ANXA2 (annexin A2) is crucial to ATG7-mediated autophagy, leading to tumor aggressiveness in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is associated with a poor prognosis and metastatic growth. TNBC cells frequently undergo macroautophagy/autophagy, contributing to tumor progression and chemotherapeutic resistance. ANXA2 (annexin A2), a potential therapeutic target for TNBC, has been reported to stimulate autophagy. In this study, we investigated the role of ANXA2 in autophagic processes in TNBC cells. TNBC patients exhibited high levels of ANXA2, which correlated with poor outcomes. ANXA2 increased LC3B-II levels following bafilomycin A1 treatment and enhanced autophagic flux in TNBC cells. Notably, ANXA2 upregulated the phosphorylation of HSF1 (heat shock transcription factor 1), resulting in the transcriptional activation of ATG7 (autophagy related 7). The mechanistic target of rapamycin kinase complex 2 (MTORC2) played an important role in ANXA2-mediated ATG7 transcription by HSF1. MTORC2 did not affect the mRNA level of ANXA2, but it was involved in the protein stability of ANXA2. HSPA (heat shock protein family A (Hsp70)) was a potential interacting protein with ANXA2, which may protect ANXA2 from lysosomal proteolysis. ANXA2 knockdown significantly increased sensitivity to doxorubicin, the first-line chemotherapeutic regimen for TNBC treatment, suggesting that the inhibition of autophagy by ANXA2 knockdown may overcome doxorubicin resistance. In a TNBC xenograft mouse model, we demonstrated that ANXA2 knockdown combined with doxorubicin administration significantly inhibited tumor growth compared to doxorubicin treatment alone, offering a promising avenue to enhance the effectiveness of chemotherapy. In summary, our study elucidated the molecular mechanism by which ANXA2 modulates autophagy, suggesting a potential therapeutic approach for TNBC treatment.Abbreviation: ATG: autophagy related; ChIP: chromatin-immunoprecipitation; HBSS: Hanks' balanced salt solution; HSF1: heat shock transcription factor 1; MTOR: mechanistic target of rapamycin kinase; TNBC: triple-negative breast cancer; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation.

OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.

CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation.

The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.

CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factor stimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

STRAP positively regulates TLR3-triggered signaling pathway.

Toll-like receptor (TLR) signaling drives the innate immune response by activating nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF). We have previously shown that STRAP interacts with TAK1 and IKKα along with NF-κB subunit p65, leading to the activation of pro-inflammatory cytokines. However, the roles of STRAP in TRIF/TBK1-mediated TLR3 activation and the subsequent type I interferon (IFN) production are not fully elucidated. Here, we demonstrate that STRAP acts as a scaffold protein in TLR3-triggered signaling. STRAP strongly interacts with TBK1 and IRF3, which enhances IFN-ß production. As a consequence, STRAP knockdown reduces the level of both pro-inflammatory cytokine and IFN in TLR3 agonist-stimulated macrophages, whereas its overexpression significantly enhances production of these cytokines. Furthermore, the C-terminus of STRAP is essential for its functional activity in TLR3-mediated IL-6 and IFN-ß production. These data suggest that STRAP is a positive regulator of the TLR3-meditated NF-κB and IRF signaling pathway.

CNBP acts as a key transcriptional regulator of sustained expression of interleukin-6.

The transcription of inflammatory genes is an essential step in host defense activation. Here, we show that cellular nucleic acid-binding protein (CNBP) acts as a transcription regulator that is required for activating the innate immune response. We identified specific CNBP-binding motifs present in the promoter region of sustained inflammatory cytokines, thus, directly inducing the expression of target genes. In particular, lipopolysaccharide (LPS) induced cnbp expression through an NF-κB-dependent manner and a positive autoregulatory mechanism, which enables prolonged il-6 gene expression. This event depends strictly on LPS-induced CNBP nuclear translocation through phosphorylation-mediated dimerization. Consequently, cnbp-depleted zebrafish are highly susceptible to Shigella flexneri infection in vivo. Collectively, these observations identify CNBP as a key transcriptional regulator required for activating and maintaining the immune response.

STRAP Acts as a Scaffolding Protein in Controlling the TLR2/4 Signaling Pathway.

The WD40-repeat protein serine/threonine kinase receptor-associated protein (STRAP) is involved in the regulation of several biological processes, including cell proliferation and apoptosis, in response to various stresses. Here, we show that STRAP is a new scaffold protein that functions in Toll-like receptor (TLR)-mediated immune responses. STRAP specifically binds transforming growth factor ß-activated kinase 1 (TAK1) and IκB kinase alpha (IKKα) along with nuclear factor-κB (NF-κB) subunit p65, leading to enhanced association between TAK1, IKKα, and p65, and subsequent facilitation of p65 phosphorylation and nuclear translocation. Consequently, the depletion of STRAP severely impairs interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and IL-1ß production, whereas its overexpression causes a significant increase in the secretion of these pro-inflammatory cytokines by TLR2 or TLR4 agonist-stimulated macrophages. Notably, STRAP translocates to the nucleus and subsequently binds to NF-κB at later times after lipopolysaccharide (LPS) stimulation, resulting in prolonged IL-6 mRNA production. Moreover, the C-terminal region of STRAP is essential for its functional activity in facilitating IL-6 production. Collectively, these observations suggest that STRAP acts as a scaffold protein that positively contributes to innate host defenses against pathogen infections.

TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells.

Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.

Hyperproduction of IL-6 caused by aberrant TDP-43 overexpression in high-fat diet-induced obese mice.

Inclusion of Tat-activating regulatory DNA-binding protein-43 (TDP-43) due to hyperphosphorylation or hyperubiquitination is a cause of neurodegenerative disease. Cellular TDP-43 expression is tightly controlled through a negative feedback loop involving its mRNA. Recently, we reported that the TDP-43-mediated sub-nuclear body is an essential site of interleukin-6 (IL-6) pre-mRNA processing. Here we show that mice fed on a high-fat diet exhibit increased TDP-43 expression in the liver and adipose tissue with a prominent increase in IL-6. TDP-43 depletion in vivo reduces IL-6 production in the liver. Overexpression or depletion of TDP-43 in pre-adipose and adipose cells causes reciprocal alteration of IL-6 expression and RNA processing. Our findings provide evidence for a link between homeostasis of TDP-43 expression and the risk of developing obesity.

InSAC: A novel sub-nuclear body essential for Interleukin-6 and -10 RNA processing and stability.

Dysregulation of cytokine expression causes inflammatory diseases or chronic infection conditions. We have identified that Tat-activating regulatory DNA-binding protein-43 (TDP-43) is involved in cytokine RNA processing in order to promote an optimal immune response. The interaction of TDP-43 with spliceosomal components from the Cajal body leads to the formation of a novel sub-nuclear body called the Interleukin (IL)-6 and IL-10 Splicing Activating Compartment (InSAC). TDP-43 binds to the IL-6 and IL-10 RNAs in a sequence-dependent manner. In cell-based studies, we observed that lipopolysaccharide (LPS) stimulation induces the formation of the InSAC through TDP-43 ubiquitination, thereby influencing the processing and expression levels of IL-6 RNA. Moreover, TDP-43 knockdown in vivo results in a decrease in IL-6 production and its RNA splicing and stability. Thus, these findings demonstrate that the InSAC is linked to the activation and modulation of the immune response.

Identification of a subnuclear body involved in sequence-specific cytokine RNA processing.

Processing of interleukin RNAs must be tightly controlled during the immune response. Here we report that a subnuclear body called the interleukin-6 and -10 splicing activating compartment (InSAC) is a nuclear site of cytokine RNA production and stability. Tat-activating regulatory DNA-binding protein-43 (TDP-43) acts as an InSAC scaffold that selectively associates with IL-6 and IL-10 RNAs in a sequence-specific manner. TDP-43 also recruits key spliceosomal components from Cajal bodies. LPS induces posttranslational modifications of TDP-43; in particular, TDP-43 ubiquitination provides a driving force for InSAC formation. As a consequence, in vivo depletion of TDP-43 leads to a dramatic reduction in the RNA processing and the protein levels of IL-6 in serum. Collectively, our findings highlight the importance of TDP-43-mediated InSAC biogenesis in immune regulation.

Differential control of interleukin-6 mRNA levels by cellular distribution of YB-1.

Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1.

Granulin is a soluble cofactor for toll-like receptor 9 signaling.

Toll-like receptor (TLR) signaling plays a critical role in innate and adaptive immune responses and must be tightly controlled. TLR4 uses LPS binding protein, MD-2, and CD14 as accessories to respond to LPS. We therefore investigated the presence of an analagous soluble cofactor that might assist in the recruitment of CpG oligonucleotides (CpG-ODNs) to TLR9. We report the identification of granulin as an essential secreted cofactor that potentiates TLR9-driven responses to CpG-ODNs. Granulin, an unusual cysteine-rich protein, bound to CpG-ODNs and interacted with TLR9. Macrophages from granulin-deficient mice showed not only impaired delivery of CpG-ODNs to endolysosomal compartments, but also decreased interaction of TLR9 with CpG-ODNs. As a consequence, granulin-deficient macrophages showed reduced responses to stimulation with CpG-ODNs, a trait corrected by provision of exogenous granulin. Thus, we propose that granulin contributes to innate immunity as a critical soluble cofactor for TLR9 signaling.

The HCMV membrane glycoprotein US10 selectively targets HLA-G for degradation.

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, US10, expressed early in the replicative cycle of HCMV as part of the same cluster that encodes the known immunoevasins US2, US3, US6, and US11. We show that US10 down-regulates cell surface expression of HLA-G, but not that of classical class I MHC molecules. The unique and short cytoplasmic tail of HLA-G (RKKSSD) is essential in its role as a US10 substrate, and a tri-leucine motif in the cytoplasmic tail of US10 is responsible for down-regulation of HLA-G. Both the kinetics of HLA-G degradation and the mechanisms responsible appear to be distinct from those used by the US2 and US11 pathways, suggesting the existence of a third route of protein dislocation from the ER. We show that US10-mediated degradation of HLA-G interferes with HLA-G-mediated NK cell inhibition. Given the role of HLA-G in protecting the fetus from attack by the maternal immune system and in directing the differentiation of human dendritic cells to promote the evolution of regulatory T cells, HCMV likely targets the HLA-G-dependent axis of immune recognition no less efficiently than it interferes with classical class I MHC-restricted antigen presentation.

Redox-regulated export of the major histocompatibility complex class I-peptide complexes from the endoplasmic reticulum.

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.

Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9.

Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9(-/-) dendritic cells, restored CpG DNA-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.

Human cytomegalovirus UL18 utilizes US6 for evading the NK and T-cell responses.

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses.

Redox regulation facilitates optimal peptide selection by MHC class I during antigen processing.

Activated CD8(+) T cells discriminate infected and tumor cells from normal self by recognizing MHC class I-bound peptides on the surface of antigen-presenting cells. The mechanism by which MHC class I molecules select optimal peptides against a background of prevailing suboptimal peptides and in a considerably proteolytic ER environment remained unknown. Here, we identify protein disulfide isomerase (PDI), an enzyme critical to the formation of correct disulfide bonds in proteins, as a component of the peptide-loading complex. We show that PDI stabilizes a peptide-receptive site by regulating the oxidation state of the disulfide bond in the MHC peptide-binding groove, a function that is essential for selecting optimal peptides. Furthermore, we demonstrate that human cytomegalovirus US3 protein inhibits CD8(+) T cell recognition by mediating PDI degradation, verifying the functional relevance of PDI-catalyzed peptide editing in controlling intracellular pathogens. These results establish a link between thiol-based redox regulation and antigen processing.

Functional dissection of HCMV US11 in mediating the degradation of MHC class I molecules.

The human cytomegalovirus (HCMV) gene product US11 dislocates MHC I heavy chains from the endoplasmic reticulum (ER) and targets them for proteasomal degradation in the cytosol. To identify the structural and functional domains of US11 that mediate MHC class I molecule degradation, we constructed truncated mutants and chimeric proteins, and analyzed these to determine their intracellular localization and their ability to degrade MHC class I molecules. We found that only the luminal domain of US11 was essential to confer ER localization to the protein but that the ability to degrade MHC class I molecules required both the transmembrane domain and the luminal domain of US11. By analyzing a series of point mutants of the transmembrane domain, we were also able to identify Gln(192) and Gly(196) as being crucial for the functioning of US11, suggesting that these residues may play a critical role in interacting with the components of the protein degradation machinery.

Promyelocytic leukemia is a direct inhibitor of SAPK2/p38 mitogen-activated protein kinase.

The promyelocytic leukemia gene (PML) encodes a growth/tumor suppressor protein that is essential for the induction of apoptosis in response to various apoptotic signals. The mechanism by which PML plays a role in the regulation of cell death is still unknown. In the current study, we demonstrate that PML negatively regulated the SAPK2/p38 signaling pathway by sequestering p38 from its upstream kinases, MKK3, MKK4, and MKK6, whereas PML did not affect the SAPK1/c-Jun NH(2)-terminal kinase pathway. PML associated with p38 both in vitro and in vivo and the carboxyl terminus of PML mediated the interaction. In contrast to other studies of PML and PML-nuclear bodies (NB), our study shows that the formation of PML-NBs was not required for PML to suppress p38 activity because PML was still able to bind and inhibit p38 activity under the conditions in which PML-NBs were disrupted. In addition, we show that the promotion of Fas-induced cell death by PML correlated with the extent of p38 inhibition by PML, suggesting that PML might regulate apoptosis through manipulating SAPK2/p38 pathways. Our findings define a novel function of PML as a negative regulator of p38 kinase and provide further understanding on the mechanism of how PML induces multiple pathways of apoptosis.