An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.

Inactivation of Interferon Receptor Promotes the Establishment of Immune Privileged Tumor Microenvironment.

Refractoriness of solid tumors, including colorectal cancers (CRCs), to immunotherapies is attributed to the immunosuppressive tumor microenvironment that protects malignant cells from cytotoxic T lymphocytes (CTLs). We found that downregulation of the type I interferon receptor chain IFNAR1 occurs in human CRC and mouse models of CRC. Downregulation of IFNAR1 in tumor stroma stimulated CRC development and growth, played a key role in formation of the immune-privileged niche, and predicted poor prognosis in human CRC patients. Genetic stabilization of IFNAR1 improved CTL survival and increased the efficacy of the chimeric antigen receptor T cell transfer and PD-1 inhibition. Likewise, pharmacologic stabilization of IFNAR1 suppressed tumor growth providing the rationale for upregulating IFNAR1 to improve anti-cancer therapies.

Type 1 interferons contribute to the clearance of senescent cell.

The major known function of cytokines that belong to type I interferons (IFN, including IFNα and IFNß) is to mount the defense against viruses. This function also protects the genetic information of host cells from alterations in the genome elicited by some of these viruses. Furthermore, recent studies demonstrated that IFN also restrict proliferation of damaged cells by inducing cell senescence. Here we investigated the subsequent role of IFN in elimination of the senescent cells. Our studies demonstrate that endogenous IFN produced by already senescent cells contribute to increased expression of the natural killer (NK) receptor ligands, including MIC-A and ULBP2. Furthermore, neutralization of endogenous IFN or genetic ablation of its receptor chain IFNAR1 compromises the recognition of senescent cells and their clearance in vitro and in vivo. We discuss the role of IFN in protecting the multi-cellular host from accumulation of damaged senescent cells and potential significance of this mechanism in human cancers.

DNA-damage-induced type I interferon promotes senescence and inhibits stem cell function.

Expression of type I interferons (IFNs) can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/ß-dependent manner, stimulates cell-autonomous IFN-ß expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-ß and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

Type I interferon controls propagation of long interspersed element-1.

Type I interferons (IFN) including IFNα and IFNß are critical for the cellular defense against viruses. Here we report that increased levels of IFNß were found in testes from mice deficient in MOV10L1, a germ cell-specific RNA helicase that plays a key role in limiting the propagation of retrotransposons including Long Interspersed Element-1 (LINE-1). Additional experiments revealed that activation of LINE-1 retrotransposons increases the expression of IFNß and of IFN-stimulated genes. Conversely, pretreatment of cells with IFN suppressed the replication of LINE-1. Furthermore, the efficacy of LINE-1 replication was increased in isogenic cell lines harboring inactivating mutations in diverse elements of the IFN signaling pathway. Knockdown of the IFN receptor chain IFNAR1 also stimulated LINE-1 propagation in vitro. Finally, a greater accumulation of LINE-1 was found in mice that lack IFNAR1 compared with wild type mice. We propose that LINE-1-induced IFN plays an important role in restricting LINE-1 propagation and discuss the putative role of IFN in preserving the genome stability.

Triggering ubiquitination of IFNAR1 protects tissues from inflammatory injury.

Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro. The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1(SA) knock-in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1(SA) mice (or their bone marrow-receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1(SA) mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation-induced tissue damage can be purposely mimicked for therapeutic benefits.

Eliminative signaling by Janus kinases: role in the downregulation of associated receptors.

Activation of cytokine receptor-associated Janus kinases (JAKs) mediates most, if not all, of the cellular responses to peptide hormones and cytokines. Consequently, JAKs play a paramount role in homeostasis and immunity. Members of this family of tyrosine kinases control the cytokine/hormone-induced alterations in cell gene expression program. This function is largely mediated through an ability to signal toward activation of the signal transducer and activator of transcription proteins (STAT), as well as toward some other pathways. Importantly, JAKs are also instrumental in tightly controlling the expression of associated cytokine and hormone receptors, and, accordingly, in regulating the cell sensitivity to these cytokines and hormones. This review highlights the enzymatic and non-enzymatic mechanisms of this regulation and discusses the importance of the ambidextrous nature of JAK as a key signaling node that integrates the combining functions of forward signaling and eliminative signaling. Attention to the latter aspect of JAK function may contribute to emancipating our approaches to the pharmacological modulation of JAKs.

A BRISC-SHMT complex deubiquitinates IFNAR1 and regulates interferon responses.

Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here, we identify serine hydroxymethyltransferase (SHMT) as a previously unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub-mediated internalization and lysosomal degradation. BRISC-deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a mechanism of DUB regulation and suggest a therapeutic use of BRISC inhibitors for treating pathophysiological processes driven by elevated IFN responses.

Protein tyrosine phosphatase 1B is a key regulator of IFNAR1 endocytosis and a target for antiviral therapies.

Type 1 interferons (IFN1) elicit antiviral defenses by activating the cognate receptor composed of IFN-α/ß receptor chain 1 (IFNAR1) and IFNAR2. Down-regulation of this receptor occurs through IFN1-stimulated IFNAR1 ubiquitination, which exposes a Y466-based linear endocytic motif within IFNAR1 to recruitment of the adaptin protein-2 complex (AP2) and ensuing receptor endocytosis. Paradoxically, IFN1-induced Janus kinase-mediated phosphorylation of Y466 is expected to decrease its affinity for AP2 and to inhibit the endocytic rate. To explain how IFN1 promotes Y466 phosphorylation yet stimulates IFNAR1 internalization, we proposed that the activity of a protein tyrosine phosphatase (PTP) is required to enable both events by dephosphorylating Y466. An RNAi-based screen identified PTP1B as a specific regulator of IFNAR1 endocytosis stimulated by IFN1, but not by ligand-independent inducers of IFNAR1 ubiquitination. PTP1B is a promising target for treatment of obesity and diabetes; numerous research programs are aimed at identification and characterization of clinically relevant inhibitors of PTP1B. PTP1B is capable of binding and dephosphorylating IFNAR1. Genetic or pharmacologic modulation of PTP1B activity regulated IFN1 signaling in a manner dependent on the integrity of Y466 within IFNAR1 in human cells. These effects were less evident in mouse cells whose IFNAR1 lacks an analogous motif. PTP1B inhibitors robustly augmented the antiviral effects of IFN1 against vesicular stomatitis and hepatitis C viruses in human cells and proved beneficial in feline stomatitis patients. The clinical significance of these findings in the context of using PTP1B inhibitors to increase the therapeutic efficacy of IFN against viral infections is discussed.

Vascular endothelial growth factor-induced elimination of the type 1 interferon receptor is required for efficient angiogenesis.

Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) and antagonized by type 1 interferons, including IFN-α/ß. On engaging their respective receptors (VEGFR2 and IFNAR), both stimuli activate protein kinase D2 (PKD2) and type 1 IFNs require PKD2 activation and recruitment to IFNAR1 to promote the phosphorylation-dependent ubiquitination, down-regulation, and degradation of the cognate receptor chain, IFNAR1. Data reveal that PKD2 activity is dispensable for VEGF-stimulated down-regulation of VEGFR2. Remarkably, VEGF treatment promotes the recruitment of PKD2 to IFNAR1 as well as ensuing phosphorylation, ubiquitination, and degradation of IFNAR1. In cells exposed to VEGF, phosphorylation-dependent degradation of IFNAR1 leads to an inhibition of type 1 IFN signaling and is required for efficient VEGF-stimulated angiogenesis. Importance of this mechanism for proangiogenic or antiangiogenic responses in cells exposed to counteracting stimuli and the potential medical significance of this regulation are discussed.

Pathogen recognition receptor signaling accelerates phosphorylation-dependent degradation of IFNAR1.

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity.

Mammalian casein kinase 1alpha and its leishmanial ortholog regulate stability of IFNAR1 and type I interferon signaling.

Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-alpha/beta signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1alpha (CK1alpha) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1alpha was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1alpha and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-alpha in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling.

Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway.

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.

Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2.

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.

Site-specific ubiquitination exposes a linear motif to promote interferon-alpha receptor endocytosis.

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)-clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-alpha/beta receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCF(betaTrcp) (Skp1-Cullin1-F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

p21 loss cooperates with INK4 inactivation facilitating immortalization and Bcl-2-mediated anchorage-independent growth of oncogene-transduced primary mouse fibroblasts.

The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21(Waf1/Cip1) in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15(INK4B)/p16(INK4A) that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-ras(V12)/c-myc-induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21(-/-); cdk4r24c MEFs expressing H-ras(V12) and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-ras(V12), and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-ras(V12)/c-myc-induced loss of contact inhibition. In addition, absence of pRb function renders H-ras(V12) + c-myc-transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein-deficient cells requires apoptotic suppression.