Autophagy in non-small cell lung carcinogenesis: A positive regulator of antitumor immunosurveillance

In a mouse model of non-small cell lung carcinogenesis, we recently found that the inactivation of the essential autophagy gene Atg5 causes an acceleration of the early phases of oncogenesis. Thus, hyperplastic lesions and adenomas are more frequent at early stages after adenoviral delivery of Cre recombinase via inhalation, when Cre—in addition to activating the KRas^G12D oncogene—inactivates both alleles of the Atg5 gene. The accelerated oncogenesis of autophagy-deficient tumors developing in KRas;Atg5^fl/fl mice (as compared with autophagy-competent KRas;Atg5^fl/+ control tumors) correlates with an increased infiltration by FOXP3⁺ regulatory T cells (Tregs). Depletion of such Tregs by means of specific monoclonal antibodies inhibits the accelerated oncogenesis of autophagy-deficient tumors down to the level observed in autophagy-competent controls. Subsequent analyses revealed that the combination of KRas activation and Atg5 inactivation favors the expression of ENTPD1/CD39, an ecto-ATPase that initiates the conversion of extracellular ATP, which is immunostimulatory, into adenosine, which is immunosuppressive. Pharmacological inhibition of ENTPD1 or blockade of adenosinergic receptors reduces the infiltration of KRas;Atg5^fl/fl tumors by Tregs and reverses accelerated oncogenesis. Altogether these data favor a model according to which autophagy deficiency favors oncogenesis via changes in the tumor microenvironment that ultimately entail the Treg-mediated inhibition of anticancer immunosurveillance.     

Autophagy is required for mitochondrial function,lipid metabolism,growth, and fate of KRASG12D-driven lung tumors

Evidence suggests that the role of autophagy in tumorigenesis is context dependent. Using genetically engineered mouse models (GEMMs) for human non-small-cell lung cancer (NSCLC), we found that deletion of the essential autophagy gene, Atg7, in KRAS^G12D-driven NSCLC inhibits tumor growth and converts adenomas and adenocarcinomas to benign oncocytomas characterized by the accumulation of respiration-defective mitochondria. Atg7 is required to preserve mitochondrial fatty acid oxidation (FAO) to maintain lipid homeostasis upon additional loss of Trp53 in NSCLC. Furthermore, cell lines derived from autophagy-deficient tumors depend on glutamine to survive starvation. This suggests that autophagy is essential for the metabolism, growth, and fate of NSCLC.     

Autophagy promotes BrafV600E-driven lung tumorigenesis by preserving mitochondrial metabolism

The role of autophagy in cancer is complex and context-dependent. Here we describe work with genetically engineered mouse models of non-small cell lung cancer (NSCLC) in which the tumor-suppressive and tumor-promoting function of autophagy can be visualized in the same system. We discovered that early tumorigenesis in Braf^V600E-driven lung cancer is accelerated by autophagy ablation due to unmitigated oxidative stress, as observed with loss of Nfe2l2/Nrf2-mediated antioxidant defense. However, this growth advantage is eventually overshadowed by progressive mitochondrial dysfunction and metabolic insufficiency, and is associated with increased survival of mice bearing autophagy-deficient tumors. Atg7 deficiency alters progression of Braf^V600E-driven tumors from adenomas (Braf^V600E; atg7^?/?) and adenocarcinomas (trp53^?/?; Braf^V600E; atg7^?/?) to benign oncocytomas that accumulated morphologically and functionally defective mitochondria, suggesting that defects in mitochondrial metabolism may compromise continued tumor growth. Analysis of tumor-derived cell lines (TDCLs) revealed that Atg7-deficient cells are significantly more sensitive to starvation than Atg7–wild-type counterparts, and are impaired in their ability to respire, phenotypes that are rescued by the addition of exogenous glutamine. Taken together, these data suggest that Braf^V600E-driven tumors become addicted to autophagy as a means to preserve mitochondrial function and glutamine metabolism, and that inhibiting autophagy may be a powerful strategy for Braf^V600E-driven malignancies.     

PDK1对生发中心的生成、发育及功能的影响

目的:研究PDK1对生发中心(GC)的生成、发育及其功能的影响。方法:通过配种小鼠得到在GC B细胞中特异性敲除PDK1的小鼠,然后采用共聚焦显微镜观察小鼠脾脏GC的大小,多色流式细胞分析方法观测PDK1的敲除是否会影响小鼠B细胞的发育,ELISA技术检测PDK1敲除的小鼠经免疫后其体内产生抗体的能力是否受影响,结合平面脂双层抗原呈递系统及全内反射荧光显微镜成像系统(TIRFM)观测PDK1的敲除是否会影响小鼠脾脏IgG细胞P85的磷酸化水平。结果:PDK1的缺失并不会影响小鼠B细胞的发育,细胞群中成熟与不成熟B细胞所占比例均无显著性变化,但在GC B细胞中条件性敲除PDK1会影响小鼠脾脏GC的生成以及T细胞依赖性抗原免疫反应,而且同等条件活化后,GC B细胞中条件性敲除PDK1的小鼠脾脏IgG细胞其胞内分子P85的磷酸化水平显著降低。结论:PDK1对GC的生成、发育及功能都具有重要作用。     

TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-chondroprogenitor cell lineage determination during mandible development

Transforming growth factor-β (TGF-β) signaling is crucial for mandible development. During its development, the majority of the mandible is formed through intramembranous ossification whereas the proximal region of the mandible undergoes endochondral ossification. Our previous work has shown that TGF-β signaling is required for the proliferation of cranial neural crest (CNC)-derived ectomesenchyme in the mandibular primordium where intramembranous ossification takes place. Here we show that conditional inactivation of Tgfbr2 in CNC cells results in accelerated osteoprogenitor differentiation and perturbed chondrogenesis in the proximal region of the mandible. Specifically, the appearance of chondrocytes in Tgfbr2^fl/fl;Wnt1-Cre mice is delayed and they are smaller in size in the condylar process and completely missing in the angular process. TGF-β signaling controls Sox9 expression in the proximal region, because Sox9 expression is delayed in condylar processes and missing in angular process in Tgfbr2^fl/fl;Wnt1-Cre mice. Moreover, exogenous TGF-β can induce Sox9 expression in the mandibular arch. In the angular processes of Tgfbr2^fl/fl;Wnt1-Cre mice, osteoblast differentiation is accelerated and Dlx5 expression is elevated. Significantly, deletion of Dlx5 in Tgfbr2^fl/fl;Wnt1-Cre mice results in the rescue of cartilage formation in the angular processes. Finally, TGF-β signaling-mediated Scleraxis expression is required for tendonogenesis in the developing skeletal muscle. Thus, CNC-derived cells in the proximal region of mandible have a cell intrinsic requirement for TGF-β signaling.     

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5^fl/fl LysM-Cre mice, referred to as atg5^−/−) and their wild-type (Atg5^fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5^−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5^−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5^−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5^−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5^−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5^−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.     

Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

Chk1 is essential for the development of murine epidermal melanocytes

Embryonic deletion of mouse Chk1 is lethal; however, whether Chk1 is essential in all individual tissues is unknown. By breeding C57Bl/ 6 mice homozygous for a conditional allele of Chk1 (Chk1fl/fl) and bearing melanocyte‐specific Tyr::Cre and DCT:: LacZ transgenes, we investigated the consequences of Chk1 deletion in the melanocytic lineage. We show that adult Tyr::Cre; Chk1fl/fl mice lack coat pigmentation and epidermal melanocytes in the hair follicles, but retain eye pigmentation in the retinal pigmented epithelium (RPE). Melanoblasts formed normally during embryogenesis in Tyr::Cre; Chk1fl/fl mice at early times (embryonic day 10.5; E10.5) but were completely absent by stage E13.5, most probably as a consequence of spontaneous DNA damage and apoptosis. By contrast, melanoblast numbers were only slightly reduced in heterozygous Tyr::Cre; Chk1fl/ + embryos, and these mice exhibited normal coat pigmentation as adults. Thus, Chk1 is essential for the developmental formation of murine epidermal melanocytes but hemizygosity has little, if any, permanent developmental consequence in this cell type.     

Intracellular Quality Control by Autophagy: How Does Autophagy Prevent Neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5^-/- mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5^flox/flox;Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2^‐/‐) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell‐types. Here, we describe Cxcr2^fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2^fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2^fl/fl mice were crossed to Mx‐Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic‐polycytidylic acid (poly(I:C)). CXCR2‐deficient neutrophils were observed in poly(I:C) treated Cxcr2^fl/fl::Mx‐Cre⁺ (Cxcr2‐CKO) mice, but not in poly(I:C) treated Cxcr2^f//+::Mx‐Cre⁺ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2‐CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2‐CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2^fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations. genesis 51:587–595. © 2013 Wiley Periodicals, Inc.     

Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1

The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8⁺ T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.     

Commentary: Autophagy promotes Braf V600E-driven lung tumorigenesis by preserving mitochondrial metabolism

The role of autophagy in cancer is complex and context-dependent. Here we describe work with genetically engineered mouse models of non-small cell lung cancer (NSCLC) in which the tumor-suppressive and tumor-promoting function of autophagy can be visualized in the same system. We discovered that early tumorigenesis in Braf ^V600E -driven lung cancer is accelerated by autophagy ablation due to unmitigated oxidative stress, as observed with loss of Nfe2l2/Nrf2-mediated antioxidant defense. However, this growth advantage is eventually overshadowed by progressive mitochondrial dysfunction and metabolic insufficiency, and is associated with increased survival of mice bearing autophagy-deficient tumors. Atg7 deficiency alters progression of Braf ^V600E-driven tumors from adenomas (Braf ^V600E ; atg7^−/−) and adenocarcinomas (trp53^−/−; Braf ^V600E ; atg7^−/−) to benign oncocytomas that accumulated morphologically and functionally defective mitochondria, suggesting that defects in mitochondrial metabolism may compromise continued tumor growth. Analysis of tumor-derived cell lines (TDCLs) revealed that Atg7-deficient cells are significantly more sensitive to starvation than Atg7–wild-type counterparts, and are impaired in their ability to respire, phenotypes that are rescued by the addition of exogenous glutamine. Taken together, these data suggest that Braf ^V600E -driven tumors become addicted to autophagy as a means to preserve mitochondrial function and glutamine metabolism, and that inhibiting autophagy may be a powerful strategy for Braf ^V600E -driven malignancies.     

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD⁺/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.     

Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide

Autophagy plays important roles in many biological processes, but our understanding of the mechanisms regulating stem cells by autophagy is limited. Interpretations of earlier studies of autophagy using knockouts of single genes are confounded by accumulating evidence for other functions of many autophagy genes. Here, we show that, in contrast to Fip200 deletion, inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 does not impair the maintenance and differentiation of postnatal neural stem cells (NSCs). Only Fip200 deletion, but not Atg5, Atg16L1, or Atg7 deletion, caused p62/sequestome1 aggregates to accumulate in NSCs. Fip200 and p62 double conditional knockout mice demonstrated that p62 aggregate formation triggers aberrant superoxide increases by impairing superoxide dismutase functions. By comparing the inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 with Fip200 deletion, we revealed a critical role of increased p62 in determining the fate of autophagy-deficient NSCs through intracellular superoxide control.     

Development of Mice with Osteoblast-Specific Connexin43 Gene Deletion

Genetic deficiency of Cx43 in vivo causes skeletal developmental defects, osteoblast dysfunction and perinatal lethality. To determine the role of Cx43 in the adult skeleton, we developed two models of osteoblast-specific Cx43 gene deletion using Cre mediated replacement of a “floxed” Cx43 allele with a LacZ reporter gene. Cre recombinase expression in osteoblasts was driven by either the osteocalcin OG2 promoter or the 2.3 kb fragment of the Colα1(I) promoter. Homozygous Cx43^fl/flmice, in which the Cx43 coding region is flanked by two loxP sites, were crossed with Cre expressing mice in a heterozygous Cx43-null background [Cx43^±; Colα1(I)-Cre or Cx43^±; OG2-Cre]. Cx43 gene ablation was demonstrated in tissues by selective X-gal staining of cells lining the endosteal surface, and in cultured osteoblastic cells from calvaria using different approaches. Although no LacZ expression was observed in proliferating calvaria cells, before osteoblast differentiation begins, post-proliferative cells isolated from conditional knockout mice [Cx43^fl/?; Colα1(I)-Cre or Cx43^fl/?; OG2-Cre] developed strong LacZ expression as they differentiated, in parallel to a progressive disappearance of Cx43 mRNA and protein abundance relative to controls. Selective Cre mediated Cx43 gene inactivation in bone forming cells will be useful to determine the role of Cx43 in adult skeletal homeostasis and overcome the perinatal lethality of the conventional null model.