RESUMO
Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.
Assuntos
Células Dendríticas/imunologia , Neoplasias/imunologia , Neoplasias/radioterapia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Apresentação Cruzada/imunologia , Imunoterapia Adotiva/métodos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli I-C/imunologia , Radioterapia/métodosRESUMO
The polypeptide hormone prolactin (PRL) stimulates breast epithelial cell growth, differentiation, and motility through its cognate receptor, PRLr. PRLr is expressed in most breast cancers; however, its exact role remains elusive. Our laboratory previously described a novel mode of PRLr signaling in which Stat5a-mediated transcription is regulated through ligand-induced phosphorylation of the PRLr transactivation domain (TAD). Herein, we used a PRLr transactivation-deficient mutant (PRLrYDmut) to identify novel TAD-specific target genes. Microarray analysis identified 120 PRL-induced genes up-regulated by wild type but not PRLrYDmut. Compared with control, PRLr expression significantly induced expression of approximately 4700 PRL-induced genes, whereas PRLrYDmut ablated induction of all but 19 of these genes. Ingenuity pathway analysis found that the PRLr TAD most profoundly affected networks involving cancer and proliferation. In support of this, PRLrYDmut expression reduced anchorage-dependent and anchorage-independent growth. In addition, pathway analysis identified a link between the PRLr TAD and the estrogen and progesterone receptors (ERα/PR). Although neither ERα nor PR was identified as a PRL target gene, a TAD mutation significantly impaired ERα/PR expression and estrogen responsiveness. TMA analysis revealed a marked increase in nuclear, but not cytoplasmic, PRLr TAD phosphorylation as a function of neoplastic progression. We propose that PRLr TAD phosphorylation contributes to breast cancer pathogenesis, in part through regulation of ERα and PR, and has potential utility as a biomarker in this disease.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/biossíntese , Receptores de Progesterona/biossíntese , Receptores da Prolactina/genética , Ativação Transcricional/genética , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/genética , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos , Humanos , Mutação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação/genética , Prognóstico , Prolactina/farmacologia , Receptores de Progesterona/genética , Receptores da Prolactina/biossíntese , Análise Serial de Tecidos/métodos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Radiation therapy induces immunogenic cell death in cancer cells, whereby released endogenous adjuvants are sensed by immune cells to direct adaptive immune responses. TLRs expressed on several immune subtypes recognize innate adjuvants to direct downstream inflammatory responses in part via the adapter protein MyD88. We generated Myd88 conditional knockout mice to interrogate its contribution to the immune response to radiation therapy in distinct immune populations in pancreatic cancer. Surprisingly, Myd88 deletion in Itgax (CD11c)-expressing dendritic cells had little discernable effects on response to RT in pancreatic cancer and elicited normal T cell responses using a prime/boost vaccination strategy. Myd88 deletion in Lck-expressing T cells resulted in similar or worsened responses to radiation therapy compared to wild-type mice and lacked antigen-specific CD8+ T cell responses from vaccination, similar to observations in Myd88-/- mice. Lyz2-specific loss of Myd88 in myeloid populations rendered tumors more susceptible to radiation therapy and elicited normal CD8+ T cell responses to vaccination. scRNAseq in Lyz2-Cre/Myd88fl/fl mice revealed gene signatures in macrophages and monocytes indicative of enhanced type I and II interferon responses, and improved responses to RT were dependent on CD8+ T cells and IFNAR1. Together, these data implicate MyD88 signaling in myeloid cells as a critical source of immunosuppression that hinders adaptive immune tumor control following radiation therapy.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias Pancreáticas , Camundongos , Animais , Fator 88 de Diferenciação Mieloide/metabolismo , Monócitos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia , Camundongos Knockout , Adjuvantes Imunológicos/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias PancreáticasRESUMO
Tissue resident memory (Trm) CD8 T cells infiltrating tumors represent an enriched population of tumor antigen-specific T cells, and their presence is associated with improved outcomes in patients. Using genetically engineered mouse pancreatic tumor models we demonstrate that tumor implantation generates a Trm niche that is dependent on direct antigen presentation by cancer cells. However, we observe that initial CCR7-mediated localization of CD8 T cells to tumor draining lymph nodes is required to subsequently generate CD103+ CD8 T cells in tumors. We observe that the formation of CD103+ CD8 T cells in tumors is dependent on CD40L but independent of CD4 T cells, and using mixed chimeras we show that CD8 T cells can provide their own CD40L to permit CD103+ CD8 T cell differentiation. Finally, we show that CD40L is required to provide systemic protection against secondary tumors. These data suggest that CD103+ CD8 T cell formation in tumors can occur independent of the two-factor authentication provided by CD4 T cells and highlight CD103+ CD8 T cells as a distinct differentiation decision from CD4-dependent central memory.
Assuntos
Memória Imunológica , Neoplasias , Animais , Camundongos , Ligante de CD40 , Neoplasias/patologia , Linfócitos T CD8-Positivos , Ativação LinfocitáriaRESUMO
Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, sphingosine-1-phosphate, prevented lung apoptosis, implying that a balance between ceramide and sphingosine-1-phosphate is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.
Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Enfisema/metabolismo , Pulmão/metabolismo , Fumar/efeitos adversos , Esfingosina/análogos & derivados , Regulação para Cima , Aciltransferases/antagonistas & inibidores , Animais , Células Cultivadas , Ceramidas/toxicidade , Relação Dose-Resposta a Droga , Enfisema/induzido quimicamente , Enfisema/patologia , Ácidos Graxos Monoinsaturados/farmacologia , Fumonisinas/farmacologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredutases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Serina C-Palmitoiltransferase , Esfingosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
In the cancer literature tumors are inconsistently labeled as 'immunogenic', and experimental results are occasionally dismissed since they are only tested in known 'responsive' tumor models. The definition of immunogenicity has moved from its classical definition based on the rejection of secondary tumors to a more nebulous definition based on immune infiltrates and response to immunotherapy interventions. This review discusses the basis behind tumor immunogenicity and the variation between tumor models, then moves to discuss how these principles apply to the response to radiation therapy. In this way we can identify radioimmunogenic tumor models that are particularly responsive to immunotherapy only when combined with radiation, and identify the interventions that can convert unresponsive tumors so that they can also respond to these treatments.
RESUMO
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate-chain length (C(8:0)) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-alpha-generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C(8:0) ceramide- and TNF-alpha (plus cycloheximide)-induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C(16:0) ceramide in response to C(8:0) and TNF-alpha treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-alpha-stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.
Assuntos
Apoptose/fisiologia , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Pulmão/citologia , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Ceramidas/química , Células Endoteliais/citologia , Ativação Enzimática , Humanos , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Complement is a critical component of humoral immunity implicated in cancer development; however, its biological contributions to tumorigenesis remain poorly understood. Using the K14-HPV16 transgenic mouse model of squamous carcinogenesis, we report that urokinase (uPA)+ macrophages regulate C3-independent release of C5a during premalignant progression, which in turn regulates protumorigenic properties of C5aR1+ mast cells and macrophages, including suppression of CD8+ T cell cytotoxicity. Therapeutic inhibition of C5aR1 via the peptide antagonist PMX-53 improved efficacy of paclitaxel chemotherapy associated with increased presence and cytotoxic properties of CXCR3+ effector memory CD8+ T cells in carcinomas, dependent on both macrophage transcriptional programming and IFNγ. Together, these data identify C5aR1-dependent signaling as an important immunomodulatory program in neoplastic tissue tractable for combinatorial cancer immunotherapy.
Assuntos
Carcinogênese/efeitos dos fármacos , Complemento C5a/efeitos dos fármacos , Tratamento Farmacológico , Receptor da Anafilatoxina C5a/efeitos dos fármacos , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Modelos Animais de Doenças , Tratamento Farmacológico/métodos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
UNLABELLED: Skin is a highly ordered immune organ that coordinates rapid responses to external insult while maintaining self-tolerance. In healthy tissue, lymphatic vessels drain fluid and coordinate local immune responses; however, environmental factors induce lymphatic vessel dysfunction, leading to lymph stasis and perturbed regional immunity. These same environmental factors drive the formation of local malignancies, which are also influenced by local inflammation. Herein, we discuss clinical and experimental evidence supporting the tenet that lymphatic vessels participate in regulation of cutaneous inflammation and immunity, and are important contributors to malignancy and potential biomarkers and targets for immunotherapy. SIGNIFICANCE: The tumor microenvironment and tumor-associated inflammation are now appreciated not only for their role in cancer progression but also for their response to therapy. The lymphatic vasculature is a less-appreciated component of this microenvironment that coordinates local inflammation and immunity and thereby critically shapes local responses. A mechanistic understanding of the complexities of lymphatic vessel function in the unique context of skin provides a model to understand how regional immune dysfunction drives cutaneous malignancies, and as such lymphatic vessels represent a biomarker of cutaneous immunity that may provide insight into cancer prognosis and effective therapy.
Assuntos
Vasos Linfáticos/imunologia , Neoplasias Cutâneas/patologia , Biomarcadores Tumorais/imunologia , Humanos , Vasos Linfáticos/patologia , Neoplasias Cutâneas/imunologia , Microambiente TumoralRESUMO
Stat5a is a transcription factor utilized by several cytokine/hormone receptor signaling pathways that promotes transcription of genes associated with proliferation, differentiation, and survival of cancer cells. However, there are currently no clinically approved therapies that directly target Stat5a, despite ample evidence that it contributes to breast cancer pathogenesis. Here, deacetylation of the Stat5a coactivator and chromatin-remodeling protein HMGN2 on lysine residue K2 by HDAC6 promotes Stat5a-mediated transcription and breast cancer growth. HDAC6 inhibition both in vitro and in vivo enhances HMGN2 acetylation with a concomitant reduction in Stat5a-mediated signaling, resulting in an inhibition of breast cancer growth. Furthermore, HMGN2 is highly acetylated at K2 in normal human breast tissue, but is deacetylated in primary breast tumors and lymph node metastases, suggesting that targeting HMGN2 deacetylation is a viable treatment for breast cancer. Together, these results reveal a novel mechanism by which HDAC6 activity promotes the transcription of Stat5a target genes and demonstrate utility of HDAC6 inhibition for breast cancer therapy. IMPLICATIONS: HMGN2 deacetylation enhances Stat5a transcriptional activity, thereby regulating prolactin-induced gene transcription and breast cancer growth. Mol Cancer Res; 14(10); 994-1008. ©2016 AACR.
Assuntos
Neoplasias da Mama/patologia , Proteína HMGN2/metabolismo , Histona Desacetilases/metabolismo , Fator de Transcrição STAT5/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Acetilação , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Desacetilase 6 de Histona , Humanos , Lisina/metabolismo , Células MCF-7 , Camundongos , Metástase Neoplásica , Transplante de NeoplasiasRESUMO
Chemotherapy and radiotherapy have been extensively used to eradicate cancer based on their direct cytocidal effects on rapidly proliferating tumor cells. Accumulating evidence indicates that these therapies also dramatically affect resident and recruited immune cells that actively support tumor growth. We now appreciate that mobilization of effector CD8+ T cells enhances efficacy of chemotherapy and radiotherapy; remarkable clinical advances have been achieved by blocking regulatory programs limiting cytotoxic CD8+ T cell activity . This review discusses immune-mediated mechanisms underlying efficacy of chemotherapy and radiotherapy, and provides a perspective on how understanding tissue-based immune mechanisms can be used to guide therapeutic approaches combining immune and cytotoxic therapies to improve outcomes for a larger subset of patients than currently achievable.
RESUMO
It is well established that cancer development ensues based on reciprocal interactions between genomically altered neoplastic cells and diverse populations of recruited "host" cells co-opted to support malignant progression. Among the host cells recruited into tumor microenvironments, several subtypes of myeloid cells, including macrophages, monocytes, dendritic cells, and granulocytes contribute to tumor development by providing tumor-promoting factors as well as a spectrum of molecules that suppress cytotoxic activities of T lymphocytes. Based on compelling preclinical data revealing that inhibition of critical myeloid-based programs leads to tumor suppression, novel immune-based therapies and approaches are now entering the clinic for evaluation. This review discusses mechanisms underlying protumorigenic programming of myeloid cells and discusses how targeting of these has potential to attenuate solid tumor progression via the induction and of mobilization CD8 cytotoxic T cell immunity.
Assuntos
Células Mieloides/imunologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/imunologia , Linfócitos B/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Granulócitos/imunologia , Humanos , Macrófagos/imunologia , Monócitos/imunologia , Neoplasias/tratamento farmacológico , Neutrófilos/imunologiaRESUMO
B cells foster squamous cell carcinoma (SCC) development through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγ receptor-dependent activation of myeloid cells. Because human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced support for SCC growth. Although ineffective as a single agent, treatment of mice bearing preexisting SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and Taxol-based chemotherapy. Improved chemoresponsiveness was dependent on altered chemokine expression by macrophages that promoted tumor infiltration of activated CD8(+) lymphocytes via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anticancer therapy in select tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/imunologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Macrófagos/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Células CHO , Carcinoma de Células Escamosas/patologia , Cricetulus , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Neoplasias/patologia , Compostos Organoplatínicos/administração & dosagem , Paclitaxel/administração & dosagem , Fenótipo , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular mechanisms that modulate the activity of the signal transducers and activators of transcription 5 (Stat5) during the progression of breast cancer remain elusive. Here, we present evidence that the calcineurin/nuclear factor of activated T cells (NFAT) pathway negatively regulates the activation of Stat5, and vice versa in breast cancer. NFAT1 interacts with Stat5 in breast cancer cells, and their physical association is mediated by the DNA binding and transactivation domains of Stat5. Ectopically expressed NFAT1 is capable of inhibiting Stat5-dependent functions, including Stat5 transactivation, Stat5-mediated transcription of the downstream target gene expression, and binding of Stat5a to the Stat5 target promoter. By contrast, overexpression of a selective NFAT inhibitor VIVIT reversed NFAT1-mediated suppression of Stat5-dependent gene expression, whereas silencing of NFAT1 through RNA interference enhanced prolactin-induced, Stat5-mediated gene transcription, and breast cancer cell proliferation. A reciprocal inhibitory effect of Stat5 activity on NFAT1 signaling was also observed, implying these two signaling cascades antagonize each other in breast cancer. Importantly, analysis of a matched breast cancer progression tissue microarray revealed a negative correlation between levels of NFAT1 and Stat5 (pY694) during the progression of breast cancer. Taken together, these studies highlight a novel negative cross talk between the NFAT1- and Stat5-signaling cascades that may affect breast tumor formation, growth, and metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptor Cross-Talk , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Fatores de Transcrição NFATC/química , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Transcrição STAT5/química , Análise Serial de Tecidos , Ativação TranscricionalRESUMO
The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.
Assuntos
Núcleo Celular/metabolismo , Proteína HMGN2/metabolismo , Receptores da Prolactina/química , Receptores da Prolactina/metabolismo , Fator de Transcrição STAT5/metabolismo , Transcrição Gênica , Ativação Transcricional/genética , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer(12:0) or Cer(8:0); 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramide's proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/toxicidade , Alvéolos Pulmonares/enzimologia , Enfisema Pulmonar/enzimologia , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/biossíntese , Animais , Apoptose/genética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Transdução de Sinais/genética , Fumar/genética , Fumar/metabolismo , Fumar/patologia , Esfingomielina Fosfodiesterase/biossíntese , Esfingomielina Fosfodiesterase/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Superóxidos/metabolismoRESUMO
alpha-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema.
Assuntos
Apoptose , Caspase 3/metabolismo , Inibidores de Caspase , Pulmão/enzimologia , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/farmacologia , Animais , Capilares/citologia , Capilares/efeitos dos fármacos , Capilares/enzimologia , Sistema Livre de Células , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Pneumopatias/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RATIONALE: There is growing evidence that alveolar cell apoptosis plays an important role in emphysema pathogenesis, a chronic inflammatory lung disease characterized by alveolar destruction. The association of alpha1-antitrypsin deficiency with the development of emphysema has supported the concept that protease/antiprotease imbalance mediates cigarette smoke-induced emphysema. OBJECTIVES: We propose that, in addition to its antielastolytic effects, alpha1-antitrypsin may have broader biological effects in the lung, preventing emphysema through inhibition of alveolar cells apoptosis. METHODS, MEASUREMENTS, AND MAIN RESULTS: Transduction of human alpha1-antitrypsin via replication-deficient adeno-associated virus attenuated airspace enlargement and emphysema caused by inhibition of vascular endothelial growth factor (VEGF) receptors with SU5416 in mice, a model of apoptosis-dependent emphysema lacking neutrophilic inflammation. The overexpressed human serine protease inhibitor accumulated in lung cells and suppressed caspase-3 activation and oxidative stress in lungs treated with the VEGF blocker or with VEGF receptor-1 and -2 antibodies. Similar results were obtained in SU5416-treated rats given human alpha1-antitrypsin intravenously. CONCLUSIONS: Our findings suggest that inhibition of structural alveolar cell apoptosis by alpha1-antitrypsin represents a novel protective mechanism of the serpin against emphysema. Further elucidation of this mechanism may extend the therapeutic options for emphysema caused by reduced level or loss of function of alpha1-antitrypsin.