Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death ...signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.
D-type cyclins form complexes with cyclin-dependent kinases (CDK4/6) and promote cell cycle progression. Although cyclin D functions appear largely tissue specific, we demonstrate that cyclin D3 has ...unique functions in lymphocyte development and cannot be replaced by cyclin D2, which is also expressed during blood differentiation. We show that only combined deletion of p27Kip1 and retinoblastoma tumor suppressor (Rb) is sufficient to rescue the development of Ccnd3−/− thymocytes. Furthermore, we show that a small molecule targeting the kinase function of cyclin D3:CDK4/6 inhibits both cell cycle entry in human T cell acute lymphoblastic leukemia (T-ALL) and disease progression in animal models of T-ALL. These studies identify unique functions for cyclin D3:CDK4/6 complexes and suggest potential therapeutic protocols for this devastating blood tumor.
► Cyclin D2 expression cannot rescue the Ccnd3 deficiency-induced developmental defects ► Deletion of p27Kip1 and Rb restores cyclin D3-deficient lymphocyte cell development ► Cyclin D:CDK activity is a potential therapeutic target in T-ALL ► The PD-0332991 CDK4/6 inhibitor efficiently targets established T-ALL
The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis ...of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.
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► TAL1, GATA3, and RUNX1 form a positive interconnected autoregulatory loop ► TAL1 core regulatory circuit controls genes involved in T cell homeostasis ► The TAL1 complex recruits MYB in a positive feed-forward regulatory loop ► TRIB2 is oppositely regulated by TAL1 and E-proteins and is crucial for cell survival
Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, ...including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf−/− macrophages, a soluble TNF antagonist was not able to do so in Tnf+/+ macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.
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•TLR4 activates CASPASE-8 to remove CYLD and disable the TNF necroptosis pathway•Autocrine TNF induces necroptosis in a cell-autonomous manner•Priming of macrophages by type I IFN is needed for necroptosis induced by LPS or TNF•Type I IFN primes expression of TNFR2 and MLKL, which are needed to induce necroptosis
Legarda et al. find that LPS engages TLR4 in macrophages to directly activate TRIF- and CASPASE-8-dependent cleavage of CYLD, thus preventing necroptosis. Blocking CASPASE-8 activity prevents cleavage of CYLD, leading to necroptosis that is primed by type I IFN and mediated by autocrine TNF.
A number of pathogens cause host cell death upon infection, and Yersinia pestis , infamous for its role in large pandemics such as the “Black Death” in medieval Europe, induces considerable ...cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinase–caspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis , influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-β (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1β, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-κB; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1β and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1–caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-κB and inflammasome activation, and host resistance after Y. pestis infection.
The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis ...and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.
Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is recruited to the TNF receptor 1 to mediate proinflammatory signaling and to regulate TNF-induced cell death. RIPK1 deficiency results ...in postnatal lethality, but precisely why Ripk1 ⁻/⁻ mice die remains unclear. To identify the lineages and cell types that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Tamoxifen administration to adult RosaCreER ᵀ²Ripk1 ᶠˡ/ᶠˡ mice results in lethality caused by cell death in the intestinal and hematopoietic lineages. Similarly, Ripk1 deletion in cells of the hematopoietic lineage stimulates proinflammatory cytokine and chemokine production and hematopoietic cell death, resulting in bone marrow failure. The cell death reflected cell-intrinsic survival roles for RIPK1 in hematopoietic stem and progenitor cells, because Vav-iCre Ripk1 ᶠˡ/ᶠˡ fetal liver cells failed to reconstitute hematopoiesis in lethally irradiated recipients. We demonstrate that RIPK3 deficiency partially rescues hematopoiesis in Vav-iCre Ripk1 ᶠˡ/ᶠˡ mice, showing that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. However, the Vav-iCre Ripk1 ᶠˡ/ᶠˡ Ripk3 ⁻/⁻ progenitors remain TNF sensitive in vitro and fail to repopulate irradiated mice. These genetic studies reveal that hematopoietic RIPK1 deficiency triggers both apoptotic and necroptotic death that is partially prevented by RIPK3 deficiency. Therefore, RIPK1 regulates hematopoiesis and prevents inflammation by suppressing RIPK3 activation.
Significance Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is involved in TNF signaling and interacts with the related RIPK3 to regulate cell death and inflammation. RIPK1 has kinase-independent prosurvival and kinase-dependent prodeath functions. To identify the lineages that depend on RIPK1 for survival, we generated conditional Ripk1 mice. Acute Ripk1 deletion results in rapid death of the animal caused by extensive cell death in the intestinal and hematopoietic lineages. A hematopoietic RIPK1 deficiency stimulates proinflammatory cytokine/chemokine production and cell death, resulting in bone marrow failure. Hematopoietic failure is partially rescued by a RIPK3 deficiency, indicating that RIPK1-deficient hematopoietic cells undergo RIPK3-mediated necroptosis. These findings show that in the hematopoietic lineage RIPK1 suppresses RIPK3 activity and suggest that RIPK-dependent necroptosis may contribute to human bone marrow failure syndromes.
Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to ...activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.
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•RIPK1 and RIPK3 kinases promote TRIF-dependent cytokine production by LPS in vitro•RIPK1 and RIPK3 kinase-dependent inflammation is independent of cell death•Erk1/2 mediates RIPK1 and RIPK3 kinase dependent inflammation•RIPK1 kinase and RIPK3 are mediators of LPS-induced cytokine production in vivo
Kinase activities of RIPK1 and RIPK3 are critical for necroptotic cell death. Degterev and colleagues demonstrate that RIPK1 and RIPK3 kinases also direct inflammatory gene expression induced by Toll-like receptor 4 ligand, lipopolysaccharide, in vitro, and in vivo. This regulation is independent of necroptosis and requires Erk1/2, cFos, and NF-κB.
The protein-tyrosine phosphatase SHP-1 has critical roles in immune signalling, but how mutations in SHP-1 cause inflammatory disease in humans remains poorly defined. Mice homozygous for the ...Tyr208Asn amino acid substitution in the carboxy terminus of SHP-1 (referred to as Ptpn6(spin) mice) spontaneously develop a severe inflammatory syndrome that resembles neutrophilic dermatosis in humans and is characterized by persistent footpad swelling and suppurative inflammation. Here we report that receptor-interacting protein 1 (RIP1)-regulated interleukin (IL)-1α production by haematopoietic cells critically mediates chronic inflammatory disease in Ptpn6(spin) mice, whereas inflammasome signalling and IL-1β-mediated events are dispensable. IL-1α was also crucial for exacerbated inflammatory responses and unremitting tissue damage upon footpad microabrasion of Ptpn6(spin) mice. Notably, pharmacological and genetic blockade of the kinase RIP1 protected against wound-induced inflammation and tissue damage in Ptpn6(spin) mice, whereas RIP3 deletion failed to do so. Moreover, RIP1-mediated inflammatory cytokine production was attenuated by NF-κB and ERK inhibition. Together, our results indicate that wound-induced tissue damage and chronic inflammation in Ptpn6(spin) mice are critically dependent on RIP1-mediated IL-1α production, whereas inflammasome signalling and RIP3-mediated necroptosis are dispensable. Thus, we have unravelled a novel inflammatory circuit in which RIP1-mediated IL-1α secretion in response to deregulated SHP-1 activity triggers an inflammatory destructive disease that proceeds independently of inflammasomes and programmed necrosis.
Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent ...manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.
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