Preparation of mesoporous silica nanocarriers targeting glucose-6-phosphate isomerase inhibition and application in the treatment of rheumatoid arthritis.

Glucose 6-phosphate isomerase (G6PI) is an indicator to assist in diagnosis of rheumatoid arthritis (RA) and monitor the disease. It also plays a key role in proliferating RA synovial tissues, pannus formation, and invasion and destruction of articular cartilage. In this study, we synthesized nanoparticles targeting G6PI (siG6PI-MSN) using mesoporous silica nanocarriers (MSN) and small interfering RNA (siRNA), followed by identifying the characteristics and functions, and preliminarily exploring their application in the treatment of RA in vivo with a type II collagen-induced arthritis (CIA) rat model. It showed that the synthetic functionalized carrier had a regular pore structure and a specific volume and surface area. No obvious hemolysis or toxicity of the carrier was found when its concentration was below 100 µg/ml. Cytological results in vitro suggested that siG6PI-MSN significantly inhibited G6PI expression and reduced the ability of proliferation, migration, and invasion of FLSs, compared with the siNC-MSN group. In vivo results in the CIA rat model showed that the arthritis index and degree of joint swelling among rats in the siG6PI-MSN-treatment group were significantly lower than those in the control group. Moreover, the number of FLSs in Synovium and the levels of TNF α and IL-1 ß were also significantly decreased in the siG6PI-MSN group. Histopathology of the synovial tissue and cartilage revealed siG6PI-MSN treatment significantly reduced the pathological manifestations of arthritis. In conclusion, siG6PI-MSN effectively suppresses the proliferation and invasive growth of synovial tissue and improve joint swelling and inflammatory infiltration, thereby preventing joint damage in RA. This carrier may be a new therapeutic measure for RA, with potential social and economic benefits.

The effect of autocrine motility factor alone and in combination with methyl jasmonate on liver cancer cell growth.

Neoplastic cells secrete autocrine motility factor (AMF) to stimulate the motility of cancer cells. In this study, AMF secreted from HT-29 colorectal cancer cells selectively suppressed liver cancer cells by downregulating pAKT and ß-catenin. In addition, HT-29 AMF significantly augmented the activity of methyl jasmonate against liver cancer cells and is a promising alternative for liver cancer therapy.

Characterization of an Expanded IL-10-Producing-Suppressive T Cell Population Associated with Immune Tolerance.

An increase in the number of glucocorticoid-induced tumor necrosis factor receptor-family related gene/protein (GITR)+CD25- (or fork-head box protein 3: Foxp3-) CD4+ T cells, after treating a mouse model of arthritis with fingolimod (FTY720), and a pathogenic antigen may play a key role in the establishment of immune tolerance. In this study, we characterized a specific expanded T cell subset in this population. Mice with glucose-6-phosphate isomerase peptide (GPI325-339)-induced arthritis were treated with FTY720 (1 mg/kg, per os) and GPI325-339 (10 µg/mouse, intravenously) for five days, starting from the onset of symptoms. The expanded GITR+CD25- (or Foxp3-) CD4+ T cell population and its cytokine production were examined using flow cytometry. Furthermore, time-dependent changes in T-bet and/or early growth response gene 2 (Egr-2) expression in this T cell subset were examined. The density of T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)+CD39+ cell subset in the GITR+Foxp3-CD4+ T cell population was significantly increased only in the combined treatment group, compared to that in the untreated and single-treatment groups. In the TIGIT+CD39+GITR+Foxp3-CD4+ T cell population, T-bet+Egr-2+/T-bet+Egr-2- cell ratio increased in the latter stage of the treatment. Furthermore, this T cell subset, which corresponded to a T helper 1 (Th1) response, produced high levels of both interleukin (IL)-10 and interferon (IFN)-γ. In conclusion, expanded TIGIT+CD39+GITR+Foxp3-CD4+ T cells shifted from an effector Th1 to IL-10-producing-suppressor T cell phenotype, which may promote an immune-tolerant state.

Autocrine motility factor promotes endometrial cancer progression by targeting GPER-1.

BACKGROUND: Autocrine motility factor (AMF) is a critical factor regulating aggressiveness of endometrial cancer (EC). Multiple pieces of evidence indicate that it is through G protein coupled estrogen receptor (GPER) signaling pathway that some growth factors promoted the migration and proliferation of tumor cells. The aim of this study is to explore the role of GPER-1 in AMF mediated regulatory mechanisms of EC recurrence and progression. METHODS: Real-Time Cell Analysis (RTCA) assays were performed to assess whether AMF depends on Autocrine motility factor recepter (AMFR) signaling in EC cells. A genome-wide expression microarray and Yeast Two-Hybrid assay were used to detect AMF and GPER-1 interaction in the context of AMFR depletion, and co-immunoprecipitation and immunofluorescence experiments were performed to confirm the physical interaction. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) analysis was used for the identification of the target pathway activated by AMF-GPER-1 interaction. Cohorts of mice harboring xenografts derived from modified SPEC2 cell lines were treated with or without exogenous AMF to validate the results of previous experiments. Immunohistochemistry was performed to assess AMF and GPER-1 expression in endometrial cancer specimens and normal endometrium. RESULTS: Our data showed that GPER-1 binds to AMF and the formed complex translocates from the plasma membrane to the cytoplasm. Mechanistic investigations demonstrated that interaction between AMF and GPER-1 triggers phosphoinositide-3-kinase signaling and promotes EC cell growth. More importantly, through animal experiments and human tissue experiments, we found that AMF contributes to GPER-1-mediated EC progression, which is consistent with the above observations. CONCLUSIONS: Our work not only delineated the regulatory mechanisms of endometrial cancer progression by AMF-GPER-1-AKT signaling cascade but also laid the foundation of targeting this pathway for treating endometrial cancer.

Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/µg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 µM; Ki = 36.33 µM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 µM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 µM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target.

Functional Mechanism(s) of the Inhibition of Disease Progression by Combination Treatment with Fingolimod Plus Pathogenic Antigen in a Glucose-6-phosphate Isomerase Peptide-Induced Arthritis Mouse Model.

We previously reported that combination treatment with fingolimod (FTY720) plus antigenic peptide of glucose-6-phosphate isomerase (residues 325-339) (GPI325-339) from the onset of symptoms significantly inhibited disease progression in a mouse model of GPI325-339-induced arthritis. In this study, we investigated the mechanism(s) involved. The model mice were treated from arthritis onset with FTY720 alone, GPI325-339 alone, or the combination of FTY720 plus GPI325-339. At the end of treatment, inguinal lymph nodes (LNs) were excised and examined histologically and in flow cytometry. Levels of apoptotic cells, programmed death-1-expressing CD4(+)forkhead box P3(-) nonregulatory T cells (non-Tregs), and cytotoxic T-lymphocyte antigen 4-expressing non-Tregs in inguinal LNs were markedly increased in the combination treatment group mice. Regulatory T cells (Tregs) were also increased. These results indicate that combination treatment with FTY720 plus GPI325-339 inhibits the progression of arthritis by inducing clonal deletion and anergy of pathogenic T cells and also by immune suppression via Tregs.

Increased migration of human mesenchymal stromal cells by autocrine motility factor (AMF) resulted in enhanced recruitment towards hepatocellular carcinoma.

BACKGROUND AND AIMS: Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC. METHODS: Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated. RESULTS: AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro. CONCLUSION: AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.

Autocrine motility factor injection for motor plate regeneration and muscle function restoration--a pilot study.

BACKGROUND: Autocrine motility factor (AMF) is a multifunctional cytokine that promotes cellular adhesion, proliferation, motility, anti-apoptosis, and tissue repair. Direct nerve implantation (DNI) is considered to be effective in peripheral motor nerve injuries with disuse of the distal nerve; however, the repaired muscle function is not satisfactory. In our study, purified AMF was injected in reinnervated muscle after DNI with the intention of assessing if AMF, as a malignant tumor-related cytokine, could improve motor plate regeneration and neuromuscular function restoration. METHODS: Purified AMF, which was extracted from AMF-transfected myoblast-conditioned medium, was regularly injected into the rat gastrocnemius in an established rat gastrocnemius denervation and reinnervation model. The nerve conduction velocity (NCV) of the tibial nerve, peak-to-peak value (PPV), area under the curve (AUC) of the compound muscle action potential (CMAP) and the Tibial Functional Index (TFI) were measured at 8, 16 and 24 weeks after injection. The regenerated endplates in gastrocnemius were examined by histochemical staining. In another group, an AMF-free solution was injected as the control. RESULTS: After the AMF injection, the direct-nerve-implanted muscle function recovery was better in terms of both the nerve velocity and the quality. The endplates in the experimental group also had a quantitative advantage in restoration. After comparing the histochemical-stained tissues, no indications of tumorigenesis were detected. CONCLUSIONS: AMF had positive effects on neuromuscular reparation and need more detailed research to determine the signalling pathways and side effects of AMF.

Autocrine motility factor promotes HER2 cleavage and signaling in breast cancer cells.

Trastuzumab (Herceptin) is an effective targeted therapy in HER2-overexpressing human breast carcinoma. However, many HER2-positive patients initially or eventually become resistant to this treatment, so elucidating mechanisms of trastuzumab resistance that emerge in breast carcinoma cells is clinically important. Here, we show that autocrine motility factor (AMF) binds to HER2 and induces cleavage to the ectodomain-deleted and constitutively active form p95HER2. Mechanistic investigations indicated that interaction of AMF with HER2 triggers HER2 phosphorylation and metalloprotease-mediated ectodomain shedding, activating phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase signaling and ablating the ability of trastuzumab to inhibit breast carcinoma cell growth. Furthermore, we found that HER2 expression and AMF secretion were inversely related in breast carcinoma cells. On the basis of this evidence that AMF may contribute to HER2-mediated breast cancer progression, our findings suggest that AMF-HER2 interaction might be a novel target for therapeutic management of patients with breast cancer, whose disease is resistant to trastuzumab.

Oxygen breathing affects 3'-deoxy-3'-18F-fluorothymidine uptake in mouse models of arthritis and cancer.

UNLABELLED: Noninvasive in vivo imaging of biologic processes using PET is an important tool in preclinical studies. We observed significant differences in 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) uptake in arthritic ankles and carcinomas between dynamic and static PET measurements when mice breathed oxygen. Thus, we suspected that air or oxygen breathing and the anesthesia protocol might influence (18)F-FLT tracer uptake. METHODS: We injected arthritic, healthy, and CT26 colon carcinoma-bearing mice with (18)F-FLT before static or dynamic small-animal PET measurements. The spontaneously oxygen- or air-breathing mice were kept conscious or anesthetized with ketamine and xylazine during (18)F-FLT uptake before the 10-min static PET measurements. For dynamic PET scans, mice were anesthetized during the entire measurement. (18)F-FLT uptake was reported in percentage injected dose per cubed centimeter by drawing regions of interest around ankles, carcinomas, and muscle tissue. Additionally, venous blood samples were collected before (18)F-FLT injection and after PET measurement to analyze pH, carbon dioxide partial pressure (pCO(2)), and lactate values. RESULTS: A significantly reduced (18)F-FLT uptake was measured in arthritic ankles and in CT26 colon carcinomas when the mice breathed oxygen and were conscious during tracer uptake, compared with mice that were anesthetized during (18)F-FLT uptake. Breathing air completely abolished this phenomenon. Analysis of blood samples that were obtained from the mice before (18)F-FLT injection and after the PET scan implicated respiratory acidosis that was induced by oxygen breathing and consciousness during tracer uptake. Acidosis was found to be the primary factor responsible for the reduced (18)F-FLT uptake, as reflected by increased pCO(2) and reduced pH and lactate values. CONCLUSION: Oxygen-breathing conscious mice sustained respiratory acidosis and, consequently, reduced cell proliferation and (18)F-FLT uptake in arthritic ankles and CT26 colon carcinomas. Thus, we suggest the use of air instead of oxygen breathing for (18)F-FLT PET measurements.

Comparative suppressive effects of tyrosine kinase inhibitors imatinib and nilotinib in models of autoimmune arthritis.

Imatinib and nilotinib are inhibitors that selectively target a set of protein tyrosine kinases, including abelson kinase (Abl), together with the chimeric oncoprotein, breakpoint cluster region-abelson kinase (Bcr-Abl), as well as stem cell factor receptor (KIT), platelet-derived growth factor receptor (PDGFR), discoidin domain receptor (DDR), and colony stimulating factor-1 receptor (CSF-1R). The aim of the present study was to investigate whether imatinib or nilotinib was effective against arthritis in the glucose-6-phosphate isomerase (GPI)-induced arthritis mouse model. Imatinib or nilotinib was administered orally to the arthritic mice at different time points. Efficacy was evaluated by visual scoring and by determining the production of anti-GPI antibody. Splenocytes from the arthritic mice were cultured with GPI in the presence of imatinib or nilotinib in vitro, and cytokine levels in the culture supernatants were analyzed. To investigate the effects of imatinib and nilotinib on T-cell proliferation, lymph node cells from the arthritic mice were cultured with GPI in the presence of imatinib or nilotinib in vitro. Interleukin (IL)-17 mRNA expression in the arthritic ankle joints from the onset of arthritis was analyzed by real-time polymerase chain reaction (PCR). The administration of imatinib from day 0 showed suppression of arthritis (P < 0.05), the administration of nilotinib from day 0 resulted in pronounced suppression of arthritis (P < 0.01), and that from day 7 showed significant inhibition of the progression of arthritis (P < 0.05). A reduction in anti-GPI antibodies was correlated with the therapeutic efficacy of imatinib, but not with that of nilotinib. Imatinib dose-dependently inhibited tumor necrosis factor (TNF)-α, IL-6, interferon (IFN)-γ, and IL-17 production by splenocytes in vitro, while nilotinib inhibited only IL-17 and IFN-γ production in a dose-dependent fashion. Imatinib at 3 µM exerted a mild antiproliferative effect on CD4+ T cells (P < 0.05), whereas imatinib at 10 µM and nilotinib at 3 and 10 µM demonstrated a marked antiproliferative effect (P < 0.01). The IL17 gene expression level on day 7 tended to be higher than that on day 14. These findings suggest that imatinib and nilotinib could prevent autoimmune arthritis, essentially via distinct mechanisms, in that imatinib inhibits both inflammatory and T-cell-derived cytokine production, whereas nilotinib suppresses T-cell-derived cytokine production. Imatinib and nilotinib could have therapeutic potential for rheumatoid arthritis (RA) and other inflammatory diseases.

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.

Novel roles of the autocrine motility factor/phosphoglucose isomerase in tumor malignancy.

Autocrine motility factor (AMF) stimulates cell motility in an autocrine manner and is related to tumor malignancy. AMF is a multifunctional molecule, also known as phosphoglucose isomerase and neuroleukin. Signal cascades of the AMF-stimulated motility and novel functions of this protein contributing to tumor malignancy have been presented recently. AMF stimulation activated small Rho-like GTPases and subsequently induced actin fiber rearrangement, which was removed by the C3 exoenzyme, a specific inhibitor of Rho. The expression of Jun N-terminal kinase (JNK)1, JNK2 and the Rho GDP dissociation inhibitor-beta was upregulated by AMF. The addition of AMF to culture medium stimulated the motility of the endothelial cells and the formation of tube-like structures in collagen gels. Highly AMF-expressing HT1080 cells induced aggressive angiogenesis in vivo. The expression of fms-like tyrosine kinase (Flt)-1, a vascular endothelial growth factor (VEGF) receptor, was enhanced in AMF-expressing tumors dependent on protein kinase C and phosphatidylinositol 3 kinase (PI3K) activation; meanwhile kinase insert domain-containing receptor, another receptor of VEGF, was not. Permeability of mesothelial and endothelial cell monolayers was increased by AMF, and numerous gaps were observed in the monolayers after treatment with AMF. AMF gene transfection transformed NIH3T3 cells to proliferate quickly and acquire anti-apoptosis ability induced by serum deprivation in a PI3K-dependent manner. The anti-apoptotic effect of AMF has been described by other authors who have shown that the AMF over-expressing cells were resistant to mitomycin-C-induced apoptosis showing regression of Apaf-1 and caspase-9 dependent on PI3K and MAP kinase. These novel functions of AMF makes it a likely target for cancer therapy.

Phosphodiesterase-I alpha/autotaxin controls cytoskeletal organization and FAK phosphorylation during myelination.

Myelination within the central nervous system (CNS) involves substantial morphogenesis of oligodendrocytes requiring plastic changes in oligodendrocyte-extracellular matrix (ECM) interactions, that is, adhesion. Our previous studies indicated that a regulator of such adhesive plasticity is oligodendrocyte-released phosphodiesterase-I alpha/autotaxin (PD-I alpha/ATX). We report here, that PD-I alpha/ATX's adhesion antagonism is mediated by a protein fragment different from the one that stimulates tumor cell motility. Furthermore, PD-I alpha/ATX's adhesion-antagonizing fragment causes a reorganized distribution of the focal adhesion components vinculin and paxillin and an integrin-dependent reduction in focal adhesion kinase (FAK) phosphorylation at tyrosine residue 925 (pFAK-925). In vivo, a similar reduction in pFAK-925 occurs at the onset of myelination when PD-I alpha/ATX expression is significantly upregulated. Most importantly, it can also be induced by the application of exogenous PD-I alpha/ATX. Our data, therefore, suggest that PD-I alpha/ATX participates in the regulation of myelination via a novel signaling pathway leading to changes in integrin-dependent focal adhesion assembly and consequently oligodendrocyte-ECM interactions.

Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate.

Autotaxin (ATX) is an exoenzyme that potently induces tumor cell motility, and enhances experimental metastasis and angiogenesis. ATX was shown recently to be identical to serum lysophospholipase D activity, producing lysophosphatidic acid (LPA) from lyso-glycerophospholipids. LPA, itself a strong chemoattractant for tumor cells, may mediate the actions of ATX. We now extend the substrate specificity to sphingosylphosphorylcholine (SPC), which ATX hydrolyzes to sphingosine-1-phosphate (S1P). Under migration assay conditions, this novel reaction for the production of S1P has a substrate (SPC) K(m) = 0.23 +/- 0.07 mM. In our responder cell lines (NIH3T3 clone7 and A2058), S1P exerts maximal biological effects at concentrations of 10-100 nM and is mimicked in its biological effects by ATX plus SPC. These effects include inhibition of ATX- and LPA-stimulated motility, and elevation of activated Rho. In NIH3T3 clone7 cells stimulated with platelet-derived growth factor and treated with 10-25 nM S1P, motility is not inhibited and activation of Rho is unaffected, indicating that S1P possesses specificity in its effects. The exoenzyme ATX can potentially regulate diverse processes such as motility and angiogenesis via the S1P family of receptors. Because ATX hydrolyzes nucleotides, lyso-glycerophospholipids, and phosphosphingolipids into bioactive products, it possesses the ability, depending on the availability of substrates, to act as positive or negative regulator of receptor-mediated activity in the cellular microenvironment.

Cdc42 and Rac1 are necessary for autotaxin-induced tumor cell motility in A2058 melanoma cells.

Autotaxin (ATX) is a strong motogen that can increase invasiveness and angiogenesis. In the present study, we investigated the signal transduction mechanism of ATX-induced tumor cell motility. Unlike N19RhoA expressing cells, the cells expressing N17Cdc42 or N17Rac1 showed reduced motility against ATX. ATX activated Cdc42 and Rac1 and increased complex formation between these small G proteins and p21-activated kinase (PAK). Furthermore, ATX phosphorylated focal adhesion kinase (FAK) that was not shown in cells expressing dominant negative mutants of Cdc42 or Rac1. Collectively, these data strongly indicate that Cdc42 and Rac1 are essential for ATX-induced tumor cell motility in A2058 melanoma cells, and that PAK and FAK might be also involved in the process.

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.

Species specificity of the cytokine function of phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) is a cytosolic glycolytic enzyme that also functions as an extracellular cytokine (neuroleukin/autocrine motility factor (AMF)/maturation factor). Contrary to mammalian PGI, bacterial PGI was not internalized by the PGI/AMF receptor (gp78/AMF-R) and neither bacterial nor yeast PGI competed with mammalian PGI for receptor binding and internalization. Furthermore, while the bacterial, yeast and mammalian preparations all exhibited isomerase activity, only mammalian PGI stimulated the motility of NIH-3T3 fibroblasts. The conserved active site of PGI is therefore not sufficient for receptor binding and cytokine activity of PGI. However, synthetic peptides corresponding to distinct peripheral mammalian PGI sequences did not inhibit internalization of mammalian PGI. Our data therefore argue that the cytokine activity of PGI is specific to mammalian PGI but cannot exclude the possibility that the receptor binding motif of PGI is complex and includes elements within and without the active site.

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.

Autotaxin (ATX) is a tumor cell motility-stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5'-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein-coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.