MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies. METHODS: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness. RESULTS: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs. CONCLUSIONS: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

Development of biomarkers for screening hepatocellular carcinoma using global data mining and multiple reaction monitoring.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases.

Tubulin beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex subunit 1 as serological diagnostic biomarkers of esophageal squamous cell carcinoma: a proteomics study.

Despite the major advances in diagnosis and treatment, esophageal squamous cell carcinoma (ESCC) remains a major life-threatening disease. Early diagnosis is critical for guiding the therapeutic management of ESCC. This case-control study focused on the proteomic analysis of serum of healthy volunteers and ESCC patients using the ClinProt profiling technology based on mass spectrometry. A total of 80 healthy volunteers and 119 ESCC patients were enrolled. We identified a pattern of proteins/peptides (including m/z 1867, 2700, and 2094) and differentiated ESCC patients from healthy volunteers with sensitivity and specificity close to 100%. Using mass spectrometry (LTQ orbitrap XL), tubulin beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex subunit 1 were identified as the three differentially expressed proteins/peptides in the patient serum. These three dysregulated proteins/peptides could be involved in the pathogenesis of ESCC and may serve as putative serological diagnostic biomarkers of ESCC. We suggest that further proteomics and multi-omics research are warranted to identify novel post-genomics diagnostics that can in the future pave the way for personalized medicine for patients with ESCC, a cancer for which we currently lack an integrated battery of diagnostics in the field of oncology.

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions.

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Novel anti-filamin-A antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma.

Identification of tumor-derived proteins in the circulation may allow for early detection of cancer and evaluation of therapeutic responses. To identify circulating tumor-derived proteins, mice were immunized with concentrated culture medium conditioned by human breast cancer cells. Antibodies generated by hybridomas were screened against conditioned media from both normal epithelial cells and tumor cells. Antibody selectively reacting with tumor cell-conditioned media was further characterized. This led to the development of a monoclonal antibody (Alper-p280) that reacts with a newly identified 280-kDa secreted variant of human filamin-A. Circulating filamin-A was detected in patient plasma samples using Alper-p280 in an ELISA assay. Human plasma samples from 134 patients with brain, breast, or ovarian cancer, 15 patients with active arthritis, and 76 healthy controls were analyzed. Filamin-A protein levels in human cell lines and tissues were analyzed by western blotting, immunohistochemistry, and electron and confocal microscopy. Circulating filamin-A was detected in the plasma of 109 of 143 patients with breast cancer and primary brain tumors. Plasma levels of filamin-A showed 89.5% sensitivity (95% confidence interval [CI] = 0.67% to 0.99%) and 97.8% specificity (95% CI = 0.88% to 0.99%) for glioblastoma at a cut-off of 21.0 ng/mL. Plasma levels of filamin-A (>36.0 ng/mL) had 96.7% sensitivity (95% CI = 0.80% to 0.99%) and 67.8% specificity (95% CI = 0.54% to 0.79%) for metastatic breast cancer. Filamin-A levels were increased in malignant breast or brain tissues, but not in normal control tissues. Filamin-A localized to lysosomes in MDA.MB.231 breast cancer cells, but not in normal human mammary epithelial cells, suggesting that filamin-A may undergo cancer-specific processing. Plasma filamin-A appears to be a specific and sensitive marker for patients with high-grade astrocytoma or metastatic breast cancer. Additional novel cancer biomarkers have been identified and are being developed alongside Alper-p280 for use in diagnosis of breast carcinoma and high-grade astrocytoma, and for use in the evaluation of therapeutic responses.

Profilin forms tetramers that bind to G-actin.

Profilin binds to G-actin and affects polymerization. However, regulation of profilin function is generally unknown and controversy exists regarding profilin effects on actin polymerization. Because protein-protein interactions are implicated in many cellular responses, human platelet profilin self-association and actin inter-action was examined. Silver stained SDS-PAGE of poly-l-proline/sepharose 4B column purified profilin revealed the presence of profilin (14.8 kD) and extraneous higher bands (primarily 30 kD and 58.5 kD). Re-electrophoretic analysis of gel electroelution purified profilin yielded predominantly 14.8 kD and 58.5 kD proteins. Rabbit IgG antibodies made against gel electroelution-purified profilin recognized all profilin sizes on immunoblots. Capillary electrophoresis of profilin in solution produced a single peak that resolved into three distinct peaks upon addition of reducing agent or high salt conditions. Further, G-actin did not bind to 14.8 kD profilin on immunoblot overlay assays, but surprisingly bound only to 58.5 kD profilin. The data indicate that monomeric profilin forms tetramers which are the relevant high affinity actin-binding form.

Purification and characterization of erythrocyte caldesmon. Hypothesis for an actin-linked regulation of a contractile activity in the red blood cell membrane.

We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.

Actin-binding protein is a component of bovine erythrocytes.

Actin-binding protein (ABP) is a well-characterized polypeptide capable of crosslinking filamentous actin. To date, this polypeptide has been shown to exist in a number of tissues and cultured cell lines. This report shows that by using a panel of three monoclonal antibodies for immunoblotting and immunofluorescence analysis, that ABP is present in bovine erythrocytes. Moreover, the data obtained suggest that this protein is a component of the erythrocyte membrane skeleton. Additionally, bovine erythrocyte ABP is shown to possess both an apparent molecular weight and an isoelectric point identical to that of bovine smooth muscle filamin, implying that these two polypeptides are identical.

Mechanism of the interaction of human platelet profilin with actin.

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.

[The locomotor-contractile system of thrombocytes--an optimal model for studying the physiological motor and contractile processes in the effector systems of the non-muscular cells]. / Oporno-sokratitel'naia sistema trombotsitov--optimal'naia model' dlia izucheniia fiziologicheskikh dvigatel'nykh i sokratitel'nykh protsessov v éffektornykh sistemakh nemyshechnykh kletok organizma.

The authors suggest a concept according to which the cytoskeleton and contractile proteins of platelets are the locomotor-contractile system playing a role of the effector apparatus of hemostatic function of these cells. Demonstrate the role of the contractile structures in leukocyte phagocytosis.

Enhancement of GTP gamma S-binding activity by cAMP-dependent phosphorylation of a filamin-like 250 kDa membrane protein in human platelets.

The treatment of human platelets with the dibutyryl cyclic AMP (dbcAMP) revealed the presence of a 250 kDa protein which enhanced its GTP-binding activity. This protein was purified from platelet membranes by successive chromatographies on DEAE-cellulose, Ultrogel AcA34, Mono Q, HCA-hydroxyapatite, and TSK-3000SW columns. The positive cross-reaction of the 250 kDa protein with the anti-filamin antibody indicated that this protein is filamin or very close to it. The GTP gamma S-binding activity of this protein, when phosphorylated with cyclic AMP-dependent protein kinase (A-kinase), showed an over tenfold increase, with the specific activity being 3.6 nmol/mg protein. Dephosphorylation of the phosphorylated protein with alkaline phosphatase reduced the GTP gamma S-binding activity to the control untreated level.

Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.

Reversible binding of actin to gelsolin and profilin in human platelet extracts.

This paper documents the reversible appearance of high-affinity complexes of profilin and gelsolin with actin in extracts of platelets undergoing activation and actin assembly. Sepharose beads coupled to either monoclonal anti-gelsolin antibodies or to polyproline were used to extract gelsolin and profilin, respectively, from EGTA-containing platelet extracts and determine the proportion of these molecules bound to actin with sufficient affinity to withstand dilution (high-affinity complexes). Resting platelets (incubated for 30 min at 37 degrees C after gel filtration) contained nearly no high-affinity actin/gelsolin or actin/profilin complexes. Thrombin, within seconds, caused quantitative conversion of platelet profilin and gelsolin to high-affinity complexes with actin, but these complexes were not present 5 min after stimulation. The calcium-dependent actin filament-severing activity of platelet extracts, a function of free gelsolin, fell in concert with the formation of EGTA-stable actin/gelsolin complexes, and rose when the adsorption experiments indicated that free gelsolin was restored. The dissociation of high-affinity complexes was temporally correlated with the accumulation of actin in the Triton-insoluble cytoskeleton.

(-)-Adrenaline-induced, calcium-dependent phosphorylation of proteins in human platelets.

In human platelets, adrenaline stimulated, approximately four-fold, as compared with controls, the phosphorylation of primarily two proteins of apparent molecular weights of 20,000 and 40,000, respectively. Maximum phosphorylation occurred after incubation for 1 min and was inhibited by the addition of either yohimbine, prostaglandin E1, or EGTA. Phosphorylation of the two proteins was accompanied by diacylglycerol formation. The (-)-adrenaline-induced phosphorylation of proteins corresponds to the activation of a calcium-dependent protein kinase partially purified by DEAE-cellulose and Sephadex G150 column chromatography. The enzymatic activity was modulated by addition of (-)-adrenaline and CaCl2, by diolein, and in the presence of membranes or phosphatidylinositol but not phosphatidylethanolamine and phosphatidylcholine. A phospholipid-dependent reaction appears to be involved in the molecular mechanism of action of adrenaline.

Hydrolysis of cytoskeletal proteins by the Ca2+-dependent protease during platelet activation.

During platelet stimulation, the cytosolic Ca2+ concentration increases to micromolar levels. One consequence of this increase is that the Ca2+-dependent protease within platelets is activated. Activation of the Ca2+-dependent protease results in hydrolysis of actin-binding protein and P235. Actin-binding protein and P235 can both affect the organization of actin, thus activation of the Ca2+-dependent protease may provide a regulatory mechanism by which stimulus-induced changes in the organization of actin filaments could be directed. Although both actin-binding protein and P235 affect actin polymerization, stimulus-induced actin polymerization occurs before hydrolysis of actin-binding protein or P235 can be detected, thus it seems unlikely that hydrolysis of these proteins affects actin polymerization. Actin-binding protein also cross-links actin filaments into networks, a function that is lost when it is hydrolyzed by the Ca2+-dependent protease. Thus, hydrolysis of actin-binding protein may result in disruption of the actin filament networks that form early during platelet activation and permit the reorganization of filaments into the bundles present at later stages of platelet activation.

The motor of leukocytes.

The movements of leukocytes involve extension, flow, and contraction of a margin of organelle-excluding cytoplasm. Actin is the principal structural component of this region. This paper reviews evidence that the expansion of cortical cytoplasm can result from the growth of actin polymers into an orthogonal network in which actin fibers branch perpendicularly under the influence of actin-binding protein. Flow occurs when actin filaments are disassembled and severed. The assembly and fragmentation of actin are regulated by actin-modulating proteins such as profilin, which sequesters actin monomers, acumentin, which binds to the slow-growing end of actin fibers, and gelsolin, a calcium-regulated protein that binds to the fast-growing end of actin polymers and severs actin filaments. Contraction of the actin network is caused by myosin, the assembly and activity of which are regulated by its state of phosphorylation, which is in turn controlled by phosphorylating and dephosphorylating enzymes and by calmodulin and calcium. Present information leads to the prediction that intracellular calcium gradients guide cytoplasmic movement and that the direction of actin assembly and therefore of cytoplasmic extension is toward regions of low cytoplasmic free calcium.