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Purpose Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear. Methods The clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells. Results We found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion. Conclusions Our data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

Background Aconitine is well-known for its potential analgesic, anti-inflammatory, and circulation promoting effects and has been widely used as a folk medicine in South Korea. Owing to its extremely toxic nature and relatively low safety margin, intoxication is sometimes fatal. The toxic compound mainly affects the central nervous system, heart, and muscle, resulting in cardiovascular complications. Purpose To determine the exact relationship between blood concentration of aconitine and clinical manifestation. Basic procedures The National Forensic Service (NFS) was commissioned to assist in a quantitative analysis of highly toxic aconitine and corresponding blood concentrations by analyzing the body fluids of three patients who were suspected of aconitine poisoning. Main findings Aconitine blood values tested by the NFS showed that patients with a blood concentration below a certain level developed symptoms slowly and showed a high severity of clinical manifestation. There was no correlation between blood concentration and symptoms or ECG results. Conclusions In case of suspected aconitine poisoning, an emergency care department should be visited, even with symptomatic improvement, and the patient should be monitored for at least 24 h, depending on the level of recovery and changes in ECG results.

Background: QT prolongation is a well-known complication when tramadol or zolpidem is ingested in large amounts acutely. However, the blood drug concentration resulting in QT prolongation or tachyarrhythmia when tramadol and zolpidem are ingested simultaneously in large amounts has not yet been reported. We report a case of acute intoxication by tramadol and zolpidem resulting in QT prolongation in a patient in whomserial blood and urine tramadol and zolpidemconcentrationswere determined.Case presentation:A 38-year-old male patient presented tothe emergency medical centrebecause ofpoisoning from 3 g of tramadol and 50 mg of zolpidemat 3 h before hisemergency department(ED) visit.During supportive treatment, he developed QT prolongation without clinical manifestations. He was discharged five days after admission without any sequelae. We measured the blood and urine concentrations of tramadol and zolpidemat various time points, which revealed ablood tramadol concentration-dependent change in QTc intervals and increasing blood tramadol concentration at 8 h after the ED visit,with a sustainedlow zolpidem concentration.Tramadol and zolpidem are metabolized by the same enzyme, cytochrome P450 3A4.Therefore, competitive inhibition bya P450 3A4isoenzyme may lead to increases indrugtoxicity. The QT interval in patients acutely intoxicated bytramadol should be evaluated carefully, particularlywhen tramadol is co-ingested with other drugs.Conclusions:Considering the half-life of tramadolor zolpidem and potential for continued absorption of drugs remaining in the gastrointestinal tract, it is necessary to observe patients viacardiac monitoring for more than 36 hafteracute intoxication.The blood concentration of tramadol may increaseand result in QT prolongationeven afterappropriate initial treatment.

In Korea, an increasing number of people attempt to evade military conscription by posing as mental health patients. To verify the authenticity of mental illness, there is a need to detect wide range of psychoactive agents in biological specimens of conscription candidates. In this study, we developed and validated a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of 18 psychoactive agents and 6 metabolites in human plasma. The method was characterized by the use of a simple, fast and cheap protein precipitation as sample preparation, a rapid run time (11 min) and a low volume of plasma sample (200 μL). The analytes were monitored under the scheduled multiple reaction monitoring (sMRM) positive and negative mode using electrospray ionization (ESI). The essential validation parameters including selectivity, linearity, accuracy, precision, matrix effect and recovery were satisfactory. The limit of detection ranged from 0.0005 to 0.001 μg/mL, and limit of quantitation ranged from 0.005 to 0.025 μg/mL. The developed method was successfully applied to 323 actual plasma samples submitted by Korea central physical examination center of military manpower administration in 2016, and is expected to contribute to the rapid and accurate disposition of military service.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Additional file 1: Figure S1. Surface localization of Epi-GV fusion protein. (A) Diagram of Epi-GV construct with C-terminal Strep tag (Epi-GV-strep). (B) Epi-GV-strep construct (or MOCK) was transfected to 293F cells with a trace amount of EGFP cDNA as a transfection marker, and its surface expression was analyzed by flow cytometry. Representative dot plots are shown. Figure S2. PMA-induced nuclear localization of epithin/PRSS14 intracellular domain. (A) 427 cells were transfected with two siRNAs against epithin/PRSS14, starved for serum, and stimulated with PMA or not. Cells were then stained with anti-N antibody (green) and DAPI (blue). Representative images are shown. Scale bars, 20 μm. (B) The computational method for determining EICD-positive pixels is illustrated. If the mean fluorescence of a particular pixel and the 8 pixels surrounding it in the nuclear image is greater than the threshold described in Methods section, the pixel was considered as an EICD-positive pixel. (C) Representative anti-N antibody-stained original image and binary image with epithin/PRSS14 positive pixels indicated in white are shown. (D) In the experiment described in A, normalized EICD nuclear localization (the sum of the fluorescence intensity of EICD-positive pixels in each cell divided by the mean fluorescence of nuclear region of the cell) was calculated and represented as bar graph. At least 57 cells from three microscopic fields were used for each condition. The error bar indicates SEM. ****p

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. βPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. βPix is expressed as alternatively spliced variants, including the ubiquitous isoform βPix-a and the neuronal isoforms βPix-b and βPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of βPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from βPix neuronal isoform knockout (βPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating βPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal βPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal βPix isoforms may promote microtubule stabilization during neurite development. βPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either βPix neuronal isoform in the βPix-NIKO neurons restores Stathmin1 phosphorylation levels, with βPix-d having a greater effect than βPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via βPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that βPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal βPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.

Background Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. Results In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. Conclusions These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

βPix is a guanine nucleotide exchange factor for the Rho family small GTPases, Rac1 and Cdc42. It is known to regulate focal adhesion dynamics and cell migration. However, the in vivo role of βPix is currently not well understood. Here, we report the production and characterization of βPix-KO mice. Loss of βPix results in embryonic lethality accompanied by abnormal developmental features, such as incomplete neural tube closure, impaired axial rotation, and failure of allantois-chorion fusion. We also generated βPix-KO mouse embryonic fibroblasts (MEFs) to examine βPix function in mouse fibroblasts. βPix-KO MEFs exhibit decreased Rac1 activity, and defects in cell spreading and platelet-derived growth factor (PDGF)-induced ruffle formation and chemotaxis. The average size of focal adhesions is increased in βPix-KO MEFs. Interestingly, βPix-KO MEFs showed increased motility in random migration and rapid wound healing with elevated levels of MLC2 phosphorylation. Taken together, our data demonstrate that βPix plays essential roles in early embryonic development, cell spreading, and cell migration in fibroblasts.

Objective— Angiogenesis, the process of building complex vascular structures, begins with sprout formation on preexisting blood vessels, followed by extension of the vessels through proliferation and migration of endothelial cells. Based on the potential therapeutic benefits of preventing angiogenesis in pathological conditions, many studies have focused on the mechanisms of its initiation as well as control. However, how the extension of vessels is terminated remains obscure. Thus, we investigated the negative regulation mechanism. Approach and Results— We report that increased intracellular calcium can induce dephosphorylation of the endothelial receptor tyrosine kinase Tie2. The calcium-mediated dephosphorylation was found to be dependent on Tie2–calmodulin interaction. The Tyr1113 residue in the C-terminal end loop of the Tie2 kinase domain was mapped and found to be required for this interaction. Moreover, mutation of this residue into Phe impaired both the Tie2-calmodulin interaction and calcium-mediated Tie2 dephosphorylation. Furthermore, expressing a mutant Tie2 incapable of binding to calmodulin or inhibiting calmodulin function in vivo causes unchecked growth of the vasculature in Xenopus . Specifically, knockdown of Tie2 in Xenopus embryo retarded the sprouting and extension of intersomitic veins. Although human Tie2 expression in the Tie2-deficient animals almost completely rescued the retardation, the Tie2(Y1113F) mutant caused overgrowth of intersomitic veins with strikingly complex and excessive branching patterns. Conclusions— We propose that the calcium/calmodulin-dependent negative regulation of Tie2 can be used as an inhibitory signal for vessel growth and branching to build proper vessel architecture during embryonic development.

PAK-interacting guanine nucleotide exchange factor (βPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which βPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced βPix isoforms in the brain. Here, we report a novel finding that the b isoform of βPix (βPix-b) mediates the regulation of spine and synapse formation. We found that βPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of βPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of βPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of βPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of βPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which βPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation. Published version

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.

The synergistic interaction of two antibodies targeting the same protein could be developed as an effective anti‐cancer therapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20–25% of breast and gastric cancer patients, and HER2‐targeted antibody therapy using trastuzumab is effective in many of these patients. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2‐positive gastric cancer. Here, we describe the development of 1E11, a HER2‐targeted humanized monoclonal antibody showing increased efficacy in a highly synergistic manner in combination with trastuzumab in the HER2‐overexpressing gastric cancer cell lines NCI‐N87 and OE‐19. The two antibodies bind to sub‐domain IV of the receptor, but have non‐overlapping epitopes, allowing them to simultaneously bind HER2. Treatment with 1E11 alone induced apoptosis in HER2‐positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. The synergistic antitumor activity of 1E11 in combination with trastuzumab indicates that it could be a novel potent therapeutic antibody for the treatment of HER2‐overexpressing gastric cancers.

DW1029M is a botanical extract consisting of Morus bark and Puerariae radix, produced by Dong-Wha Pharmaceutical, for nephroprotective drug development; it has been in phase II clinical trials in Korea. In our mechanistic investigations, we found that DW1029M inhibits advanced glycation end products (AGEs), rat lens aldose reductase (RLAR), and transforming growth factor (TGF)-β1signaling, all of which are implicated in diabetic complications such as diabetic nephropathy and diabetic retinopathy. DW1029M inhibits AGE formation via Fe2+chelation. The extract contains 13 active constituents that inhibit AGE formation, 8 active constituents that inhibit RLAR activity, and 1 inhibitor of TGF-β1signaling. Our results suggest DW1029M protects against diabetic nephropathy via blockade of AGE formation, RLAR activity, and TGF-β1signaling.

Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.

Cardiovascular disease, which is caused by unregulated platelet aggregation, is one of the main causes of deaths worldwide. Many studies have focused on natural products with antiplatelet effects as a safe alternative therapy to prevent the disease. In this context, an in-house chemical library was screened to find natural products capable of inhibiting the interaction between platelet integrin αIIbβ3 and fibrinogen, which is an essential step in platelet aggregation. On the basis of the screening results, indothiazinone, an alkaloid found in microbial cultures, was identified as a potential antiplatelet agent. Specifically, indothiazinone treatment significantly inhibited the binding of fibrinogen to Chinese hamster ovary cells expressing integrin αIIbβ3. It also restricted thrombin- and adenosine diphosphate-dependent spreading of human platelets on a fibrinogen matrix. More importantly, surface plasmon resonance and molecular dynamics studies suggested that indothiazinone suppressed talin-induced activation of integrin αIIbβ3 presumably by inhibiting talin-integrin interaction. In conclusion, these results suggest that indothiazinone can be used as a lead compound for the development of antiplatelet drugs with a novel mode of action.

βPix is a Rac/Cdc42 guanine nucleotide exchange factor (GEF) that is known to be a regulator of actin cytoskeleton remodeling. Recently, a novel splicing isoform, βPix-b(L), was identified as an alternative translational product of the βPix-b mRNA with an extended N-terminus comprising a partial calponin homology (CH) domain and a serine-rich (SR) domain. However, the cellular function of βPix-b(L) is largely unknown. In the current study, we analyzed the genomic DNA structure and cellular functions of βPix-b(L). The results of this study demonstrate that βPix is composed of 24 exons and 21 introns spanning around 100 kb. RT-PCR experiments revealed that there are two forms of βPix mRNA with distinct 5' UTRs that are the result of alternative splicing of exon 1 and 2 from βPix genomic DNA. In addition, affinity chromatography analysis and a pull-down assay with the N-terminal region of βPix-b(L) revealed that βPix-b(L) interacts with tubulin and actin via its N-terminal CH and SR domains, respectively. Interaction with tubulin enabled βPix-b(L) to bundle the microtubule and form membrane protrusions. Furthermore, the N-terminus of βPix-b(L) was also critical for its localization to cellular vesicles. Functionally, βPix-b(L) induced pinocytosis through cooperative action of the CH and Dbl homology (DH) domains, demonstrating the role of βPix-b(L) in the regulation of membrane dynamics.

The epithelial-derived type II transmembrane serine protease epithin has been shown to be upregulated in a variety of cancer cell lines and tumor tissues, and its upregulation correlates well with tumor progression in many cases. However, little is known regarding the regulation of its expression and the mechanism of its roles in tumor progression. Here, we show that transforming growth factor-beta (TGF-beta), a potent inducer of epithelial-mesenchymal transition (EMT) in tumor progression, upregulates epithin, and that epithin plays a critical role in TGF-beta-induced EMT. Forced overexpression of epithin induced EMT to exhibit characteristic morphological changes, alternations in EMT-related proteins and enhanced cell motility. Conversely, shRNA-mediated knockdown of endogenous epithin inhibited TGF-beta-induced expression of mesenchymal markers and morphological changes. Furthermore, TGF-beta-induced cell migration and invasion were significantly impaired by epithin knockdown. In addition, we demonstrate that TGF-beta upregulates epithin transcriptionally via the Smad2/Smad4-mediated pathway. These results suggest that epithin is a key mediator of TGF-beta-induced EMT in tumor progression.

betaPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of betaPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of betaPIX, which is important for its physiological functions. We have solved the crystal structure of the betaPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The betaPIX CC forms a 76-A-long parallel CC trimer. Despite the fact that the betaPIX CC exposes three PDZ binding motifs in the C-termini, the betaPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive beta-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of betaPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the betaPIX trimer. The structure shows canonical class I PDZ binding with a beta-sheet interaction extending to position -6 of betaPIX. The betaB-betaC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the beta-sheet interaction and to accommodate the bulky side chain of Trp -5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of betaPIX CC and Shank PDZ.

The epithelial-derived, type II transmembrane serine protease matriptase, the mouse homologue of which is epithin, has been shown to be involved in epidermal differentiation, hair formation, and thymus function. We show in this study that epithin/matriptase (Epi/MTP) plays a significant role in mammary epithelial cell growth and morphogenesis. Epi/MTP is expressed at low level in the mouse mammary epithelium of young animals and it accumulates at the terminal end-bud of the growing ducts. The level of Epi/MTP is elevated in the mammary glands at stages when epithelial proliferation and modeling occur. It is primarily present in the luminal epithelial cells of mouse mammary ducts and lobules. Using an ex vivo three-dimensional culture system for mammary epithelial functional assays, we show that mammary epithelial growth and morphogenesis in the presence of the latent form hepatocyte growth factor (pro-HGF) are blocked either by an inhibitor of the Epi/MTP protease activity or by siRNA knockdown of the Epi/MTP expression. These studies demonstrate that Epi/MTP participates in mammary epithelial growth and modeling through activation of pro-HGF. Our findings reveal an important pathway in normal mammary epithelial morphogenesis which may participate in breast cancer progression.

Crystal structure of the dimerization domain of human filamin A Min-Duk Seo,1y Seung-Hyeon Seok,1y Hookang Im, Ae-Ran Kwon, Sang Jae Lee, Hyung-Ryong Kim, Yongcheol Cho, Dongeun Park, and Bong-Jin Lee* 1 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea 2 Promeditech Ltd., Seoul 151-010, Korea 3Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk 570-749, Korea 4 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Vimentin, an intermediate filament protein induced during epithelial-to-mesenchymal transition, is known to regulate cell migration and invasion. However, it is still unclear how vimentin controls such behaviors. In this study, we aimed to find a new integrin regulator by investigating the H-Ras-mediated integrin suppression mechanism. Through a proteomic screen using the integrin β3 cytoplasmic tail protein, we found that vimentin might work as an effector of H-Ras signaling. H-Ras converted filamentous vimentin into aggregates near the nucleus, where no integrin binding can occur. In addition, an increase in the amount of vimentin filaments accessible to the integrin β3 tail enhanced talin-induced integrin binding to its ligands by inducing integrin clustering. In contrast, the vimentin head domain, which was found to bind directly to the integrin β3 tail and compete with endogenous vimentin filaments for integrin binding, induced nuclear accumulation of vimentin filaments and reduced the amount of integrin-ligand binding. Finally, we found that expression of the vimentin head domain can reduce cell migration and metastasis. From these data, we suggest that filamentous vimentin underneath the plasma membrane is involved in increasing integrin adhesiveness, and thus regulation of the vimentin-integrin interaction might control cell adhesion.

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.

The preimplantation development of mammalian embryo after fertilization encompasses a series of events including cleavage, compaction, and differentiation into blastocyst. These events are likely to be associated with substantial changes in embryonic gene expression. In the present study, we explored the expression patterns and function of epithin, a mouse type II transmembrane serine protease, during preimplantation embryo development. RT-PCR analysis showed that epithin mRNAs were detectable during the cleavage stages from a 1-cell zygote to the blastocyst. Immunocytochemical studies revealed that epithin protein was expressed at blastomere contacts of the compacted 8-cell and later embryonic stages. Epithin colocalized with E-cadherin at the membrane contacts of the compacted morula-stage embryo as revealed by double-staining immunocytochemistry and confocal microscopy, respectively. Post-transcriptional epithin gene silencing by RNA interference (RNAi) resulted in the blockade of 8-cell in vitro-stage embryo compaction and subsequent embryonic deaths after several rounds of cell division. These results strongly suggest that epithin plays an important role in the compaction processes that elicit the signal for the differentiation into trophectoderm and inner cell mass.

Phospholipase C-γ1 (PLCγ1) plays a critical role in cell growth and proliferation by generating the second messengers, diacylglycerol and 1, 4, 5-inositol triphosphate. To investigate the roles of Src homology domain 2 and domain 3 of PLCγ1 in PLCγ1-mediated cell signaling, we characterized some proteins binding to these domains in the MCF7 and MDA-MB-231 breast cancer cell lines. Of the several proteins that bind to glutathione-S-transferase-SH2/SH2/SH3, we identified an 85 kDa protein that binds to the SH3 domain of PLCγ1 as the guanine nucleotide exchange factor, p21-activated protein kinase-interacting exchange factor-a (βPix-a). βPix-a co-immunoprecipitated with PLCγ1 in breast cancer tissues extracts and in MCF7 and MDA-MB-231 cell extracts. In addition, PDGF-stimulated PLCγ1 activity was elevated in βPix-a-overexpressing NIH3T3 cells. Our results suggest that βPix-a binds to the Src homology domain 3 of PLCγ1 and promotes tumor growth in breast cancer by enhancing the activity PLCγ1. J. Cell. Biochem. 94: 1010–1016, 2005. © 2004 Wiley-Liss, Inc.

The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOH-terminal region of Nox1, a protein related to gp91(phox) (Nox2) of phagocytic cells, is constitutively associated with beta Pix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of beta Pix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, beta Pix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.

Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.

Myristoylated alanine-rich C kinase substrate (MARCKS) translocates from the cytosol to the plasma membrane while mononucleated myoblasts fuse to form multinucleated myotubes. Here, we show that protein phosphatase-1-mediated dephosphorylation of MARCKS largely influences its subcellular localization and the fusion process. Treatment with okadaic acid or tautomycin, which are potent inhibitors of protein phosphatases and cell fusion, was found to reversibly block the MARCKS translocation. Moreover, the dephosphorylating activity against MARCKS markedly increased during myogenesis, and this increase was closely correlated with the membrane fusion of the cells. In addition, protein phosphatase-1 was identified as a major enzyme that is responsible for dephosphorylation of MARCKS. Furthermore, a mutation preventing MARCKS phosphorylation and thus facilitating MARCKS translocation resulted in promotion of the cell fusion. In contrast, overexpression of MARCKS carrying a mutation that blocks myristoylation and thus prevents the MARCKS translocation impaired the myoblast fusion. Together with the fact that MARCKS regulates the cytoskeleton dynamics by crosslinking the actin filaments in the plasma membrane and that myoblast fusion accompanies massive cytoskeleton reorganization, these results suggest that protein phosphatase-1-mediated MARCKS localization at the membrane is required for the fusion of embryonic muscle cells.

c-Cbl, a RING-type ubiquitin E3 ligase, down-regulates receptor tyrosine kinases, including EGF receptor, and inhibits cell proliferation. Moreover, c-Cbl mutations are frequently found in patients with myeloid neoplasm. Therefore, c-Cbl is known as a tumor suppressor. αPix is expressed only in highly proliferative and mobile cells, including immune cells, and up-regulated in certain invasive tumors, such as glioblastoma multiforme. Here, we showed that c-Cbl serves as an ubiquitin E3 ligase for proteasome-mediated degradation of αPix, but not βPix. Remarkably, the rat C6 and human A172 glioma cells were unable to express c-Cbl, which leads to a dramatic accumulation of αPix. Depletion of αPix by shRNA markedly reduced the ability of the glioma cells to migrate and invade, whereas complementation of shRNA-insensitive αPix promoted it. These results indicate that c-Cbl negatively regulates αPix-mediated cell migration and invasion and the lack of c-Cbl in the C6 and A172 glioma cells is responsible for their malignant behavior.

Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-β (TGF-β) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-β-induced epithin/PRSS14 shedding. TGF-β treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-β induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 ProteinInteracting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa βPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, βPix-a also contains a putative leucine zipper domain at the C-terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of βPix-a homodimers. Using in vitro andin vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish βPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type βPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant could not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes were completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables βPix-a to homodimerize, and homodimerization is essential for βPix-a signaling functions leading to the cytoskeletal reorganization.

βPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. We examined the effect of βPix on MAP kinase signaling and cytoskeletal rearrangement in NIH3T3 fibroblast cells. Overexpression of βPix enhanced the activation of p38 in the absence of other stimuli and also induced translocation of p38 to the nucleus. This βPix-induced p38 activation was blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of βPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of βPix in growth factor-stimulated p38 activation was evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing βPix SH3m (W43K) and βPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) blocked membrane ruffling induced by βPix, suggesting that p38 might be involved in mediating βPix-induced membrane ruffling. The results in this study suggest that βPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase.

The cellular localization of alpha3beta1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of alpha3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and alpha3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. Alpha-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the alpha3A subunit and other myofibrillar proteins into sarcomeres revealed that alpha3A was integrated into sarcomeres following incorporation of alpha-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that alpha3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that alpha3A integrins participate in the initial assembly of myofibrillar proteins. The alpha3B, beta1A and beta1D subunits were also localized in costameres, where they formed alpha3Abeta1A, alpha3Abeta1D and alpha3Bbeta1A heterodimers. The alpha3Bbeta1D heterodimer, however, was not found in cardiac myocytes. The antisera raised against the cytoplasmic domains of alpha3A, alpha3B, beta1A and beta1D caused disruption of sarcomere structure. Thus, the myofibril-extracellular matrix linkages mediated by isoforms of alpha3beta1 integrin may play a crucial role in the stabilization of myofibril assembly and in the maintenance of sarcomere structure. Co-immunoprecipitation experiments revealed that beta1A, but not beta1D, interacts with the Nck signaling protein, suggesting that Nck participates in downstream signaling triggered by beta1A and that the beta1A-mediated signaling pathway is distinct from that of beta1D.

The present study investigated the cellular localization of α‐subunit of Gq (Gαq) protein in developing L8E63, rat skeletal muscle cell line. The colocalization of Gαq with actin cytoskeleton was demonstrated by double‐labeling experiments. In mononucleated myoblasts, the immune‐fluorescence staining pattern of Gαq was almost identical with that of F‐actin visualized with rhodamine‐conjugated phalloidin. However, this colocalization of Gαq with cytoskeleton was not maintained in multinucleated myotubes. The staining pattern of Gαq in myotubes did not match with any specific subcellular structure, but appeared as a uniformly distributed diffuse staining throughout the whole cell surface. Interestingly, change in the expression level of Gαq was not detected during myoblast differentiation, suggesting that actin‐associated Gαq protein might dissociate from the cytoskeleton as cells differentiate. Immunocytochemical experiments using specific antibodies directed against several G proteins indicated that the s...

The phospholipase C (PLC)-beta isozymes differ from the PLC-gamma and PLC-delta isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by alpha subunits of the Gq class of G proteins, associate with the particulate subcellular fraction, and are present in the nucleus. Most of the COOH-terminal domain of PLC-beta isozymes is predicted to be helical, and three regions in this domain, PLC-beta1 residues 911-928 (region 1), 1055-1072 (region 2), and 1109-1126 (region 3), contain a high proportion of basic residues that are highly conserved. Projection of the sequences of these three regions in helical wheels reveals clustering of the basic residues. The role of the COOH terminus and the clustered basic residues in PLC-beta1 was investigated by either truncating the entire COOH-terminal domain (mutant DeltaC) or replacing two or three clustered basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The DeltaC mutant no longer showed the ability to be activated by Gqalpha, to translocate to the nucleus, or to associate with the particulate fraction. Substitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by Gqalpha, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the cluster of lysine residues 914, 921, and 925 in region 1 had the most marked effect, reducing Gqalpha-dependent activity to 10% of that of wild type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted with isoleucine, behaved like the wild-type enzyme in showing an approximately equal distribution between cytoplasm and nucleus; only 12% of the region 2 mutant was present in the nucleus. None of the basic clusters appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-beta1 in the particulate fraction by some extent, suggesting that all the basic residues contribute to the association, presumably by interacting with acidic residues in the particulate fraction. Membrane localization of PLC-beta isozymes is therefore likely mediated by both the COOH-terminal domain and the pleckstrin homology domain, the latter of which is known to bind phosphatidylinositol 4,5-biphosphate.

Remodeling of dendritic spines through regulation of actin dynamics is a key event in activity-dependent structural plasticity. However, the molecular mechanism underlying this process is poorly understood. Here, we show that activity-dependent modulation of Abl interactor 1–Ca2+/calmodulin-dependent kinase IIα (Abi1–CaMKIIα) interaction, and thereby their activity, is important for regulation of spine morphology in cultured rat hippocampal neurons. Abi1 interacts with CaMKIIα at resting conditions through Abi1's tSNARE (target membrane-associated SNARE), which harbors striking homology with CaMKIIα regulatory domain. The interaction of the two proteins, Abi1 and CaMKIIα, results in their simultaneous inhibition, inhibition of CaMKIIα activity, and also inhibition of Abi1-dependent Rac activation. Their functional impediment is released when they dissociate from each other by calmodulin binding through glutamate receptor activation. Before dissociation, Abi1 is phosphorylated by CaMKIIα at serine 88, which may involve in regulation of Rac activation and spine maturation. Our results suggest that modulation of the interaction between Abi1 and CaMKIIα, through the glutamate receptor pathway, may be a molecular mechanism underlying activity-regulated structural plasticity in rat hippocamapal neurons.

The fourth member of mammalian beta-type phospholipase C isozymes, PLC-beta 4, was recently purified from bovine retina, and the corresponding cDNA was cloned from rat brain and sequenced. PLC-beta 4 has now been shown to differ from the other three mammalian beta-type isozymes (PLC-beta 1, -beta 2, and -beta 3) in that it is selectively inhibited by ribonucleotides. The inhibition requires the 5'-phosphate and 2'-hydroxyl groups of ribose as well as the base moiety. Thus, deoxyribonucleotides and ribose 5-phosphate were not inhibitory. The monophosphate, diphosphate, and triphosphate nucleoside derivatives were all inhibitory, whereas cyclic nucleotides were ineffective. Purine nucleotides were more potent inhibitors than pyrimidine nucleotides; the 50% inhibitory concentrations were 20-30 microM for AMP and GMP, and 100-200 microM for UMP and CMP. Unlike the other beta-type isozymes, PLC-beta 4 contains the GX4GKS consensus sequence for the recognition of the phosphoryl group of nucleotides. In the absence of ribonucleotides, the specific activity of PLC-beta 4 toward phosphatidyl-inositol 4,5-bisphosphate was four to five times the average specific activity of PLC-beta 1 and PLC-beta 3. Thus, nucleotide-dependent inhibition may serve to reduce the activity of PLC-beta 4 in the absence of a hormonal signal. The regulation of PLC-beta 4 by G-proteins was also studied. Similar to the other three PLC-beta isozymes, PLC-beta 4 was activated by the alpha subunit of Gq but not by the transducin alpha subunit. However, unlike other PLC-beta isozymes, PLC-beta 4 was not responsive to activation by G beta gamma subunits.

βPix, a Pak-interacting nucleotide exchange factor (Cool-1/p85SPR), is a Cdc42/Rac1-specific guanine nucleotide exchange factor (GEF) involved in various actin-related processes. Many previous studies have focused on ubiquitously expressed βPix-a, while the role of the neuronal-specific isoform βPix-b is still unknown, especially whether its role is distinct from or similar to βPix-a. Here we show that unlike βPix-a, overexpression of βPix-b stimulates actin-dependent comet formation in BHK21 cells. This effect is attributed to the interaction between its proline-rich domain (PRD) and the WH1 domain of N-WASP. In addition, we show that overexpression of βPix-b stimulates actin-dependent dendritic spine formation in rat hippocampal neurons in culture, a formation that is blocked by co-expression of the WH1 domain of N-WASP or the PRD of βPix-b. Knocking-down endogenous expression of βPix-b by shRNA reduced the number of dendritic spines, which were rescued only by PRD-containing βPix-b mutants. GEF activity of βPix-b is also required for these effects. The results show that neuronal-specific βPix-b stimulates actin-dependent processes in cells via the interaction between its PRD and the WH1 domain of N-WASP. Our results identify N-WASP as the first protein shown to interact with the PRD of βPix-b, raising the possibility that, as an N-WASP WH1-binding protein, βPix-b may regulate N-WASP's activity in cells. J. Cell. Physiol. 227: 1476–1484, 2012. © 2011 Wiley Periodicals, Inc.

PRSS14/Epithin (also known as matriptase and ST14), a member of the type II transmembrane serine proteases, is primarily found in a subpopulation of normal epithelial cells and in epithelial cancers. Its known functions include maintaining the epithelial barrier, thymic development, and cancer progression. In this study, we show that several macrophage cell lines and activated bone marrow-derived macrophages also express PRSS14/Epithin. Surface expression, as well as cytoplasmic expression, was detectable upon activation by IFN-γ, but not TNF-α or TGF-β. Induction of the protein appeared to be restricted to macrophages. IFN-γ showed a biphasic regulation in RAW264.7 cells, and upregulated expression was sustained for several days. This induction by IFN-γ was partially through the increase of PRSS14/Epithin mRNA production, which is downstream of the JAK pathway, shown by the inhibition by tyrphostin AG490. Using chromatin immunoprecipitation, we verified that two sites among six putative STAT1 binding sites in the PRSS14/Epithin promoter were occupied by STAT1 upon activation. Treatment with IFN-γ enhanced the serum-triggered transendothelial migration of RAW264.7 cells, but not that of PRSS14/Epithin knock-down RAW264.7 cells, although they express multiple markers such as ICAM1, CD80, and CD40 at normal levels. These data strongly suggest that PRSS14/Epithin plays an important role in the transendothelial migration of activated macrophages in the inflammatory microenvironment, and the mode of action is similar to the events in cancer metastasis.