NF-κB pathway controls mitochondrial dynamics

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-β, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.Mitochondria perform multiple functions that are critical to the maintenance of cellular homeostasis. Mitochondrial dysfunctions have been linked to the development of degenerative diseases and aging. Damaged mitochondria are removed by mitophagy, a process partially regulated by the PARK2-encoded E3 ubiquitin ligase (Parkin) in a PTEN-induced putative protein kinase 1 (PINK1)-dependent manner.^{1, 2, 3, 4} During mitophagy, the phosphorylation of mitofusin (Mfn) 2 by PINK1 has been suggested to induce the recruitment of Parkin to the mitochondria in cardiomyocytes.⁵ However, previous groups have shown that that Mfn 1 and 2 are dispensable for Parkin-dependent mitophagy in fibroblasts, whereas the Parkin-dependent degradation of these proteins may impair fusion of damaged mitochondria with the healthy network.^{6, 7, 8} PINK1 and Parkin thus act as a quality control machinery on the outer mitochondrial membrane (OMM) to preserve mitochondrial integrity through the ubiquitination of OMM proteins.^{9, 10} Moreover, through its E3 ubiquitin ligase activity,^{11, 12} Parkin was reported to bind to the linear ubiquitin chain assembly complex (LUBAC) and to increase the ubiquitination of NF-κB essential modulator (NEMO),¹³ a component of the classical NF-κB signaling pathway.¹⁴ Müller–Rischart et al. also proposed that Parkin positively regulates the expression of the mitochondrial guanosine triphosphatase Optic atrophy 1 protein (OPA1) through linear ubiquitination of NEMO.¹³ OPA1 is a regulator of mitochondrial inner membrane fusion and cristae remodeling.^{15, 16, 17} A defect in OPA1 expression is associated with mitochondrial network fragmentation and enhanced sensitivity of the cells to undergo apoptosis by promoting cytochrome c release from the mitochondria.^{18, 19, 20} Because NEMO-deficient mouse embryonic fibroblast (MEF) cells display a normal mitochondrial network morphology, we decided to re-examine the role of Parkin in regulating OPA1 expression through the NF-κB signaling pathway.     

cFLIP is critical for oligodendrocyte protection from inflammation

Neuroinflammation associated with degenerative central nervous system disease and injury frequently results in oligodendrocyte death. While promoting oligodendrocyte viability is a major therapeutic goal, little is known about protective signaling strategies. We report that in highly purified rat oligodendrocytes, interferon gamma (IFNγ) activates a signaling pathway that protects these cells from tumor necrosis factor alpha (TNFα)-induced cytotoxicity. IFNγ protection requires Jak (Janus kinase) activation, components of the integrated stress response and NF-κB activation. Although NF-κB activation also occurred transiently in the absence of IFNγ and presence of TNFα, this activation was not sufficient to prevent induction of the TNFα-responsive cell death pathway. Genetic inhibition of NF-κB translocation to the nucleus abrogated IFNγ-mediated protection and did not change the cell death induced by TNFα, suggesting that NF-κB activation via IFNγ induces a different set of responses than activation of NF-κB via TNFα. A promising candidate is the NF-κB target cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which is protease-deficient caspase homolog that inhibits caspase-3 activation. We show that IFNγ-mediated protection led to upregulation of cFLIP. Overexpression of cFLIP was sufficient for oligodendrocyte protection from TNFα and short hairpin RNA knockdown of cFLIP-abrogated IFNγ -mediated protection. To determine the relevance of our in vitro finding to the more complex in vivo situation, we determined the impact on oligodendrocyte death of regional cFLIP loss of function in a murine model of neuroinflammation. Our data show that downregulation of cFLIP during inflammation leads to death of oligodendrocytes and decrease of myelin in vivo. Taken together, we show that IFNγ-mediated induction of cFLIP expression provides a new mechanism by which this cytokine can protect oligodendrocytes from TNFα-induced cell death.Interferon gamma (IFN-γ), the only type-II class IFN, has a paradoxical role in modulating cell function. It is critical for innate and adaptive immunity, but has multiple other functions. In the central nervous system (CNS), IFNγ has contrasting effects on the oligodendrocyte progenitor cells (O-2A/OPCs) that generate myelin-producing oligodendrocytes. O-2A/OPCs show suppressed division when exposed to IFNγ.^{1, 2, 3} However, when O-2A/OPCs differentiate into oligodendrocytes, IFNγ becomes pro-apoptotic.^{4, 5, 6, 7} Although IFNγ has a critical role in the pathogenesis of immune-mediated demyelinating disease;^{8, 9} the response of committed oligodendrocytes to IFNγ is more complex. For example, tumor necrosis factor alpha (TNFα) can show enhanced cytotoxicity in oligodendrocytes and transformed human neural cell lines when co-exposed with IFNγ.^{3, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}In contrast with reported toxic effects of IFNγ on oligodendrocytes, other studies did not see negative effects on mature oligodendrocytes^{5, 9, 20} or saw protection of glial lineage cells. IFNγ protects the Oli-neu oligodendrocyte-like cell line from reactive oxygen and nitrogen species,²¹ and overexpression of IFNγ before the induction of experimental autoimmune encephalomyelitis (EAE) protected oligodendrocytes from immune-mediated damage.⁹ The mechanism of such protection remains elusive.We now report that IFNγ protects purified, committed oligodendrocytes from TNFα-mediated apoptosis via Janus kinase (Jak)-mediated activation of the stress kinase PKR (double-stranded RNA-dependent protein kinase) and NF-κB-induced expression of cFLIP (cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein), which inhibits caspase activation. Moreover, gain-of-function and loss-of-function experiments show that cFLIP is necessary and sufficient for oligodendrocyte protection from TNFα. These results demonstrate induction of cFLIP in a stress response and NF-κB-dependent manner, leading to inhibition of caspase-mediated apoptosis, and reveal an important role for cFLIP in oligodendrocyte protection in vivo.     

Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout

Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO^LPC-KO mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. To address potential functional redundancies between death receptors we generated and analysed NEMO^LPC-KO mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO^LPC-KO mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO^LPC-KO mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO^LPC-KO mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.Liver cancer is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide.^{1, 2} Liver cancer predominantly arises in the context of chronic inflammatory conditions, most notably in virus hepatitis (HBV and HCV).^{1, 2} Although infectious agents are the primary cause of liver cancer worldwide, the incidence in western countries is rising due to the increase in obesity and non-alcoholic steatohepatitis.³ The pathogenesis of hepatocellular carcinoma (HCC) is incompletely understood and it is plausible that the different underlying aetiologies determine a distinct context for liver carcinogenesis. However, the prevailing universal concept is that continuous liver parenchymal damage and hepatocyte cell death drive compensatory proliferation and within the context of a chronically inflamed liver tissue mutations and epigenetic changes accumulate eventually transforming hepatocytes into malignant cells. Therefore, understanding the tissue-intrinsic processes that determine cell death and chronic inflammation resulting in hepatocarcinogenesis is a critical need in order to design more effective therapeutic strategies.The nuclear factor κB (NF-κB) pathway is implicated in cancer development in particular in the context of chronic inflammation.^{4, 5} In relation to liver cancer, NF-κB signalling has been implicated in the pathogenesis of hepatitis, liver fibrosis, cirrhosis and HCC.^{6, 7} The IKK complex, composed of two catalytic subunits, IKK1/IKKα and IKK2/IKKβ, and a regulatory subunit termed NEMO/IKKγ, activates NF-κB by phosphorylating inhibitor of NF-κB (IκB) proteins targeting them for degradation by the proteasome and thus allowing the nuclear accumulation of NF-κB dimers.⁵ IKK2 is primarily responsible for targeting and degrading IκBα thus inducing canonical NF-κB activation, although the two kinases show some degree of functional redundancy in controlling canonical NF-κB signalling.^{5, 8} NEMO/IKKγ is indispensable for activation of canonical NF-κB signalling.^{9, 10, 11}NF-κB signalling was proposed to exhibit tumour promoter or tumour suppressor properties in different models of liver cancer. In the Mdr2^−/− mouse model of inflammation-driven liver carcinogenesis, NF-κB inhibition caused by transgenic IκBα super–repressor expression in hepatocytes inhibited HCC progression.¹² Moreover, hepatocyte-restricted ablation of IKK2 prevented hepatitis and liver tumorigenesis induced by overexpression of lymphotoxins α and β in hepatocytes.¹³ However, mice with hepatocyte-specific IKK2 ablation developed more tumours induced by a single injection of the chemical carcinogen diethylnitrosamine,¹⁴ revealing a tumour suppressor role of NF-κB in this context.Studies in mice lacking NEMO specifically in liver parenchymal cells (LPCs) further supported a tumour suppressor function of IKK/NF-κB signalling in liver cancer. NEMO^LPC-KO mice showed spontaneous hepatocyte apoptosis resulting in chronic steatohepatitis and the development of HCC by the age of 1 year.¹⁵ LPC-specific ablation of Fas-Associated with Death Domain (FADD or MORT1), an adapter protein essential for the recruitment of caspase-8 to the Death Inducing Signalling Complex and the induction of death receptor-mediated apoptosis,¹⁶ prevented both spontaneous and LPS-induced apoptosis of NEMO-deficient hepatocytes and the development of steatohepatitis.¹⁵ In addition, LPC-specific knockout of caspase-8 inhibited spontaneous hepatocyte apoptosis and HCC development in NEMO^LPC-KO mice, although it caused non-apoptotic hepatocyte death and cholestasis.¹⁷ Given the essential role of FADD and caspase-8 in mediating apoptosis downstream of death receptors,¹⁶ we hypothesized that death receptor-mediated apoptosis of NEMO-deficient hepatocytes drives the development of hepatitis and HCC in NEMO^LPC-KO mice. The three main death receptors of the TNF receptor superfamily that are capable of inducing caspase-8-mediated apoptosis are TNFR1, Fas/CD95 and TRAIL-R/DR5.¹⁶ To address the role of death receptor-induced apoptosis in triggering the spontaneous death of NEMO-deficient hepatocytes and the development of steatohepatitis and HCC, we generated and analysed NEMO^LPC-KO mice lacking TNFR1, Fas or TRAIL-R specifically in LPCs. Surprisingly, we found that LPC-specific knockout of each of the death receptors alone but also combined deficiency of TNFR1, Fas and TRAIL-R in LPCs did not prevent spontaneous hepatocyte apoptosis, hepatitis and HCC development in NEMO^LPC-KO mice. In addition, knockout of TNF in all cells also did not protect NEMO^LPC-KO mice from hepatocyte death, hepatitis and HCC. Collectively, these results demonstrate that TNFR1, Fas and TRAIL-R are not required for the development of chronic liver damage and HCC in NEMO^LPC-KO mice.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.