High-fat diet alters N-glycosylation of PTPRJ in murine liver.

Protein tyrosine phosphatases (PTPs) regulate multiple signaling pathways. Disruption of tyrosine phosphorylation through imbalanced action between protein tyrosine kinases (RTKs) and PTPs is a hallmark of metabolic disorders, including insulin resistance. A representative member of the receptor-type PTP family, PTPRJ (DEP-1), was previously identified as a negative regulator of insulin signaling and possesses post-translational glycosylation sites. In this regard, it seems of great importance to decipher the structure of PTPRJ's glycosylation, particularly in the context of metabolic disturbances, but this has not been done in detail. Thus, here we aimed at characterizing the glycosylation pattern of PTPRJ in liver. We show that N-glycosylation accounts for up to half of PTPRJ's molecular weight. Applying mass spectrometry, we detected increased levels of high-mannose structures in PTPRJ in liver tissue of obese mice compared to lean littermates. In addition, complex neutral structures without fucose were also elevated in PTPRJ of high-fat diet (HFD) mice. Conversely, complex fucosylated N-glycans as well as sialylated bi- and triantennary N-glycans, were significantly reduced in PTPRJ of HFD-derived liver tissue compared to LFD by ∼two fold (P≤.01, P≤.0001 and P≤.001, respectively). In congruence with these findings, the mannosidase MAN2A1, responsible for the conversion of high-mannose to complex N-glycans, was significantly downregulated under HFD conditions. Here we present for the first time that HFD-induced obesity impacts on the glycosylation pattern of the insulin signaling component PTPRJ in liver. These findings may inspire new research on the glycosylation of PTPs in metabolic diseases and may open up new therapeutic approaches.

Increased cFLIP expression in thymic epithelial tumors blocks autophagy via NF-κB signalling.

The anti-apoptotic cellular FLICE-like inhibitory protein cFLIP plays a pivotal role in normal tissues homoeostasis and the development of many tumors, but its role in normal thymus (NT), thymomas and thymic carcinomas (TC) is largely unknown. Expression, regulation and function of cFLIP were analyzed in biopsies of NT, thymomas, thymic squamous cell carcinomas (TSCC), thymic epithelial cells (TECs) derived thereof and in the TC line 1889c by qRT-PCR, western blot, shRNA techniques, and functional assays addressing survival, senescence and autophagy. More than 90% of thymomas and TSCCs showed increased cFLIP expression compared to NT. cFLIP expression declined with age in NTs but not in thymomas. During short term culture cFLIP expression levels declined significantly slower in neoplastic than non-neoplastic primary TECs. Down-regulation of cFLIP by shRNA or NF-κB inhibition accelerated senescence and induced autophagy and cell death in neoplastic TECs. The results suggest a role of cFLIP in the involution of normal thymus and the development of thymomas and TSCC. Since increased expression of cFLIP is a known tumor escape mechanism, it may serve as tissue-based biomarker in future clinical trials, including immune checkpoint inhibitor trials in the commonly PD-L1high thymomas and TCs.

Crystal Violet Assay for Determining Viability of Cultured Cells.

Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.

Techniques to Distinguish Apoptosis from Necroptosis.

The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis.

Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation.

The biochemical signaling of cell death pathways is executed at a number of different intracellular and/or membrane-bound high-molecular mass complexes. It is crucial to be able to detect the formation, differences in assembly, and differential composition of such complexes to understand their contribution to the execution phase of apoptotic or necroptotic cell death. We describe here the use of caspase-8 coimmunoprecipitation in the spontaneously transformed keratinocyte cell line, HaCaT, to study the formation and composition of the Ripoptosome, a complex that is based on the serine-threonine kinase receptor-interacting protein 1 (RIPK1). However, the method can be adapted for use with other antibodies and cell lines. This protocol determines whether cells form the Ripoptosome complex, which is important for both apoptosis and necroptosis execution. Caspase-8 is an indispensible Ripoptosome component; therefore, caspase-8 antibodies are used to pull down the respective complex. However, the method cannot discriminate whether this complex triggers apoptosis (through the RIPK1 → FADD → caspase-8 activation pathway), necroptosis (through the RIPK1 → RIPK3 → MLKL activation pathway) or nondeath signaling. The actual signaling output (death or nondeath signaling) depends on the stoichiometry of the respective molecules as well as on the activity of FLIP, caspase-8, or other factors.

Characterization of the ripoptosome and its components: implications for anti-inflammatory and cancer therapy.

Most intracellular signaling cascades rely on the formation of multiprotein signaling complexes assembled in large protein signaling platforms. Especially in cell death signaling, there is a large variety of these complexes, including the apoptosome, the necrosome, or the death-inducing signaling complex (DISC), to name only a few. During the last years, a number of cellular conditions were identified that lead to the formation of another signaling platform, the so-called ripoptosome. Diverse stimuli such as genotoxic stress, death receptor or Toll-like-receptor (TLR) ligation, or degradation of cellular inhibitor of apoptosis proteins (cIAPs) are able to induce ripoptosome formation. The ripoptosome is tightly regulated by cIAPs that control intracellular RIP1 assembly and the association with other cell death-regulating proteins, most likely by ubiquitin linkage. The suppression of cIAP activity results in accumulation of RIP1 platforms that ultimately triggers necroptosis by activation of RIP3-MLKL-dependent necrosis signaling pathways. The ripoptosome is a 2-MDa protein complex, which consists of the core components caspase-8, FADD, different cFLIP isoforms, and RIP1. It represents one of the rheostats in cell death signaling, as it can activate apoptotic and necroptotic cell death responses. The specific formation and activation of the ripoptosome in cancer but not in primary cells suggests that this complex is a potential novel target for cancer or anti-inflammatory therapy, as suggested by the potential proinflammatory effects of necroptosis. Therefore, the better understanding and characterization of this signaling platform is of enormous importance for the development of novel cancer therapeutics. In this chapter, we describe several methods for purification and investigation of the ripoptosome in human cells. We also describe methods for monitoring apoptotic as well as necroptotic cell death.

On the TRAIL to overcome BRAF-inhibitor resistance.

BRAF inhibition has been an instant, although short-lasting, success in BRAF-mutated melanoma treatment. Novel data by Berger et al. now suggest that BRAF-inhibitor-mediated "priming to death" facilitates tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis. We give an overview about the importance of the crosstalk of extrinsic and mitochondrial apoptotic signaling and propose other combination therapies that may prevent or overcome secondary resistance in melanoma.

cFLIP regulates skin homeostasis and protects against TNF-induced keratinocyte apoptosis.

FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.

Cell death in the skin: how to study its quality and quantity?

The characterization of the quality and quantity of cell death has gained substantial interest over the past decades. More recently necroptosis as a programmed form of necrosis has been identified as an important additional form of cell death with relevance in the skin. Understanding how to assay cell death in specific is of critical importance for cancer research and treatment. Here we describe six different methods that can be used to assay cell viability and to study the quality or quantity of cultured human keratinocytes in vitro. These methods include crystal violet assay, hypodiploidy analysis, caspase-8 cleavage, release of HMGB1, annexin V/propidium iodide co-staining, and Hoechst/SYTOX green co-staining.

Pick your poison: the Ripoptosome, a cell death platform regulating apoptosis and necroptosis.

At an unbelievable pace, recent evidence has emerged that demonstrates the importance of a programmed form of necrosis (necroptosis) in physiology, pathophysiology and embryonic development. It is clear that the understanding of the intracellular control of necroptosis as compared to caspase-dependent apoptosis is of paramount importance. Tumorigenesis, immune surveillance of cancer and pathogen-induced disease, to name only a few, appear to be affected by the mode of cell death in vivo. Here, we discuss the Ripoptosome, a newly defined 2 MDa intracellular signalling complex that can be formed upon genotoxic stress or loss of inhibitor-of apoptosis proteins (IAPs). The Ripoptosome is a signaling platform that can switch modes between apoptotic and necroptotic cell death. In this report, we extend our recent studies and further the notion that the stoichiometric balance between RIP1 and cIAPs is critical for Ripoptosome formation. Furthermore, we demonstrate the critical relevance of the balance of expression levels of short (cFLIPS) or viral (vFLIP) forms of FLIP and RIP3 kinase for the spontaneous execution of necroptosis whenever cIAPs are absent in the cells. Our study thus supports and extends the intriguing role of the Ripoptosome for the regulation of apoptosis and necroptosis.

cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms.

The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate "Ripoptosome" is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIP(L) prevents Ripoptosome formation, whereas, intriguingly, cFLIP(S) promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the "rheostat" that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.

Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.

The contact allergen nickel sensitizes primary human endothelial cells and keratinocytes to TRAIL-mediated apoptosis.

Primary endothelial cells are fully resistant to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Here, we demonstrate that certain environmental conditions, such as exposure to the widespread allergen nickel, can dramatically increase the susceptibility of naturally resistant primary endothelial cells or keratinocytes to TRAIL-induced apoptosis. While nickel treatment increased surface expression of the apoptosis-inducing TRAIL receptors TRAIL-R1 and TRAIL-R2, it also up-regulated the apoptosis-deficient TRAIL-R4, suggesting that modulation of TRAIL receptor expression alone is unlikely to fully account for the dramatic sensitization effect of nickel. Further analysis of candidate mediators revealed that nickel strongly repressed c-FLIP at mRNA and protein levels. Accordingly, increased activation of Caspase-8 and Caspase-3 following nickel treatment was observed. Importantly, depletion of c-FLIP by RNA interference could largely recapitulate the effect of nickel and sensitize endothelial cells to TRAIL-dependent apoptosis in the absence of nickel pre-treatment. Conversely, ectopic expression of c-FLIP(L) largely protected nickel-treated cells from TRAIL-mediated apoptosis. Our data demonstrate that one key mechanism of sensitization of primary human endothelial cells or keratinocytes is transcriptional down-regulation of c-FLIP. We hypothesize that environmental factors, exemplified by the contact allergen nickel, strongly modulate death ligand sensitivity of endothelial cells and keratinocytes thus influencing vascular and epidermal function and integrity under physiological and pathophysiological conditions.

Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment.

A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP(L) and not cFLIP(S) interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.

FLIP ing the coin? Death receptor-mediated signals during skin tumorigenesis.

Keratinocyte skin cancer is a multi-step process, during which a number of obstacles have to be overcome by the tumor cell to allow the development of a manifest tumor. Beside proliferation and immortality, apoptosis resistance is one additional and critical step during skin carcinogenesis. Over the past two decades, much has been learned about the prototypical membrane-bound inducers of apoptosis, namely the death receptors and their ligands, and the apoptosis signalling pathways activated by death receptors have been elucidated in great detail. In contrast, much less is known about the tissue-specific role of the death receptor/ligands systems during the development of skin cancer. Here, we summarize and discuss the role of this intriguing receptor family and the potential mechanistical impact of the intracellular caspase-8 inhibitor cFLIP for keratinocyte skin cancer. Given more recent data about cFLIP and its isoforms, a more complex regulatory role of cFLIP can be suspected. Indeed, cFLIP may not solely interfere with death receptor-mediated apoptosis signalling pathways, but may positively or negatively influence other, potential harmful signalling pathways such as the production of inflammatory cytokines, tumor cell migration or the activation of transcription factors such as NF-kappaB, considered crucial during skin tumorigenesis. In this respect, cFLIP may act to 'FLIP the coin' during the development of keratinocyte skin cancer.

NF-kappaB inhibition reveals differential mechanisms of TNF versus TRAIL-induced apoptosis upstream or at the level of caspase-8 activation independent of cIAP2.

Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-kappaB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-kappaB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-kappaB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-kappaB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-kappaB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-kappaB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.

Modulation of T cell development and activation by novel members of the Schlafen (slfn) gene family harbouring an RNA helicase-like motif.

The regulatory networks governing development and differentiation of hematopoietic cells are incompletely understood. Members of the Schlafen (Slfn) protein family have been implicated in the regulation of cell growth and T cell development. We have identified and chromosomally mapped four new members, slfn5, slfn8, slfn9 and slfn10, which belong to a distinct subgroup within this gene family. The characteristic feature of these proteins is the presence of sequence motifs identifying them as distinct members of the superfamily I of DNA/RNA helicases. A significant role of these newly identified members in hematopoietic cell differentiation is suggested based on their differential regulation (i) in developing and activated T cells, (ii) in LPS or IFNgamma activated macrophages, (iii) upon IL6 or LIF driven terminal differentiation of myeloblastic M1 cells into macrophage-like cells, and (iv) in splenocytes of mice infected with Listeria monocytogenes. In contrast to wild-type cells, IRF-1 and IFNalpha/betaR deficient macrophages, although undergoing growth arrest, fail to upregulate slfn gene expression upon IFNgamma or LPS stimulation, respectively. Therefore, an essential participation in IFNgamma or LPS induced growth arrest appears unlikely. Likewise, ectopic expression of the newly identified slfn family members in fibroblasts did not reveal a general impact on growth control. In contrast, transgenic T-cell specific expression of a representative member of this new subfamily, slfn8, resulted in profoundly impaired T cell development and peripheral T cells showed a reduced proliferative potential. Thus, functional participation of slfn8 in the regulatory networks governing T cell development and growth appears to be cell type specific.