Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs.

The mechanisms whereby neutrophils respond differentially to live and dead organisms are unknown. We show here that neutrophils produce 5- to 30-fold higher levels of the Cxcl2 chemokine in response to live bacteria, compared with killed bacteria or isolated bacterial components, despite producing similar levels of Cxcl1 or pro-inflammatory cytokines. Secretion of high levels of Cxcl2, which potently activates neutrophils by an autocrine mechanism, requires three signals. The first two signals are provided by two different sets of signal peptides released by live bacteria, which selectively activate formylated peptide receptor 1 (Fpr1) and Fpr2, respectively. Signal 3 originates from Toll-like receptor activation by microbial components present in both live and killed bacteria. Mechanistically, these signaling pathways converge at the level of the p38 MAP kinase, leading to activation of the AP-1 transcription factor and to Cxcl2 induction. Collectively, our data demonstrate that the simultaneous presence of agonists for Fpr1, Fpr2, and Toll-like receptors represents a unique signature associated with viable bacteria, which is sensed by neutrophils and induces Cxcl2-dependent autocrine cell activation.

Constitutive MHC class I molecules negatively regulate TLR-triggered inflammatory responses via the Fps-SHP-2 pathway.

The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions. Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in crosstalk with TLR signaling via the Fps-SHP-2 pathway and control TLR-triggered innate inflammatory responses.

The FES Gene at the 15q26 Coronary-Artery-Disease Locus Inhibits Atherosclerosis.

BACKGROUND: Genome-wide association studies have discovered a link between genetic variants on human chromosome 15q26.1 and increased coronary artery disease (CAD) susceptibility; however, the underlying pathobiological mechanism is unclear. This genetic locus contains the FES (FES proto-oncogene, tyrosine kinase) gene encoding a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. We investigated the effect of the 15q26.1 variants on FES expression and whether FES plays a role in atherosclerosis. METHODS AND RESULTS: Analyses of isogenic monocytic cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression. Small-interfering-RNA-mediated knockdown of FES promoted migration of monocytes and vascular smooth muscle cells. A phosphoproteomics analysis showed that FES knockdown altered phosphorylation of a number of proteins known to regulate cell migration. Single-cell RNA-sequencing revealed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages, although several other cell types including smooth muscle cells also expressed FES. There was an association between the 15q26.1 CAD risk genotype and greater numbers of monocytes/macrophage in human atherosclerotic plaques. An animal model study demonstrated that Fes knockout increased atherosclerotic plaque size and within-plaque content of monocytes/macrophages and smooth muscle cells, in apolipoprotein E-deficient mice fed a high fat diet. CONCLUSIONS: We provide substantial evidence that the CAD risk variants at the 15q26.1 locus reduce FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages and smooth muscle cells. This study is the first demonstration that FES plays a protective role against atherosclerosis and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Epigenomic profiling of DNA methylation for hepatocellular carcinoma diagnosis and prognosis prediction.

BACKGROUND AND AIM: DNA hypermethylation has emerged as a novel molecular biomarker for the diagnosis and prognosis prediction of many cancers. We aimed to identify clinically useful biomarkers regulated by DNA methylation in hepatocellular carcinoma (HCC). METHODS: Genome-wide methylation analysis in HCCs and paired noncancerous tissues was performed using an Illumina Infinium HumanMethylation 450K BeadChip array. Methylation-specific polymerase chain reaction and pyrosequencing were used to validate the methylation status of selected genes in 100 paired HCCs and noncancerous samples. RESULTS: A total of 97 027 (20.0%) out of 485 577 CpG sites significantly were differed between HCC and noncancerous tissues. Among all the significant CpG sites, 48.8% are hypermethylated and 51.2% are hypomethylated in HCCs. Multiple signaling pathways (AMP-activated protein kinase, estrogen, and adipocytokine) involved in gene methylation were identified in HCC. FES was selected for further analysis based on its high level of methylation confirmed by polymerase chain reaction and pyrosequencing. The result showed that FES hypermethylation was correlated with tumor size (0.001), serum alpha fetoprotein (0.023), and tumor differentiation (0.006). FES protein was significantly downregulated in 51/100 (51%) HCCs, and 94.12% (48/51) of them were due to promoter hypermethylation. Both FES hypermethylation and protein downregulation were associated with the progression-free survival and overall survival of HCC patients. Overexpressed and knockdown of FES confirmed its inhibitory effect on the proliferation and migration of HCC cells. CONCLUSIONS: We identified many new differentially methylated CpGs in HCCs and demonstrate that FES functions as a tumor suppressor gene in HCC and its methylation status could be used as an indicator for prognosis of HCC.

A new signaling pathway (JAK-Fes-phospholipase D) that is enhanced in highly proliferative breast cancer cells.

The products of the oncogene Fes and JAK3 are tyrosine kinases, whose expressions are elevated in tumor growth, angiogenesis, and metastasis. Phosphatidic acid, as synthesized by phospholipase D (PLD), enhances cancer cell survival. We report a new signaling pathway that integrates the two kinases with the lipase. A new JAK3-Fes-PLD2 axis is responsible for the highly proliferative phenotype of MDA-MB-231 breast cancer cells. Conversely, this pathway is maintained at a low rate of expression and activity levels in untransformed cells such as MCF10A. We also deciphered the inter-regulation that exists between the two kinases (JAK3 and the oncogene Fes) and between these two kinases and the lipase (PLD2). Whereas JAK3 and Fes marginally activate PLD2 in non-transformed cells, these kinases greatly enhance (>200%) PLD activity following protein-protein interaction through the SH2 domain and the Tyr-415 residue of PLD2. We also found that phosphatidic acid enhances Fes activity in MDA-MB-231 cells providing a positive activation loop between Fes and PLD2. In summary, the JAK3, Fes and PLD2 interactions in transformed cells maintain PLD2 at an enhanced level that leads to abnormal cell growth. Modulating this new JAK3-Fes-PLD2 pathway could be important to control the highly invasive phenotype of breast cancer cells.

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action.

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.

MiR-424 regulates monocytic differentiation of human leukemia U937 cells by directly targeting CDX2.

MiR-424 plays an important role via promoting the monocytic differentiation in many human leukemia cell lines. Here, we report that miR-424 decreased miR-125b expression to 36 % by directly targeting caudal type homeobox 2. However, miR-424 also decreased expression of Fes, PU.1 and colony-stimulating factor receptor (MCSFR). As Fes, PU.1 and MCSFR were down-regulated by over-expression of miR-125b (unpublished work), a similar effect of miR-424 and Fes siRNA on CD64, Egr-1, Egr-2 and CEBPA indicates that Fes may be an important downstream target of miR-424. We hypothesize that miR-424 promotes monocytic differentiation by regulating other critical factors and miR-424 has high affinity for these factors. For the first time, the molecular mechanism of miR-424 during monocytic differentiation of U937 cells has been elucidated in this study.

[Detection of fps tumor antigen with mono-specific anti-fps serum in tumors induced by acute transforming ALV].

OBJECTIVE: To prepare anti-fps mono-specific serum, and detect the fps antigen in tumors induced by acute transforming avian leukosis/sarcoma virus containing v-fps oncogene. METHODS: Two part of v-fps gene was amplified by RT-PCR using the Fu-J viral RNA as the template. Mono-specific serum was prepared by immuning Kunming white mouse with both two recombinant infusion proteins expressed by the prokaryotic expression system. Indirect immunofluorescent assay was used to detect fps antigen in tumor tissue suspension cells and CEF infected by sarcoma supernatant. Immunohistochemical method was used to detect fps antigen in tumor tissue. RESULTS: The mouse mono-specific serum was specific as it had no cross reaction with classical ALV-J strains. The result reveals that the tumor tissue suspension cells, the CEF infected by sarcoma supernatant, and the slice immunohistochemistry of the sarcoma showed positive results. CONCLUSION: The anti-fps mono-specific serum was prepared, and the detection method was established, which laid the foundation for the study of viral biological characteristics and mechanism of tumourgenesis of acute transforming avian leukosis/sarcoma virus containing v-fps oncogene.

Pathological significance and predictive value for biochemical recurrence of c-Fes expression in prostate cancer.

BACKGROUND: c-Fes is a proto-oncogene encoded non-receptor protein-tyrosine kinase (PTK). However, genetic studies have indicated that it has anti-tumorigenic effects in certain cancers. The pathological and clinical significance of c-Fes in prostate cancer are unknown. METHODS: Expression of c-Fes was evaluated in normal glands, prostatic intraepithelial neoplasia (PIN), cancer cells in tissues of knock-in mouse adenocarcinoma prostate (KIMAP) model, and prostate cancer patients free of metastasis. Expression of c-Fes was analyzed by immunohistochemistry, and quantified by using the immunoreactivity score (IRS) (staining intensity × percentage of positive cells). Relationships between c-Fes expression and pT stage, Gleason's score (GS), and biochemical recurrence in patients who underwent radical surgery were also investigated. RESULTS: In KIMAP, the percentage in normal glands, PIN and cancer cells positive for c-Fes expression were 0 (0/7), 25.0 (2/8), and 100% (7/7), respectively. In human tissues, c-Fes expression was also significantly higher in cancer cells than in normal cells and PIN, and it correlated with pT stage (P < 0.001) and GS (P = 0.047). Multivariate analysis showed that c-Fes expression was an independent predictor of poor outcome poor prognosis (hazard ratio = 3.21, 95% confidence interval = 1.11-9.37, P = 0.032). CONCLUSION: The results suggested that c-Fes expression is a useful predictor of biochemical recurrence after radical surgery. The results also suggested that c-Fes is a potentially useful therapeutic target in prostate cancer and a predictor of biochemical recurrence after radical prostatectomy.

Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering.

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.

Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH.

Pulmonary arterial hypertension (PAH) is up to threefold more prevalent in women than men. Female mice overexpressing the serotonin transporter (SERT; SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH, whereas male SERT+ mice remain unaffected. To further investigate these sex differences, microarray analysis was performed in the pulmonary arteries of normoxic and chronically hypoxic female and male SERT+ mice. Quantitative RT-PCR analysis was employed for validation of the microarray data. In relevant groups, immunoblotting was performed for genes of interest (CEBPß, CYP1B1, and FOS). To translate clinical relevance to our findings, CEBPß, CYP1B1, and FOS mRNA and protein expression was assessed in pulmonary artery smooth muscle cells (PASMCs) derived from idiopathic PAH (IPAH) patients and controls. In female SERT+ mice, multiple pathways with relevance to PAH were altered. This was also observed in chronically hypoxic female SERT+ mice. We selected 10 genes of interest for qRT-PCR analysis (FOS, CEBPß, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1, and C1S), and 100% concordance was reported. Protein expression of three selected genes, CEBPß, CYP1B1, FOS, was also upregulated in female SERT+ mice. Serotonin and 17ß-estradiol increased CEBPß, CYP1B1, and FOS protein expression in PASMCs. In addition, CEBPß, CYP1B1, and FOS mRNA and protein expression was also increased in PASMCs derived from IPAH patients. Here, we have identified a number of genes that may predispose female SERT+ mice to PAH, and these findings may also be relevant to human PAH.

Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.

FES-Cre targets phosphatidylinositol glycan class A (PIGA) inactivation to hematopoietic stem cells in the bone marrow.

A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.

FES kinase participates in KIT-ligand induced chemotaxis.

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicated in cell migration.

fps/fes knockout mice display a lactation defect and the fps/fes tyrosine kinase is a component of E-cadherin-based adherens junctions in breast epithelial cells during lactation.

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase implicated in vesicular trafficking and cytokine and growth factor signaling in hematopoietic, neuronal, vascular endothelial and epithelial lineages. Genetic evidence has suggested a tumor suppressor role for Fps/Fes in breast and colon. Here we used fps/fes knockout mice to investigate potential roles for this kinase in development and function of the mammary gland. Fps/Fes expression was induced during pregnancy and lactation, and its kinase activity was dramatically enhanced. Milk protein and fat composition from nursing fps/fes-null mothers was normal; however, pups reared by them gained weight more slowly than pups reared by wild-type mothers. Fps/Fes displayed a predominantly dispersed punctate intracellular distribution which was consistent with vesicles within the luminal epithelial cells of lactating breast, while a small fraction co-localized with beta-catenin and E-cadherin on their basolateral surfaces. Fps/Fes was found to be a component of the E-cadherin adherens junction (AJ) complex; however, the phosphotyrosine status of beta-catenin and core AJ components in fps/fes-null breast tissue was unaltered, and epithelial cell AJs and gland morphology were intact. We conclude that Fps/Fes is not essential for the maintenance of epithelial cell AJs in the lactating breast but may instead play important roles in vesicular trafficking and milk secretion.

Promoter methylation blocks FES protein-tyrosine kinase gene expression in colorectal cancer.

The FES locus encodes a unique nonreceptor protein-tyrosine kinase (FES) traditionally viewed as a proto-oncogene but more recently implicated as a tumor suppressor in colorectal cancer (CRC). Recent studies have demonstrated that while FES is expressed in normal colonic epithelium, expression is lost in tumor tissue and colorectal cancer cell lines, a finding common among tumor suppressors. Here we provide compelling evidence that promoter methylation is an important mechanism responsible for downregulation of FES gene expression in colorectal cancer cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in the expression of functional FES transcripts in all CRC cell lines examined, including Caco-2, COLO 320, DLD-1, HCT 116, SNU-1040, SW-480, and HT-29. Bisulfite sequencing of genomic DNA isolated from 5-aza-2'-deoxycytidine-treated HT-29 cells identified methylated CpG dinucleotides immediately upstream from the FES transcription initiation sites. In contrast, this region of the FES promoter was hypomethylated in genomic DNA from normal colonic epithelium. In addition, methylation completely blocked the activity of the FES promoter in reporter gene assays. Promoter methylation is a previously unrecognized mechanism by which FES expression is suppressed in CRC cell lines, and is consistent with a tumor suppressor role for FES in this tumor site despite its tyrosine kinase activity.

Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis.

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.