The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

p57Kip2 is an essential regulator of vitamin D receptor-dependent mechanisms.

A cyclin-dependent kinase (CDK) inhibitor, p57Kip2, is an important molecule involved in bone development; p57Kip2-deficient (p57-/-) mice display neonatal lethality resulting from abnormal bone formation and cleft palate. The modulator 1α,25-dihydroxyvitamin D3 (l,25-(OH)2VD3) has shown the potential to suppress the proliferation and induce the differentiation of normal and tumor cells. The current study assessed the role of p57Kip2 in the 1,25-(OH)2VD3-regulated differentiation of osteoblasts because p57Kip2 is associated with the vitamin D receptor (VDR). Additionally, 1,25-(OH)2VD3 treatment increased p57KIP2 expression and induced the colocalization of p57KIP2 with VDR in the osteoblast nucleus. Primary p57-/- osteoblasts exhibited higher proliferation rates with Cdk activation than p57+/+ cells. A lower level of nodule mineralization was observed in p57-/- osteoblasts than in p57+/+ cells. In p57+/+ osteoblasts, 1,25-(OH)2VD3 upregulated the p57Kip2 and opn mRNA expression levels, while the opn expression levels were significantly decreased in p57-/- cells. The osteoclastogenesis assay performed using bone marrow cocultured with 1,25-(OH)2VD3-treated osteoblasts revealed a decreased efficiency of 1,25-(OH)2VD3-stimulated osteoclastogenesis in p57-/- cells. Based on these results, p57Kip2 might function as a mediator of 1,25-(OH)2VD3 signaling, thereby enabling sufficient VDR activation for osteoblast maturation.

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells.

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (-1)- and (-2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.

Expression of p57KIP2 reduces growth and invasion, and induces syncytialization in a human placental choriocarcinoma cell line, BeWo.

INTRODUCTION: Syncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. METHODS: We generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. RESULTS: The human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. DISCUSSION: These results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

Chondroitin sulfate E blocks enzymatic action of heparanase and heparanase-induced cellular responses.

We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.

Incorporation, intracellular trafficking and processing of extracellular heparanase by mast cells: Involvement of syndecan-4-dependent pathway.

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Food polyphenols targeting peptidyl prolyl cis/trans isomerase Pin1.

We searched for inhibitors against prolyl isomerase Pin1 in order to develop functional foods to prevent and cure various Pin1 related diseases such as cancer, diabetes, cardiovascular disease, Alzheimers's disease, and so on. We created a polyphenol library consisting of ingredients in healthy foods and beverages, since polyphenols like epigallocatechin gallate (EGCG) in green tea and 974B in brown algae had been identified as its Pin1 inhibitors. Several polyphenols such as EGCG derivatives, caffeic acid derivatives and tannic acid inhibited Pin1 activity. These results provide a first step in development of the functional foods and beverage targeting Pin1 and its related diseases.

Lectin microarray analysis of isolated polysaccharides from Sasa veitchii.

We report lectin microarray profile of the polysaccharide fraction derived from Sasa veitchii leaf that exhibits anti-influenza activity. This fraction showed higher reactivities with lectins known as binders to oligo-mannose, fucose, or galactose. Our findings along with previously reported monosaccharide components suggest that the polysaccharide can be cross-reactive with cell surface receptors involved in immune system, thereby exerting anti-influenza activity.

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells.

BACKGROUND: Activation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. METHODS: Immunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. RESULTS: We found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. CONCLUSIONS: These results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. GENERAL SIGNIFICANCE: Our evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

Retinoylation (covalent modification by retinoic acid) of Rho-GDIß in the human myeloid leukemia cell line HL60 and its functional significance.

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIß (28kDa) by TOF-MS and co-migration with Rho-GDIß (28kDa). Truncated Rho-GDIß (23kDa, N∆19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIß (5kDa), which is the second product resulting from the cleavage of Rho-GDIß (28kDa) by caspase-3. RA treatment increased the level of Rho-GDIß (28kDa) and decreased the level of Rho-GDIß (23kDa). RA did not induce caspase-3 activity or Rho-GDIß mRNA expression. It is likely that retinoylation of Rho-GDIß increases its metabolic stability.

Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

Inhibition of ASCT2 is essential in all-trans retinoic acid-induced reduction of adipogenesis in 3T3-L1 cells.

Vitamin A has preventive effects on obesity. All-trans retinoic acid (ATRA), the active form of vitamin A, inhibits lipid accumulation in 3T3-L1 cells in an experimental adipogenesis model. We found that ATRA suppressed up-regulation of the amino acid transporter, Asct2, in adipogenerating 3T3-L1 cells. We observed that Asct2 was up-regulated at 1 day after adipogenesis stimuli. The Asct2 inhibitor l-γ-glutamyl-p-nitroanilide (GPNA) decreased lipid accumulation. Glutamine-free conditions also suppressed adipogenesis. Suppression of adipogenesis by ATRA may be through Asct2 reduction. These results indicate that Asct2 could be a target for obesity prevention and treatment.

Role of prolyl isomerase pin1 in pathogenesis of diseases and remedy for the diseases from natural products.

The peptidyl prolyl cis/trans isomerase Pin1, the human ortholog of yeast Ess1 specifically isomerizes peptide bindings of pSer/pThr-Pro residues in various proteins, and regulates the expression levels and functions of phosphorylated proteins. Activation of Pin1 is associated with pathology of a variety of diseases, such as cancer, Alzheimer's disease, infectious diseases and so on. Therefore, regulatory compounds for Pin1 can be applied as a clinical medicine against these diseases. Many chemists have exerted themselves to synthesize the inhibitors based on the 3D structure of Pin1. We have screened for the inhibitors against Pin1 from the natural products including the functional foods. Here we review the Pin1-associated pathology and the known inhibitors identified from natural products. And we introduce the screening methods targeting Pin1 activity.

15-deoxy-?(12,14)-prostaglandin J2 as a potential TRPV1-dependent atopic dermatitis enhancer.

Atopic dermatitis (AD) is a recurrent chronic inflammatory skin condition characterized by a complex pathogenesis, including skin barrier dysfunctions, allergy/immunology, and pruritus.In AD lesions, mast cells migrate into the epidermis, and exert their biological effects by releasing paracrine mediators. TRPV1, a non-selective cation channel widely expressed in skin tissues, has been shown to contribute to the development of diverse dermatoses and pruritus. In the present study, we identified a TRPV1 agonist as a neuritogenic enhancer produced from mast cells and characterized a possible molecular mechanism for the TRPV1-dependent neuritogenesis in AD. Based on the hypothesis that activated mast cells produce a TRPV1-dependent neuritogenic enhancer,we screened a number of inflammatory mediators for the neuritogenic-promoting activityand identified a PGD2 metabolite, 15-deoxy-?(12,14)-prostaglandin J2 (15d-PGJ2), as a potential neuritogenic enhancer derived from mast cells. 15d-PGJ2 significantly enhanced the nerve growth factor (NGF)-induced neuritogenesis in PC12 cells, and its enhancing potency was attributed to the electrophilic center of 15d-PGJ2. 15d-PGJ2 indeed activated TRPV1, leading to a significant increase in the intracellular Ca(2) level. In addition, the treatment of PC12 cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-TRPV1 adduct, indicating that 15d-PGJ2 directly modified the TRPV1 in the cells. Furthermore, 15d-PGJ2 facilitated the NGF-dependent signal transductions including ERK and JNK pathways in a Ca(2 ?)-dependent manner. These findings suggest that 15d-PGJ2 enhances NGF signaling via TRPV1-dependent Ca(2) influx, thereby acting as a potential neuritogenic enhancer in AD.

Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells.

Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor α (RXRα) with peroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA's inhibitory effects on adipogenesis, but not on the intracellular levels of both RXRα and PPARγ. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPARγs levels in a RXR activation-independent manner.

p53 Retards cell-growth and suppresses etoposide-induced apoptosis in Pin1-deficient mouse embryonic fibroblasts.

We studied the effects of Pin1, a regulatory molecule of the oncosuppressor p53, on both cell cycle arrest and apoptosis by treating primary mouse embryonic fibroblasts (MEFs) with etoposide. Etoposide induced G1 arrest in both wild-type and Pin1 null (pin1(-/-)) MEFs, and G2/M arrest and apoptotic cell death in MEFs lacking either p53 only (p53(-/-)) or both Pin1 and p53 (pin1(-/-)p53(-/-)). Both pin1(-/-) and pin1(-/-)p53(-/-) MEFs were enhanced the release of cytochrome c from the mitochondria, which might induce apoptosis. In response to etoposide treatment, apoptotic cell death was displayed in pin1(-/-)p53(-/-) MEFs but not in pin1(-/-) MEFs. These results suggest that p53 retards growth and suppresses etoposide-induced apoptosis in pin1(-/-) MEFs.

[A study of the transport of three dimensional medical images to remote institutions for telediagnosis].

Using a 3D-imaging-create-function server and network services by IP-VPN, we began to deliver 3D images to the remote institution. An indication trial of the primary image, a rotary trial of a 3D image, and a reproducibility trial were studied in order to examine the practicality of using the system in a real network between Hakodate and Sapporo (communication distance of about 150 km). In these trials, basic data (time and receiving data volume) were measured for every variation of QF (quality factor) or monitor resolution. Analyzing the results of the system using a 3D image delivery server of our hospital with variations in the setting of QF and monitor resolutions, we concluded that this system has practicality in the remote interpretation-of-radiogram work, even if the access point of the region has a line speed of 6 Mbps.

The anti-tumor agent, p-DDAP potently suppresses proliferation through apoptosis in human neuroblastoma NB-39-nu cells.

Retinoic acid (RA) is a chemotherapeutic agent used to induce neuronal cellular differentiation of neuroblastoma. However, because treatment with RA is associated with the side-effect of nyctalopia, efforts have been underway to identify new compounds that could potentially overcome these drawbacks. As part of these studies we have examined anti-cancer effects on the neuroblastoma NB-39-nu cells of p-dodecylaminophenol (p-DDAP), a novel derivative of N-(4-hydroxyphenyl) retinamide (4-HPR). p-DDAP suppresses proliferation, and induces G(0)/G(1) arrest and apoptosis to a greater extent than RA and 4-HPR. Neuronal differentiation was not detected in p-DDAP-treated cells. Since p-DDAP is not toxic and does not reduce blood retinol levels, p-DDAP might be a useful anti-neuroblastoma drug having reduced side-effects.

Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver.

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.