Inhibition of aberrant tissue remodelling by mesenchymal stromal cells singly coated with soft gels presenting defined chemomechanical cues.

The precise understanding and control of microenvironmental cues could be used to optimize the efficacy of cell therapeutics. Here, we show that mesenchymal stromal cells (MSCs) singly coated with a soft conformal gel presenting defined chemomechanical cues promote matrix remodelling by secreting soluble interstitial collagenases in response to the presence of tumour necrosis factor alpha (TNF-α). In mice with fibrotic lung injury, treatment with the coated MSCs maintained normal collagen levels, fibre density and microelasticity in lung tissue, and the continuous presentation of recombinant TNF-α in the gel facilitated the reversal of aberrant tissue remodelling by the cells when inflammation subsided in the host. Gel coatings with predefined chemomechanical cues could be used to tailor cells with specific mechanisms of action for desired therapeutic outcomes.

Nrf2 Regulates Anti-Inflammatory A20 Deubiquitinase Induction by LPS in Macrophages in Contextual Manner.

The aberrant regulation of inflammatory gene transcription following oxidant and inflammatory stimuli can culminate in unchecked systemic inflammation leading to organ dysfunction. The Nrf2 transcription factor dampens cellular stress and controls inflammation by upregulating antioxidant gene expression and TNFα-induced Protein 3 (TNFAIP3, aka A20) deubiquitinase by controlling NF-kB signaling dampens tissue inflammation. Here, we report that Nrf2 is required for A20 induction by inflammatory stimuli LPS in monocyte/bone marrow derived macrophages (MDMΦs) but not in lung-macrophages (LDMΦs). LPS-induced A20 expression was significantly lower in Nrf2-/- MDMΦs and was not restored by antioxidant supplementation. Nrf2 deficiency markedly impaired LPS-stimulated A20 mRNA expression Nrf2-/- MDMΦs and ChIP assays showed Nrf2 enrichment at the promoter Nrf2-/- MDMΦs upon LPS stimulation, demonstrating that Nrf2 directly regulates A20 expression. Contrary to MDMΦs, LPS-stimulated A20 expression was not largely impaired in Nrf2-/- LDMΦs ex vivo and in vivo and ChIP assays showed lack of increased Nrf2 binding at the A20 promoter in LDMΦ following LPS treatment. Collectively, these results demonstrate a crucial role for Nrf2 in optimal A20 transcriptional induction in macrophages by endotoxin, and this regulation occurs in a contextual manner.

Nrf2 mediates hypoxia-inducible HIF1α activation in kidney tubular epithelial cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2-/-) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2-/- RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2-/- RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI.

FOSL1 Promotes Kras-induced Lung Cancer through Amphiregulin and Cell Survival Gene Regulation.

The FOSL1/AP-1 transcription factor regulates gene expression, thereby controlling various pathophysiological processes. It is a major effector of RAS-ERK1/2 signaling and is activated in human lung epithelia by tumorigenic stimuli. Recent evidence shows an inverse correlation between FOSL1 expression and the survival of patients with lung cancer and adenocarcinomas; however, its role in lung tumorigenesis remains elusive. In this work, we sought to determine the role of FOSL1 in Kras-induced lung adenocarcinoma in vivo and its downstream effector mechanisms. We used mice expressing the Kras oncogene in the lung with concomitant Fosl1 deletion, Kras-activated murine alveolar epithelial cells (mAECs) with Fosl1 deletion, and KRAS mutant human lung adenocarcinoma (HLAC) cells with FOSL1 deficiency, and performed cell proliferation and gene expression analyses. Mutant Kras induced Fosl1 expression in vitro (mAECs) and in vivo (lung tissue), and mice with Fosl1 deletion showed reduced levels of mutant Kras-induced lung tumorigenesis and survived longer than Fosl1-sufficient mice. Studies with mutant Kras-activated mAECs and KRAS-mutant HLAC cells revealed that FOSL1 regulates mutant KRAS-induced gene expression, thereby controlling cell proliferation and survival. In contrast, FOSL1 depletion in non-KRAS-mutant HLAC cells and nonmalignant human lung epithelia had no effect. Our data support the notion that FOSL1-mediated expression of amphiregulin and apoptotic and antioxidative genes plays a role in regulating HLAC cell proliferation and survival. FOSL1 is a determinant of lung cancer in vivo and regulates HLAC cell proliferation and survival, largely in the context of KRAS mutations. Activation of FOSL1 in adenocarcinomas may be a prognostic marker and potential target for human lung cancer with KRAS mutations.

Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation.

Ischemia-reperfusion (IR)-induced kidney injury is a major clinical problem, but its underlying mechanisms remain unclear. The transcription factor known as nuclear factor, erythroid 2-like 2 (NFE2L2 or Nrf2) is crucial for protection against oxidative stress generated by pro-oxidant insults. We have previously shown that Nrf2 deficiency enhances susceptibility to IR-induced kidney injury in mice and that its upregulation is protective. Here, we examined Nrf2 target antioxidant gene expression and the mechanisms of its activation in both human and murine kidney epithelia following acute (2 h) and chronic (12 h) hypoxia and reoxygenation conditions. We found that acute hypoxia modestly stimulates and chronic hypoxia strongly stimulates Nrf2 putative target HMOX1 expression, but not that of other antioxidant genes. Inhibition of AKT1/2 or ERK1/2 signaling blocked this induction; AKT1/2 but not ERK1/2 inhibition affected Nrf2 levels in basal and acute hypoxia-reoxygenation states. Unexpectedly, chromatin immunoprecipitation assays revealed reduced levels of Nrf2 binding at the distal AB1 and SX2 enhancers and proximal promoter of HMOX1 in acute hypoxia, accompanied by diminished levels of nuclear Nrf2. In contrast, Nrf2 binding at the AB1 and SX2 enhancers significantly but differentially increased during chronic hypoxia and reoxygenation, with reaccumulation of nuclear Nrf2 levels. Small interfering-RNA-mediated Nrf2 depletion attenuated acute and chronic hypoxia-inducible HMOX1 expression, and primary Nrf2-null kidney epithelia showed reduced levels of HMOX1 induction in response to both acute and chronic hypoxia. Collectively, our data demonstrate that Nrf2 upregulates HMOX1 expression in kidney epithelia through a distinct mechanism during acute and chronic hypoxia reoxygenation, and that both AKT1/2 and ERK1/2 signaling are required for this process.

c-Jun Is Required for Nuclear Factor-κB-Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages.

Previously, we have reported that Fos-related antigen-1 (Fra-1) transcription factor promotes LPS-induced acute lung injury and mortality, and that LPS-induced Fra-1 expression in the lung occurs predominantly in alveolar macrophages. Nuclear factor-κB (NF-κB) and c-Jun transcription factors play key roles in modulating inflammatory and immune responses induced by infectious and non-infectious insults. Here, we report that NF-κB and c-Jun coregulate Fra-1 induction by LPS in alveolar macrophages and that this regulation occurs through both the NF-κB and the extracellular signal-regulated protein kinase (ERK) signaling pathways. Transient transfections with Fra-1 promoter-reporter constructs and inhibitor studies revealed that the transcriptional activation of Fra-1 by LPS in alveolar macrophages is mediated by NF-κB and ERK1/2 signaling. Importantly, chromatin immunoprecipitation assays revealed the recruitment of c-Jun and NF-κB to the endogenous Fra-1 promoter after LPS stimulation. We found that inhibition of ERK1/2 signaling reduced LPS-stimulated c-Jun and NF-κB recruitment to the promoter. Likewise, NF-κB inhibitor blocked LPS-induced NF-κB and c-Jun binding to the promoter. ERK1/2 inhibition had no effect on c-Jun activation but suppressed LPS-stimulated NF-κB phosphorylation. Finally, functional assays showed reduced levels of LPS-stimulated NF-κB regulated proinflammatory IL-1ß and macrophage inflammatory protein-1α expression and increased antiinflammatory IL-10 expression in lung alveolar macrophages of Fra-1-null mice in vivo. Thus, our studies indicate that NF-κB and c-Jun coregulate LPS-induced Fra-1 transcription and that Fra-1 selectively modulates LPS-stimulated inflammatory cytokine expression in lung alveolar macrophages during inflammatory lung injury.

Sirtuin 1 Promotes Hyperoxia-Induced Lung Epithelial Cell Death Independent of NF-E2-Related Factor 2 Activation.

Lung epithelial cell damage accompanied by death is a cardinal feature of toxicant- and prooxidant-induced acute lung injury. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NEF2L2 or NRF2) activates several antioxidant enzymes (AOEs) and prosurvival genes in response to oxidant stress, and its deficiency enhances susceptibility to hyperoxic lung injury and other oxidant-induced lung pathologies. Sirtuin 1 (SIRT1) regulates cell growth and survival in response to both physiological and pathological stresses by selectively deacetylating multiple proteins required for chromatin remodeling and transcription; therefore, we sought to examine potential SIRT1-NRF2 cross-talk in the regulation of AOE expression during hyperoxia-induced lung epithelial cell death. Unexpectedly, pharmacological inhibition or small interfering RNA-mediated depletion of SIRT1 caused a reduction in cell death, accompanied by reduced levels of NRF2-dependent AOE expression in chronic hyperoxia. NRF2 acetylation was markedly and transiently higher in cells exposed to acute (6 h) hyperoxia. Sirtinol blocked this acute effect, but NRF2 acetylation was low or undetectable in cells exposed to chronic hyperoxia (24-36 h) both with and without sirtinol. SIRT1 activation by resveratrol augmented hyperoxia-induced death in cells with NRF2 deficiency. SIRT1 inhibition or depletion led to a reduced activation of the cell-death executioner caspase 3, whereas caspase inhibition prevented death. Consistent with these results, sirtinol attenuated hyperoxia-induced lung alveolar permeability and toxicity in vivo. Collectively, these results reveal that, in chronic hyperoxia, SIRT1 promotes hyperoxia-induced lung epithelial cell damage and death by altering pro- and antiapoptotic balance, not by dampening optimal NRF2-dependent AOE expression.

Visualization of Fra-1/AP-1 activation during LPS-induced inflammatory lung injury using fluorescence optical imaging.

Inappropriate lung inflammatory response following oxidant and toxicant exposure can lead to abnormal repair and disease pathogenesis, including fibrosis. Thus early detection of molecular and cellular processes and mediators promoting lung inflammation is necessary to develop better strategies for therapeutic intervention and disease management. Previously, we have shown that transcription factor Fra-1/AP-1 plays key roles in lung inflammatory response, as Fra-1-null mice are less susceptible than wild-type mice to LPS-induced lung injury and mortality. Herein, we developed a transgenic reporter mouse model expressing tdTomato under the control of FRA-1 (human) promoter (referred to as FRA-1(TdTg) mice) to monitor its activation during inflammatory lung injury using fluorescence protein-based optical imaging and molecular analysis in vivo and ex vivo. A higher red fluorescent signal was observed in the lungs of LPS-treated FRA-1(TdTg) mice compared with vehicle controls, and Western blot and qRT-PCR analyses revealed a significant correlation with the FRA-1-tdTomato reporter expression. Immunocolocalization demonstrated expression of FRA-1-tdTomato largely in lung alveolar macrophages and to some extent in epithelial cells. Moreover, we validated these results with a second reporter mouse model that expressed green fluorescent protein upon activation of endogenous Fra-1 promoter. Additionally, we demonstrated increased expression of FRA-1 in alveolar macrophages in human lung instilled with Escherichia coli ex vivo. Collectively, our data obtained from two independent reporter mouse models and from human samples underscore the significance of Fra-1 activation in alveolar macrophages during inflammatory lung injury and may aid in developing strategies to target this transcription factor in lung injury and repair.