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Scatter plots of the diagnostic values of the constructed diagnostic model and tumor marker levels in all 191 cases.

PDF - 146KB, Scatter plots of the diagnostic values of the constructed diagnostic model and tumor marker levels in all 191 cases.

PDF - 142KB, Supplementary Table 1: Additional information of the study subjects. Supplementary Table 2: Additional information of the subjects used for the comparisons between the healthy volunteers and chronic pancreatitis patients. Supplementary Table 3: All compounds detected in the subjects� serum. Supplementary Table 4: The diagnostic performance of the metabolites as individual biomarkers for pancreatic cancer in the training set study. Supplementary Table 5: Variance inflation factors of the variables in the diagnostic model. Supplementary Table 6: Variables selected by the stepwise method. Supplementary Table 7: Serum metabolites examined in the study of chronic pancreatitis.

Background: To improve the prognosis of patients with pancreatic cancer, more accurate serum diagnostic methods are required. We used serum metabolomics as a diagnostic method for pancreatic cancer.Methods: Sera from patients with pancreatic cancer, healthy volunteers, and chronic pancreatitis were collected at multiple institutions. The pancreatic cancer and healthy volunteers were randomly allocated to the training or the validation set. All of the chronic pancreatitis cases were included in the validation set. In each study, the subjects' serum metabolites were analyzed by gas chromatography mass spectrometry (GC/MS) and a data processing system using an in-house library. The diagnostic model constructed via multiple logistic regression analysis in the training set study was evaluated on the basis of its sensitivity and specificity, and the results were confirmed by the validation set study.Results: In the training set study, which included 43 patients with pancreatic cancer and 42 healthy volunteers, the model possessed high sensitivity (86.0%) and specificity (88.1%) for pancreatic cancer. The use of the model was confirmed in the validation set study, which included 42 pancreatic cancer, 41 healthy volunteers, and 23 chronic pancreatitis; that is, it displayed high sensitivity (71.4%) and specificity (78.1%); and furthermore, it displayed higher sensitivity (77.8%) in resectable pancreatic cancer and lower false-positive rate (17.4%) in chronic pancreatitis than conventional markers.Conclusions: Our model possessed higher accuracy than conventional tumor markers at detecting the resectable patients with pancreatic cancer in cohort including patients with chronic pancreatitis.Impact: It is a promising method for improving the prognosis of pancreatic cancer via its early detection and accurate discrimination from chronic pancreatitis. Cancer Epidemiol Biomarkers Prev; 22(4); 571–9. ©2013 AACR.

Additional information of the study subjects

PDF file - 263K, Supplementary Figure 6. Knockdown of SAC1, PI4KIIIbeta and GOLPH3.

Supplementary Figure Legends 1-5 from Casein Kinase 2–Interacting Protein-1, a Novel Akt Pleckstrin Homology Domain-Interacting Protein, Down-regulates PI3K/Akt Signaling and Suppresses Tumor Growth In vivo

PDF file - 263K, Supplementary Figure 4. Knockdown of PI4K variants.

PDF file - 194K, Supplementary Figure 2. Quantification of phosphoinositides.

PDF file - 274K, Supplementary Figure 5. Knockdown of PI4KIIIbeta in Hs578t cells.

PDF file - 114K

Supplementary Figures 1-5 from Casein Kinase 2–Interacting Protein-1, a Novel Akt Pleckstrin Homology Domain-Interacting Protein, Down-regulates PI3K/Akt Signaling and Suppresses Tumor Growth In vivo

The serine/threonine kinase Akt plays a central role in cell survival and proliferation. Its activation is linked to tumorigenesis in several human cancers. Although many Akt substrates have been elucidated, the Akt-binding proteins that regulate Akt function remain unclear. We report herein having identified casein kinase 2–interacting protein-1 (CKIP-1) as an Akt pleckstrin homology (PH) domain-binding protein with Akt inhibitory function. CKIP-1 formed a complex with each Akt isoform (Akt1, Akt2, and Akt3) via its NH2 terminus. Dimerization of CKIP-1 via its leucine zipper (LZ) motif at the COOH terminus was found to be associated with Akt inactivation because deletion of the LZ motif eliminated Akt inhibitory function, although it could still bind to Akt. Expression of the NH2 terminus–deleted CKIP-1 mutant containing the LZ motif, but lacking Akt-binding ability, induced Akt phosphorylation and activation by sequestering the ability of endogenous CKIP-1 to bind to Akt. Stable CKIP-1 expression caused Akt inactivation and cell growth inhibition in vitro. In addition, the growth of stable CKIP-1 transfectants xenografted into nude mice was slower than that of mock transfectants. These results indicate that CKIP-1, a novel Akt PH domain-interacting protein, would be a candidate of tumor suppressor with an Akt inhibitory function. [Cancer Res 2007;67(20):9666–76]

PDF file - 229K, Supplementary Figure 3. Knockdown of SAC1 and PI4KIIIbeta in MCF7 cells.

PDF file - 296K, Supplementary Table 1. SAC1 expression in cancer.

PDF file - 235K, Supplementary Figure 1. Knockdown of SAC1 in T47D cells.

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2 ) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P2 on focal adhesion dynamics in MDA-MB-231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5-trisphosphatase (PIP3 ) 5-phosphatase that generates PI(3,4)P2 , in MDA-MB-231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3-phosphatase that de-phosphorylates PIP3 and PI(3,4)P2 , induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P2 , and knockdown of Lpd, a downstream effector of PI(3,4)P2 , resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis.

Skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP), a PIP3 phosphatase, has been implicated in the regulation of insulin signaling in skeletal muscle. SKIP interacts with Pak1 and glucose-regulated protein 78 (GRP78), both of which are necessary for the regulation of insulin signaling. In this study, we showed that GRP78 directly binds to the SKIP C-terminal homology (SKICH) domain of SKIP and that this binding is necessary for the localization of SKIP at the ER. In addition, in vitro binding analysis showed that GRP78 and Pak1 competitively bind to SKIP. Taken together, these findings suggest a model by which GRP78 regulates intracellular localization of SKIP and how SKIP binds to Pak1 on insulin stimulation.

Insulin resistance is critical in the pathogenesis of type 2 diabetes. Endoplasmic reticulum (ER) stress in liver and adipose tissues plays an important role in the development of insulin resistance. Although skeletal muscle is a primary site for insulin-dependent glucose disposal, it is unclear if ER stress in those tissues contributes to insulin resistance. In this study, we show that skeletal muscle kidney-enriched inositol polyphosphate phosphatase (SKIP), a PIP3 (phosphatidylinositol-3,4,5-trisphosphate) phosphatase, links ER stress to insulin resistance in skeletal muscle. SKIP expression was increased due to ER stress and was higher in the skeletal muscle isolated from high-fat-diet-fed mice and db/db mice than in that from wild-type mice. Mechanistically, ER stress promotes activating transcription factor 6 (ATF6) and X-box binding protein 1 (XBP1)-dependent expression of SKIP. These findings underscore the specific and prominent role of SKIP in the development of insulin resistance in skeletal muscle.

Phosphofructokinase (PFK) 1 is a glycolytic enzyme, and its abnormality contributes to the development of multiple human diseases, such as cancer. Here, we report that nucleoredoxin (NRX), a thioredoxin-related oxidoreductase, is a novel interacting partner of PFK1. NRX binds directly to PFK1, and endogenous NRX and PFK1 interact in vivo. In NRX−/− mouse embryonic fibroblasts (MEFs), the oligomerization status of PFK1 is altered and the catalytic activity of PFK1 is decreased. NRX deficiency augmented levels of NADPH and reduced glutathione, two major cellular antioxidants generated through the pentose phosphate pathway. Indeed, NRX−/− MEFs are significantly more resistant to oxidative stress than NRX+/+ MEFs. These results reveal a novel role of NRX in the regulation of PFK1 activity and in the balance between glycolysis and the pentose phosphate pathway.

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

srGAP1 limits Rac1 activity at lamellipodia in a negative feedback manner, allowing concomitant activation of Rac1 and RhoA at lamellipodia. Rho signaling causes membrane ruffling through actomyosin contractility and removes the protrusive structures. Such coordination of Rac and Rho determines migratory behavior through lamellipodial dynamics., The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

Skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP) has previously been implicated in the regulation of insulin signaling in skeletal muscle. Here, we present the first report of the mechanisms by which SKIP specifically suppresses insulin signaling and the subsequent glucose uptake. Upon insulin stimulation, SKIP is translocated to the membrane ruffles, where it binds to the active form of Pak1, which mediates multiple protein complex formation with phosphatidylinositol 3,4,5-triphosphate (PIP(3)) effectors such as Akt2, PDK1, and Rac1; this leads to inactivation of these proteins. SKIP also promotes the inhibition of Rac1-dependent kinase activity and the scaffolding function of Pak1, which results in the dissociation of Akt2 and PDK1 from Pak1. Thus, specific suppression of insulin signaling is achieved via the spatiotemporal regulation of SKIP through the scaffolding function of Pak1. These interactions are the foundation of the specific and prominent role of SKIP in the regulation of insulin signaling.

Insulin-like growth factors (IGFs) are essential for the development, regeneration, and hypertrophy of skeletal muscles. IGF-II promotes myoblast differentiation through phosphatidylinositol 3-kinase (PI 3-kinase), Akt, and mTOR signaling. Here, we report that skeletal muscle- and kidney-enriched inositol polyphosphate phosphatase (SKIP) negatively regulates myogenesis through inhibition of IGF-II production and attenuation of the IGF-II-Akt-mTOR signaling pathway. We also demonstrate that SKIP expression, which was markedly elevated during differentiation, was controlled by MyoD in C2C12 cells. Expression of SKIP inhibited IGF-II at the transcription level. These results indicate that SKIP regulates MyoD-mediated muscle differentiation. Silencing of SKIP increased IGF-II transcription and myoblast differentiation. Furthermore, knockdown of SKIP resulted in thick myotubes with a larger number of nuclei than that in control C2C12 cells. Taken together, these data indicate that SKIP controls the IGF-II-PI 3-kinase-Akt-mTOR auto-regulation loop during myogenesis. Our findings identify SKIP as a key regulator of muscle cell differentiation.

The small GTPase RhoD, activated by fibroblast growth factor (FGF) signaling, forms actin-based, cytoneme-like, thin and long cellular protrusions through activating mDia3C. These protrusions transmit FGF receptors toward the cell body. They are likely to be responsible for intercellular communication between FGF-producing cells and target cells., The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8–coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation.

Circular dorsal ruffles (CDRs) are highly dynamic F-actin–based membrane structures involved in bulk endocytosis of membrane receptors and macropinocytosis. A key role of ARAP1, an Arf GAP protein, is found to be in the control of the ring size of CDRs., Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity–dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1–Arf1/5 pathway.

Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. It is found that SHIP2 interacts with RhoA in a GTP-dependent manner and this interaction is required for proper localization of PI(3,4,5)P3 and regulation of cell polarization and migration., Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front–rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2–containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and migration.

Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry, and matrix assisted laser desorption ionization-mass spectrometry. Then, the findings of studies about GC-MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases.

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and l-serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3-hydroxypropionic acid and pyruvic acid; and l-alanine, glucuronoic lactone and l-glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer. Copyright © 2011 John Wiley & Sons, Ltd.

We have previously shown that SGIP1α is an endocytic protein specifically expressed in neural tissues. SGIP1α has a lipid-binding domain called the MP domain, which shows no significant homology to any other domains. In this study, we characterized FCHO2, a protein with a high level of homology to SGIP1α. FCHO2 lacks the MP domain but has another lipid-binding domain, the EFC/F-BAR domain. FCHO2 was ubiquitously expressed. The FCHO2 EFC domain bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubes. FCHO2 localized to clathrin-coated pits at the plasma membrane and bound to Eps15, an important adaptor protein in clathrin-mediated endocytosis. FCHO2 knockdown reduced transferrin endocytosis. These results suggest that FCHO2 regulates clathrin-mediated endocytosis through its interactions with membranes and Eps15. These properties of FCHO2 are similar to those of SGIP1α. FCHO2 is likely to be a ubiquitous homologue of SGIP1α. We furthermore found that FCHO2 was subjected to monoubiquitination, and gel filtration analysis showed that FCHO2 formed an oligomer. These new properties might also contribute to the role of FCHO2 in clathrin-mediated endocytosis.

The roles that amino acids play in immunity and inflammation are well defined, and the relationship between inflammatory bowel disease (IBD) and certain amino acids has recently attracted attention. In this study, the levels of amino acids and trichloroacetic acid (TCA) cycle-related molecules in the colonic tissues and sera of patients with ulcerative colitis (UC) were profiled by gas chromatography/mass spectrometry (GC/MS), with the aim of evaluating whether the clinical state induced by UC leads to variations in the amino acid profile. Colonic biopsy samples from 22 UC patients were used, as well as serum samples from UC patients (n = 13), Crohn’s disease (CD) patients (n = 21), and healthy volunteers (n = 17). In the GC/MS-based profiling of amino acids and TCA cycle-related molecules, lower levels of 16 amino acids and 5 TCA cycle-related molecules were observed in the colonic lesion tissues of the UC patients, and the serum profiles of amino acids and TCA cycle-related molecules of the UC patients were different from those of the CD patients and healthy volunteers. Our study raises the possibility that GC/MS-based profiling of amino acids and TCA cycle-related molecules is a useful early diagnostic tool for UC.

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.

Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.

Muscle Building The signaling mechanisms involved in actin filament formation for myofibril formation, which is required for growth factor-induced muscle maturation and hypertrophy, remain unclear. Takano et al. (p. 1536 ; see the Perspective by Gautel and Ehler ) now show that the mechanism involves the interaction of nebulin and N-WASP. N-WASP is an activator of the Arp2/3 complex, which induces branched actin filaments in nonmuscle cells. The nebulin–N-WASP complex formed in muscle, however, causes nucleation of unbranched actin filaments within myofibrils without the Arp2/3 complex. Nebulin–N-WASP–mediated myofibrillar actin filament formation is required for muscle hypertrophy and might explain a congenital hereditary neuromuscular disorder caused by nebulin gene mutation: nemaline myopathy.

Pancreatic cancer is one of the leading causes of cancer-related death, and there is currently little hope of a cure because there are no effective biomarkers for its early detection. Therefore, the search for novel biomarkers that would allow the early detection of pancreatic cancer is ongoing. In this study, the differences between the metabolomes of pancreatic cancer patients with Stage III, Stage IVa, or Stage IVb disease (n = 20) and healthy volunteers (n = 9) were evaluated by metabolomics, which is the endpoint of the Omics cascade and therefore the last step in the cascade before the phenotype. In our experimental conditions using gas chromatography mass spectrometry (GC/MS), a total of 60 metabolites were detected in serum, and the levels of 18 of the 60 metabolites were significantly changed in pancreatic cancer patients compared with those in healthy volunteers. Then, Principal Component Analysis (PCA), which is a basic form of Multiple Classification Analysis, was performed, and the PCA scores plots based on the 60 metabolites highlighted the metabolomic differences between the pancreatic cancer patients and healthy volunteers. The differences between different stages of pancreatic cancer were also assessed by Partial Least Squares Discriminant Analysis (PLS-DA), which is one of Multiple Classification Analysis, and we found that it was possible to discriminate among the Stage III, Stage IVa, and Stage IVb groups. In addition, values of the 9 metabolites in 1 Stage I pancreatic cancer patient were similar to those obtained from the Stage III, Stage IVa, and Stage IVb pancreatic cancer patients. Our findings will aid the discovery of novel biomarkers that allow the early detection of pancreatic cancer by metabolomic approaches.

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Whereas Coronin 1A co-localizes with PI(4,5)P2 at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P2 signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P2-containing liposomes and that PI(4,5)P2 antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.

Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and PRIP-2 [officially designated PLCL1 and PLCL2]) was first identified in our laboratory, but the biological functions have remained elusive. Therefore, we generated Plcl1 and Plcl2 double-knockout mice (Plcl1tm1Mh; Plcl2tm1Tta) to gain insight into the biological function. Double-knockout mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments to discriminate whether males or females were defective indicated that the cause appeared to be on the female side. Mutant female mice had an apparently smaller uterus by gross anatomical observation and had more estrous days during the cycles. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone were measured for 5–6 consecutive days and were significantly higher in the mutant, wh...

Among an increasing number of lipid-binding domains, a group that not only binds to membrane lipids but also changes the shape of the membrane has been found. These domains are characterized by their strong ability to transform globular liposomes as well as flat plasma membranes into elongated membrane tubules both in vitro and in vivo. Biochemical studies on the structures of these proteins have revealed the importance of the amphipathic helix, which potentially intercalates into the lipid bilayer to induce and/or sense membrane curvature. Among such membrane-deforming domains, BAR and F-BAR/EFC domains form crescent-shaped dimers, suggesting a preference for a curved membrane, which is important for curvature sensing. Bioinformatics in combination with structural analyses has been identifying an increasing number of novel families of lipid-binding domains. This review attempts to summarize the evidence obtained by recent studies in order to gain general insights into the roles of membrane-deforming domains in a variety of biological events.

Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P2-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P2-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P2 biosynthesis by allowing optimal PIKfyve-ArPIKfyve association. These opposing inputs raise the key question as to whether reduced Sac3 protein levels and/or hydrolyzing activity will produce gain in insulin responsiveness. Here we report that small interfering RNA-mediated knockdown of endogenous Sac3 by ∼60%, which resulted in a slight but significant elevation of PtdIns(3,5)P2 in 3T3L1 adipocytes, increased GLUT4 translocation and glucose entry in response to insulin. In contrast, ectopic expression of Sac3WT, but not phosphatase-deficient Sac3D488A, reduced GLUT4 surface abundance in the presence of insulin. Endogenous Sac3 physically assembled with PIKfyve and ArPIKfyve in both membrane and soluble fractions of 3T3L1 adipocytes, but this remained insulin-insensitive. Importantly, acute insulin markedly reduced the in vitro C8-PtdIns(3,5)P2 hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P2 subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [3H]inositol-PtdIns(3,5)P2 was minor. Together, our data identify Sac3 as an insulin-sensitive phosphatase whose down-regulation increases insulin responsiveness, thus implicating Sac3 as a novel drug target in insulin resistance.

The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal-amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.

Insulin controls glucose homeostasis and lipid metabolism, and insulin impairment plays a critical role in the pathogenesis of diabetes mellitus. Human skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) is a member of the phosphatidylinositol 3,4,5-trisphosphate phosphatase family (T. Ijuin et al. J. Biol. Chem. 275:10870-10875, 2000; T. Ijuin and T. Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). Previous studies showed that SKIP negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling (Ijuin and Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). We now have generated mice with a targeted mutation of the mouse ortholog of the human SKIP gene, Pps. Adult heterozygous Pps mutant mice show increased insulin sensitivity and reduced diet-induced obesity with increased Akt/protein kinase B (PKB) phosphorylation in skeletal muscle but not in adipose tissue. The insulin-induced uptake of 2-deoxyglucose into the isolated soleus muscle was significantly enhanced in Pps mutant mice. A hyperinsulinemic-euglycemic clamp study also revealed a significant increase in the rate of systemic glucose disposal in Pps mutant mice without any abnormalities in hepatic glucose production. Furthermore, in vitro knockdown studies in L6 myoblast cells revealed that reduction of SKIP expression level increased insulin-stimulated Akt/PKB phosphorylation and 2-deoxyglucose uptake. These results imply that SKIP regulates insulin signaling in skeletal muscle. Thus, SKIP may be a promising pharmacologic target for the treatment of insulin resistance and diabetes.

The BUF/Mna strain of rat is a model of focal and segmental glomerulosclerosis (FSGS) in which a quantitative trait locus (QTL) for proteinuria, Pur1, has been identified. The aim of the present study was to identify candidates for the Pur1 gene. To narrow the Pur1 QTL, we performed fine QTL mapping and single nucleotide polymorphism (SNP) genotyping. To identify candidate genes, sequencing and gene-expression analyses of all genes contained in the narrowed locus were conducted. The narrowed Pur1 region contained 25 genes. Among these genes, only the Arp3 gene was mutated in the BUF/Mna strain; it contained a missense mutation that caused an (L)111(F) substitution. This leucine is conserved across species. Gene-expression analysis failed to identify any other candidate genes for Pur1. Arp3-mediated actin assembly abnormalities were visible in immunohistochemical and electron microscopic examinations of podocytes in old BUF/Mna rats. Taken together, these data suggest that Arp3 is a candidate for the Pur1 gene. This observation is consistent with our growing recognition that abnormal signaling-induced assembly of actin in podocytes leads to the development of FSGS.