Multiple mechanisms modulate distinct cellular susceptibilities toward apoptosis in the developing Drosophila eye
Although apoptosis is mechanistically well understood, a comprehensive understanding of how cells modulate their susceptibility toward apoptosis in a developing tissue is lacking. Here, we reveal striking dynamics in the apoptotic susceptibilities of different cell types in the Drosophila retina over a period of only 24 hr. Mitotic cells are extremely susceptible to apoptotic signals, while postmitotic cells have developed several strategies to promote survival. For example, photoreceptor neurons accumulate the inhibitor of apoptosis, Diap1. In unspecified cells, Cullin-3-mediated degradation keeps Diap1 levels low. These cells depend on EGFR signaling for survival. As development proceeds, developmentally older photoreceptors degrade Diap1, resulting in increased apoptosis susceptibility. Finally, R8 photoreceptors have very efficient survival mechanisms independent of EGFR or Diap1. These examples illustrate how complex cellular susceptibility toward apoptosis is regulated in a developing organ. Similar complexities may regulate apoptosis susceptibilities in mammalian development, and tumor cells may take advantage of it.
Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.
Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages
Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.
The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-beta to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.
Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila
Caspases are the executioners of apoptosis. Although much is known about their physiological roles and structures, detailed analyses of missense mutations of caspases are lacking. As mutations within caspases are identified in various human diseases, the study of caspase mutants will help to elucidate how caspases interact with other components of the apoptosis pathway and how they may contribute to disease. DrICE is the major effector caspase in Drosophila required for developmental and stress-induced cell death. Here, we report the isolation and characterization of six de novo drICE mutants, all of which carry point mutations affecting amino acids conserved among caspases in various species. These six mutants behave as recessive loss-of-function mutants in a homozygous condition. Surprisingly, however, two of the newly isolated drICE alleles are gain-of-function mutants in a heterozygous condition, although they are loss-of-function mutants homozygously. Interestingly, they only behave as gain-of-function mutants in the presence of an apoptotic signal. These two alleles carry missense mutations affecting conserved amino acids in close proximity to the catalytic cysteine residue. This is the first time that viable gain-of-function alleles of caspases are described in any intact organism and provides a significant exception to the expectation that mutations of conserved amino acids always abolish the pro-apoptotic activity of caspases. We discuss models about how these mutations cause the gain-of-function character of these alleles.
The beneficial role of extracellular reactive oxygen species in apoptosis-induced compensatory proliferation
Apoptosis-induced proliferation (AiP) maintains tissue homeostasis following massive stress-induced cell death. During this phenomenon, dying cells induce proliferation of the surviving cells to compensate for the tissue loss, and thus restore organ size. Along with wound healing and tissue regeneration, AiP also contributes to tumor repopulation following radiation or chemotherapy. There are several models of AiP. Using an "undead" AiP model that causes hyperplastic overgrowth of Drosophila epithelial tissue, we recently demonstrated that extracellular reactive oxygen species (eROS) are produced by undead epithelial cells, and are necessary for inducing AiP and overgrowth. Furthermore, hemocytes, the Drosophila blood cells, are seen adjacent to the undead epithelial tissue, and may secrete the TNF ortholog Eiger that signals through the TNF receptor to active Jun-N-terminal kinase (JNK) in the undead tissue and induce proliferation. We propose that undead epithelial tissue triggers an inflammatory response that resembles recruitment of macrophages to human epithelial tumors, and that these tumor-associated macrophages release signals for proliferation and tumor growth of the epithelium. This Extra View article summarizes these recent findings with a focus on the role of eROS for promoting regeneration and inflammation-induced tumorigenesis.
BACKGROUND: ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer.
RESULTS: Here, we show that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development.
CONCLUSION: In addition to a role of dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP.
The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila
A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired.
Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix-attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix-associated genome is highly cell-context dependent.
Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia.
Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease
The spectrum of alcoholic liver disease (ALD) is a major cause of mortality with limited therapies available. Because alcohol targets numerous signaling pathways in hepatocytes and in immune cells, the identification of a master regulatory target that modulates multiple signaling processes is attractive. In this report, we assessed the role of spleen tyrosine kinase (SYK), a nonreceptor tyrosine kinase, which has a central modulatory role in multiple proinflammatory signaling pathways involved in the pathomechanism of ALD. Using mouse disease models that represent various phases in the progression of human ALD, we found that alcohol, in all of these models, induced SYK activation in the liver, both in hepatocytes and liver mononuclear cells. Furthermore, significant SYK activation also occurred in liver samples and peripheral blood mononuclear cells of patients with ALD/alcoholic hepatitis compared to controls. Functional inhibition of SYK activation in vivo abrogated alcohol-induced hepatic neutrophil infiltration, resident immune cell activation, as well as inflammasome and extracellular signal-regulated kinase 1 and 2-mediated nuclear factor kappa B activation in mice. Strikingly, inhibition of SYK activation diminished alcohol-induced hepatic steatosis and interferon regulatory factor 3-mediated apoptosis.
CONCLUSION: Our data demonstrate a novel, functional, and multicellular role for SYK phosphorylation in modulating immune cell-driven liver inflammation, hepatocyte cell death, and steatosis at different stages of ALD. These novel findings highlight SYK as a potential multifunctional target in the treatment of alcoholic steatohepatitis.
Stress-signaling pathways are evolutionarily conserved and play an important role in the maintenance of homeostasis. These pathways are also critical for adaptation to new cellular environments. The endoplasmic reticulum (ER) unfolded protein response (UPR) is activated by biosynthetic stress and leads to a compensatory increase in ER function. The JNK and p38 MAPK signaling pathways control adaptive responses to intracellular and extracellular stresses, including environmental changes such as UV light, heat, and hyperosmotic conditions, and exposure to inflammatory cytokines. Metabolic stress caused by a high-fat diet represents an example of a stimulus that coordinately activates both the UPR and JNK/p38 signaling pathways. Chronic activation of these stress-response pathways ultimately causes metabolic changes associated with obesity and altered insulin sensitivity. Stress-signaling pathways, therefore, represent potential targets for therapeutic intervention in the metabolic stress response and other disease processes.
Summary: Reversible protein phosphorylation plays a fundamental role in signal transduction networks. Phosphorylation alters protein function by regulating enzymatic activity, stability, cellular localization, or binding partners. Over three-quarters of human proteins may be phosphorylated, with many targeted at multiple sites. Such multisite phosphorylation substantially increases the scope for modulating protein function—a protein with n phosphorylation sites has the potential to exist in 2n distinct phosphorylation states, each of which could, in theory, display modified functionality. Proteins can be substrates for several protein kinases, thereby integrating distinct signals to provide a coherent biological response. However, they can also be phosphorylated at multiple sites by a single protein kinase to promote a specific functional output that can be reversed by dephosphorylation by protein phosphatases. On page 233 of this issue, Mylona et al. (1) reveal an unexpected role for multisite phosphorylation, whereby a protein kinase progressively phosphorylates sites on a transcription factor to promote and then subsequently limit its activity independently of dephosphorylation.
Obese, insulin-resistant states are characterized by a paradoxical pathogenic condition in which the liver appears to be selectively insulin resistant. Specifically, insulin fails to suppress glucose production, yet successfully stimulates de novo lipogenesis. The mechanisms underlying this dysregulation remain controversial. Here, we hypothesized that carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, plays a central role in this paradox. Administration of fructose increased hepatic hexose-phosphate levels, activated ChREBP, and caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis in mice. Activation of ChREBP was required for the increased expression of glycolytic and lipogenic genes as well as glucose-6-phosphatase (G6pc) that was associated with the effects of fructose administration. We found that fructose-induced G6PC activity is a major determinant of hepatic glucose production and reduces hepatic glucose-6-phosphate levels to complete a homeostatic loop. Moreover, fructose activated ChREBP and induced G6pc in the absence of Foxo1a, indicating that carbohydrate-induced activation of ChREBP and G6PC dominates over the suppressive effects of insulin to enhance glucose production. This ChREBP/G6PC signaling axis is conserved in humans. Together, these findings support a carbohydrate-mediated, ChREBP-driven mechanism that contributes to hepatic insulin resistance.
Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging-associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18-mo-old transgenic mice with muscle-specific overexpression of IL-10 (MIL10) and in wild-type mice during hyperinsulinemic-euglycemic clamping. Despite similar fat mass and energy balance, MIL10 mice were protected from aging-associated insulin resistance with significant increases in glucose infusion rates, whole-body glucose turnover, and skeletal muscle glucose uptake ( approximately 60%; P < 0.05), as compared to age-matched WT mice. This protective effect was associated with decreased muscle inflammation, but no changes in adipose tissue inflammation in aging MIL10 mice. These results demonstrate the importance of skeletal muscle inflammation in aging-mediated insulin resistance, and our findings further implicate a potential therapeutic role of anti-inflammatory cytokine in the treatment of aging-mediated insulin resistance.
Altered Interleukin-10 Signaling in Skeletal Muscle Regulates Obesity-Mediated Inflammation and Insulin Resistance
Skeletal muscle insulin resistance is a major characteristic of obesity and type 2 diabetes. Although obesity-mediated inflammation is causally associated with insulin resistance, the underlying mechanism is unclear. Here, we examined the effects of chronic obesity in mice with muscle-specific overexpression of interleukin-10 (MIL10). After 16 weeks of a high-fat diet (HFD), MIL10 mice became markedly obese but showed improved insulin action compared to that of wild-type mice, which was largely due to increased glucose metabolism and reduced inflammation in skeletal muscle. Since leptin regulates inflammation, the beneficial effects of interleukin-10 (IL-10) were further examined in leptin-deficient ob/ob mice. Muscle-specific overexpression of IL-10 in ob/ob mice (MCK-IL10ob/ob) did not affect spontaneous obesity, but MCK-IL10ob/ob mice showed increased glucose turnover compared to that in ob/ob mice. Last, mice with muscle-specific ablation of IL-10 receptor (M-IL10R-/-) were generated to determine whether IL-10 signaling in skeletal muscle is involved in IL-10 effects on glucose metabolism. After an HFD, M-IL10R-/- mice developed insulin resistance with reduced glucose metabolism compared to that in wild-type mice. Overall, these results demonstrate IL-10 effects to attenuate obesity-mediated inflammation and improve insulin sensitivity in skeletal muscle, and our findings implicate a potential therapeutic role of anti-inflammatory cytokines in treating insulin resistance and type 2 diabetes.
During embryonic development, adipocytes emerge from microvasculature. Lineage-‐tracing studies in mice have shown that adipocyte progenitors reside in the adipose tissue capillaries. However, the direct evidence of an association between adipocyte progenitors and vasculature in humans is lacking. A specific class of adipocytes (brown and beige/brite) expresses the uncoupling protein 1 (UCP1), which consumes glucose and fatty acids to generate heat. The abundance of UCP1- containing adipocytes correlates with a lean metabolically healthy phenotype in human. However, a causal relationship between the presence of these cells and metabolic benefits in human is not clear.
In this thesis, I report human adipocyte progenitors proliferate in response to pro-angiogenic factors in association with adipose capillary networks in-vitro. The capillary-derived adipocytes transform from being UCP1-negative to positive upon adenylate cyclase activation, a defining feature of the brite/beige phenotype. Activated cells have denser, round mitochondria with UCP1 protein, and display uncoupled respiration. When implanted into NOD-scid IL2rgnull (NSG) mice, the adipocytes can form a vascularized fat pad that induces vascularization and becomes integrated into mouse circulatory system. In normal or high fat diet-fed NSG mice, activated brite/beige adipocytes enhance systemic glucose tolerance and improved hepatic steatosis, thus providing evidence for their potential therapeutic use. The adipocytes also express neuroendocrine and secretory factors such as Interleukin-33, proprotein convertase PCSK1 and proenkephalin PENK, which are correlated with human obesity. Finally, analyses on single-cell clones of capillary-sprout cells reveal the existence of diverse adipogenic progenitor populations. Further characterization of the clones will define the identifying features of the diverse adipocyte progenitor types that exist in human adipose tissue.
HPV Vaccine Reminders at the Point of Service: Efficacy and Missed Opportunities. A Claims Based Study within One Health Plan
Introduction: Our objective is to assess HPV vaccine series completion rates, whether on-screen Point of Service reminders (POS) make a difference, and missed opportunities for reminders to have an effect.
Methods: Retrospective, claims-based study of females aged 9-26 receiving an initial dose of HPV vaccine during 2 periods: before (period 1) and after (period 2) implementation of a POS reminder system in 1(“Change Group”) of 2 physician groups using EHRs for both periods. Completion rates, and missed opportunities during eligible periods were calculated for those with continuous enrollment in the health plan investigated.
Results: Completion rates within 1 year of the 1st dose were Period 1: 47% Change Group vs. 46% Control Group (p=0.847). Period 2: 50% Change group vs. 57% Control Group (p=0.158). No significant improvement occurred between the 2 periods in either group. Differences in 1 year completion rates by specialty of initiating provider or age group (<18 or ≥18) were not significant.
During period 2, among those with continuous insurance plan enrollment in the Change Group, 43 patients received 1 dose and 46 received 2 doses. Of those receiving 1 dose, 30 (70%) had a visit to the same group within an eligible time period (median # visits: 2, range 1-20); of those completing 2 doses, 4 (9%) had a visit to the same group within an eligible period (median # visits: 1, range: 1-3). Among those receiving 1 dose, 25 (58%) had a visit to the same group and same specialty as the initial dose (median # visits: 1, range 1-8); for those having received 2 doses, 3 (6%) had a subsequent visit to same group and specialty (median # visits: 1, range 1-3).
Conclusion: POS reminder systems was not associated with improved completion rates. POS reminders are limited by infrequent visits among non-completers in an eligible period.
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Endoplasmic reticulum stress-induced hepatocellular death pathways mediate liver injury and fibrosis via Stimulator of Interferon Genes.
Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbontetrachloride (CCl4) administration to WT, IRF3-, TRAM-, TRIF-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3 and Type-I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4. Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4. In contrast, mice deficient in Type-I IFN receptors or in TLR4-signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis, and indicate that innate immune signaling modulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.