported that SIRT3 deficient (Sirt3−/−) mice have increased lung fibrosis following asbestos exposure associated with exaggerated AEC mtDNA damage and apoptosis. Herein, we determined whether SIRT3 deficiency augments bleomycin-induced lung fibrosis and whether AEC acetylation is increased in lung biopsy samples from patients with idiopathic pulmonary fibrosis (IPF). Methods Male 8- to 10- week-old 129SJ (Sirt3+/+) and Sirt3−/− mice were treated with a single intratracheal instillation of saline or bleomycin (0.01U). At 3 weeks, the lungs were harvested for various endpoints including Sircol collagen assay, fibrosis scoring and measurement of lung compliance. Specimens from explanted lungs of patients with IPF were subject to immunohistochemistry with antibodies to MnSODK68, Ac-OGG1 and IgG (negative control) to assess acetylation. Results Compared to wild type, Sirt3−/− mice developed increased pulmonary fibrosis following bleomycin exposure as measured by fibrosis score (7.8 vs. 11.25, p<0.05), Sircol assay (0.90 vs. 1.35, p<0.05) and lung compliance (0.0688 mL/cmH2O vs. 0.0449 mL/cm H2O, p<0.05). Notably, increased expression of MnSODK68 and Ac-OGG1 was evident in the lungs of patients with IPF. Co-localization studies evaluating MnSODK68 and SFPTC are ongoing. Conclusions SIRT3 deficiency enhances bleomycin-induced pulmonary fibrosis in a manner similar to asbestos fibers. An important role for augmented human IPF lung parenchymal cell mitochondrial acetylation is suggested by our pilot studies. Taken together, this suggests that SIRT3 plays a key role in the pathogenesis of IPF in part by preserving AEC mitochondrial function and mtDNA through modulation of the SIRT3/ACO-2/OGG1/apoptosis axis. Given the crucial role for aging in IPF as well as changes in SIRT3 expression with aging, our findings suggest a novel therapeutic target for modulating lung fibrosis. Funding VA Merit and NIH R01 ES02037-01A1 (DK), NIH/NHLBI T32 HL076139-11A1 (RJ).