Crystallization and preliminary X-ray crystallographic analysis of human autotaxin.

Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Šresolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, ß = 122.6°.

Silencing of autocrine motility factor induces mesenchymal-to-epithelial transition and suppression of osteosarcoma pulmonary metastasis.

Phosphoglucose isomerase (PGI) is a multifunctional enzyme that functions in glucose metabolism as a glycolytic enzyme catalyzing an interconversion between glucose and fructose inside the cell, while it acts as cytokine outside the cell, with properties that include autocrine motility factor (AMF)-regulating tumor cell motility. Overexpression of AMF/PGI induces epithelial-to-mesenchymal transition with enhanced malignancy. Recent studies have revealed that silencing of AMF/PGI resulted in mesenchymal-to-epithelial transition (MET) of human lung fibrosarcoma cells and breast cancer cells with reduced malignancy. Here, we constructed a hammerhead ribozyme specific against GUC triplet at the position G390 in the human, mouse, and rat AMF/PGI mRNA sequence. Mesenchymal human osteosarcoma MG-63, HS-Os-1, and murine LM8 cells were stably transfected with the ribozyme specific for AMF/PGI. The stable transfectant cells showed effective downregulation of AMF/PGI expression and subsequent abrogation of AMF/PGI secretion, which resulted in morphologic change with reduced growth, motility, and invasion. Silencing of AMF/PGI induced MET, in which upregulation of E-cadherin and cytokeratins, as well as downregulation of vimentin, were noted. The MET guided by AMF/PGI gene silencing induced osteosarcoma MG-63 to terminally differentiate into mature osteoblasts. Furthermore, MET completely suppressed the tumor growth and pulmonary metastasis of LM8 cells in nude mice. Thus, acquisition of malignancy might be completed in part by upregulation of AMF/PGI, and waiver of malignancy might also be controlled by downregulation of AMF/PGI.

Crystallization and preliminary X-ray crystallographic study of phosphoglucose isomerase from Plasmodium falciparum.

Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 A resolution were collected from an orthorhombic crystal form belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 A. Structural analysis by molecular replacement is in progress.

Autotaxin promotes the expression of matrix metalloproteinase-3 via activation of the MAPK cascade in human fibrosarcoma HT-1080 cells.

Autotaxin (ATX) is an approximately 125-kDa transmembrane protein that is considered to be a tumor progression factor based on its lysophospholipase D activity. Here, we report that lysophosphatidic acid produced by ATX promotes the secretion of matrix metalloproteinase-3 (MMP3) from the human fibrosarcoma cell line HT-1080. The c-Jun N-terminal kinases (JNKs) and c-Jun of HT-1080 cells were rapidly phosphorylated after ATX treatment. A specific JNK inhibitor also exhibited this activation of signaling molecules and MMP3 expression. The present results suggest a novel function of ATX in promoting MMP3 production via the mitogen-activated protein kinase cascade, thereby stimulating tumor cell invasiveness.

Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway.

The autocrine motility factor (AMF) is a multifunctional protein that is involved in tumor progression including enhanced invasiveness via induction of matrix metalloproteinase-3 (MMP3). The increase in MMP3 was found in an AMF-high production tumor cell line, and c-Jun, c-Fos and mitogen-activated protein kinases (MAPKs) were also highly phosphorylated compared with the parent line. AMF stimulation induced the rapid phosphorylation of the cellular MAPK cascade and MMP3 secretion, which was blocked using a specific MAPK inhibitor. Results of this study suggest that AMF stimulation stimulates MMP3 expression via a MAPK signaling pathway.

Scalable purification and characterization of the extracellular domain of human autotaxin from prokaryotic cells.

Autotaxin (ATX) is an approximately 125kDa transmembrane protein known as a tumor progression factor based on its lysophospholipase D (lysoPLD) activity. There are many reports of the biological and biochemical properties of ATX, but crystallographic or structural studies have not been reported because a large-scale production process using prokaryotic cells has not been established. Here we report a bulk purification process and soluble expression of the recombinant human ATX (rhATX S48) from prokaryotic cells. The extracellular domain of human ATX cDNA was cloned into a pET101/D-TOPO vector and transformed to an Escherichia coliBL21 strain which was co-transformed with a pTF16 chaperone plasmid. The rhATX S48 was purified with chaperone and it was removed by Mg(2+)-ATP treatment. The final yield of purified rhATX S48 was approximately 3.5mg/l culture of recombinant strain. The rhATX S48 shows lysoPLD enzymatic activity and effectively stimulates the growth and motile activity of the human tumor cells as well as native ATX. This is a first report for scalable purification of the ATX molecule and the rhATX S48 should be a good tool for immunization of anti-ATX or crystallographic analysis of ATX.

AMF/G6PI induces differentiation of leukemic cells via an unknown receptor that differs from gp78.

Autocrine Motility Factor (AMF)/maturation factor (MF)/neuroleukin (NLK) is a multifunctional protein, which acts as a glucose 6-phosphate isomerase (G6PI) intracellularly. Exto-G6PI stimulates invasion and metastasis of tumor cells, neurotropic growth and differentiation of leukemic cells. The cell motility and proliferation receptor is known to be gp78 (78 kilo-Dalton glycoprotein), which has seven transmembrane domains in its N-terminal region, but the maturation factor receptor remains unclear. The human acute monocytic leukemia line does not express gp78 and its motile activity is not enhanced by AMF though it is well differentiated by AMF exposure. The forced expression of gp78 in leukemic cells recovered acceptable motile stimulation, concomitant with reduced differentiation ability. Two unknown proteins were detected by crosslinking between AMF and leukemic cells. The results of this report suggest that the receptor molecule for AMF/NLK/MF in leukemic differentiation is not gp78.

The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF.

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.

Crystal structures of mouse autocrine motility factor in complex with carbohydrate phosphate inhibitors provide insight into structure-activity relationship of the inhibitors.

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is identical to the extracellular cytokines neuroleukin and maturation factor and, interestingly, to the intracellular enzyme phosphoglucose isomerase. The cytokine activity of AMF is inhibited by carbohydrate phosphate compounds as they compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven transmembrane helix protein. Here, we report the first comprehensive high-resolution crystal structure analyses of the inhibitor-free form and the eight types of inhibitor (phosphate, erythrose 4-phosphate (E4P), arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P), 6-phosphogluconic acid (6PGA), fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), or mannose 6-phosphate (M6P)) complexes of mouse AMF (mAMF). We assayed the inhibitory activities of these inhibitors against the cytokine activity of mAMF. The inhibitory activities of the six-carbon sugars (G6P, F6P, M6P, and 6PGA) were found to be significantly higher than those of the four or five-carbon sugars (E4P or A5P). The inhibitory activities clearly depend on the length of the inhibitor molecules. A structural comparison revealed that a water-mediated hydrogen bond between one end of the inhibitor and a rigid portion of the protein surface in the shorter-chain inhibitor (E4P) complex is replaced by a direct hydrogen bond in the longer-chain inhibitor (6PGA) complex. Thus, to obtain a new compound with higher inhibitory activities against AMF, water molecules at the inhibitor binding site of AMF should be replaced by a functional group of inhibitors in order to introduce direct interactions with the protein surface. The present structure-activity relationship studies will be valuable not only for designing more effective AMF inhibitors but also for studying general protein-inhibitor interactions.

Hypoxia-inducible factor-1 drives the motility of the erythroid progenitor cell line, UT-7/Epo, via autocrine motility factor.

OBJECTIVE: It is well known that hypoxic stress strongly enhances erythropoiesis, but the effect of hypoxia on erythroid progenitors has not been examined precisely. In the present study, using the erythropoietin-dependent cell line UT-7/Epo, which has characteristics of erythroid progenitors, we investigated a novel role of hypoxia in erythropoiesis. METHODS: UT-7/Epo and four other hematopoietic and lymphoid cell lines (HL-60, THP-1, Raji, and CEM) were cultured in 20%, 5%, or 1% O2. Morphology was observed under a phase-contrast microscope. Cell motility was evaluated using the Transwell migration assay. An analysis of the protein level of hypoxia-inducible factor-1 (HIF-1) alpha and autocrine motility factor (AMF) was conducted using Western blotting and immunocytochemistry, respectively. Reverse transcription polymerase chain reaction was performed to evaluate the expression of AMF mRNA. Human bone marrow stromal cells were used in cocultures with UT-7/Epo. Apoptosis of UT-7/Epo was examined by immunocytochemistry using an antiactive form of caspase 3 antibody. RESULTS: Among the five cell lines, UT-7/Epo exhibited active pseudopodial extension in hypoxia (1% O2), and cell motility was increased. HL-60, THP-1, Raji, and CEM did not show an increase in cell motility even in 1% O2. In addition, expression of the alpha-subunit of HIF-1 was activated by hypoxia, and expression of the mRNA and protein of AMF induced by HIF-1, increasing cell motility, was promoted. The addition of an HIF-1 inhibitor, cadmium chloride (CdCl2), or alpha-ketoglutarate (2-oxoglutarate) decreased the AMF mRNA expression, and an AMF inhibitor, erythrose 4-phosphate, decreased the cell motility. When UT-7/Epo was cocultured with human bone marrow-derived stromal cells that significantly inhibit the apoptosis of UT-7/Epo, the migration of UT-7/Epo under the stromal cells (pseudoemperipolesis) was increased in hypoxia. CONCLUSION: Under hypoxic conditions, erythroid progenitors may exhibit active migration in the bone marrow and the opportunity for contact with stromal cells increases, inhibiting apoptosis.

[Possibility that AMF will serve as a target molecule for the diagnosis and treatment of a metastatic neoplasm].

The Autocrine Motility Factor (AMF) identified as a tumor cell motile stimulation factor is a key molecule of invasion and metastasis. The AMF is also identified as neuroleukin (NLK) and maturation factor (MF) which are secreted phosphohexose isomerase (PHI, PGI) from anaplastic cells. Tumor AMF promotes cellular locomotion or invasion, and regulates tumor MMPs secretion or apoptotic resistance. The AMF was thought to be an autocrine factor as the name shows it, and it is peculiar to malignant cells. However we found paracrine effect of AMF against tumor surrounding host tissues. Especially, endothelial cells which are essential parts of tumor induced angiogenesis or ascites accumulation express the AMF-receptor and they responded to AMF stimulation. Metastasis is a most complicated biological phenomenon that a large number of molecules or factors induced by tumor and host are related, thus AMF is also unusual molecule reacting between tumor and host tissues, and therefore AMF should be a target of treatment or diagnosis of cancer.

Crystallization and preliminary X-ray crystallographic studies of mouse autocrine motility factor.

Mouse autocrine motility factor (mAMF), a tumour-secreted cytokine that stimulates cell migration in vitro and metastasis in vivo, has been crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 69.97, b = 115.88, c = 73.27 A, beta = 101.76 degrees . There are two subunits (one dimer) per asymmetric unit. Complexes with four-, five- and six-carbon carbohydrate phosphate inhibitors have also been crystallized. The crystals diffract to at least 1.8 A resolution and are suitable for X-ray structure analyses at high resolution.

Autocrine motility factor signaling induces tumor apoptotic resistance by regulations Apaf-1 and Caspase-9 apoptosome expression.

Autocrine motility factor (AMF) is a cytokine that regulates locomotion and metastasis of tumor cells. It is well known that expression levels of AMF secretion and its receptor (AMF R) are closely related to tumor malignancy and rheumatoid arthritis. We have established that AMF signaling induced anti-apoptotic activity and that human fibrosarcoma HT-1080 line that secreted high levels of AMF were resistant to drug-induced apoptosis. These cells did not express the apoptotic protease activating factor-1 (Apaf-1) and Caspase-9 genes that encode for the proteins that form the "apoptosome" complex. The disappearance of the Apaf-1 and Caspase-9 gene was recovered by a cellular signaling inhibitor of protein kinase C, phosphatidylinositol 3-phosphate kinase and mitogen-activated protein kinase of the in vitro cultured human fibrosarcoma HT-1080 line. Treatment with these inhibitors favored apoptotic cell death induced by anti-cancer drugs of the murine ascites Ehrlich line. Apoptotic resistance of tumor cells allows them to escape death from cancer chemotherapy, so an understanding of malignant anti-apoptotic activities is important. Antibodies against AMF induced Ehrlich ascites apoptosis in vitro, and effectively aided in vivo apoptosis induced by anti-cancer drugs. The results might indicate a novel route by which tumor cells protect themselves with products, such as AMF, and proliferate despite various stresses and chemical insults; AMF regulates expression of Apaf-1 and caspase-9 genes via a complex signaling pathway and indirectly regulates formation of the apoptosome.

Autocrine motility factor secreted by tumor cells upregulates vascular endothelial growth factor receptor (Flt-1) expression in endothelial cells.

The autocrine motility factor (AMF) is known as a cytokine regulating tumor cells motility via AMF receptor (AMFR) and promotes their metastasis. Recently, AMFRs have been found on the surface of host cells and it was showed that AMF possibly affects them. The signaling of AMF-AMFR in the host endothelial cells induces expression of a vascular endothelial growth factor receptor (VEGFR) Flt-1 and AMFR feedback that is regulated at the transcriptional level. AMF-exposure stimulated the Flt-1 expression on human umbilical vein endothelial cells (HUVECs) surface and this AMF-treated cells exhibited high-responsibility against VEGF. The protein kinase C (PKC) and phosphatidylinositol 3 kinase (PI3K) play an important role in this signal transduction. The findings of our study suggest the possibility of "tumor AMF-->host AMFR-->PKC, PI3K-->-->VEGFR or AMFR-->angiogenesis, metastasis" as a new signal cross talk between the tumor and the host.

Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid.

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.

Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies.

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical with the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, the intracellular enzyme phosphohexose isomerase (PHI). The crystal structures of the inhibitor-free open form and the inhibitor (erythrose 4-phosphate, E4P, a strong inhibitor of AMF's cytokine activity)-bound closed form of human AMF have been determined at 1.9 A and 2.4 A resolution, respectively. Upon E4P binding, local conformation changes (open to closed) occur around the inhibitor-binding site. The E4P-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PHIs. The present study shows clearly that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF and PHI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate group. One likely scenario is that the compound could compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven-transmembrane helix protein. The other scenario is that the local conformation changes upon inhibitor binding may affect the AMF-AMFR interactions. To examine roles of the residues in the inhibitor-binding site, two mutant AMFs were prepared. Replacements of His389, which is hydrogen-bonded to the hydroxyl group of E4P by Phe, and Thr215, which is hydrogen-bonded to the phosphate group of E4P by Asp, result in mutant AMFs that are impaired in cytokine activity. These results suggest a role for these amino acids in recognition of a carbohydrate moiety of the AMFR. Since the E4P is one of the smallest compounds having AMF inhibitor activity, knowledge of the present crystal structure would provide an insight into the lead compound design of more effective AMF inhibitors.