Transmembrane 4 L six family member 5 (TM4SF5) enhances migration and invasion of hepatocytes for effective metastasis

Overexpression of transmembrane 4 L six family member 5 (TM4SF5), a four‐transmembrane L6 family member, causes aberrant cell proliferation and angiogenesis, but the roles of TM4SF5 in migration, invasion, and tumor metastasis remain unknown. Using in vitro hepatocarcinoma cells that ectopically or endogenously express TM4SF5 and in vivo mouse systems, roles of TM4SF5 in metastatic potentials were examined. We found that TM4SF5 expression facilitated migration, invadopodia formation, MMP activation, invasion, and eventually lung metastasis in nude mice, but suppression of TM4SF5 with its shRNA blocked the effects. Altogether, TM4SF5‐mediated migration and invasion suggest that TM4SF5 may be therapeutically targeted to deal with TM4SF5‐mediated hepatocellular cancers. J. Cell. Biochem. 111: 59–66, 2010. © 2010 Wiley‐Liss, Inc.     

Spatial restriction of alpha4 integrin phosphorylation regulates lamellipodial stability and alpha4beta1-dependent cell migration

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.     

TM4SF1, a binding protein of DVL2 in hepatocellular carcinoma,positively regulates beta-catenin/TCF signalling

The interaction between Axin and DVL2 is critical for the breaking down of the beta-catenin destruction complex and the activation of the Wnt/beta-catenin cascade. However, this biological process remains poorly understood. In the present study, TM4SF1 was identified as the interacting partner of DVL2 and positively regulated as Wnt/beta-catenin signalling by strengthening the DVL2-Axin interaction. The expression levels of TM4SF1 were elevated in hepatocellular carcinoma (HCC) and were induced by Kras signalling. The overexpression of TM4SF1 promoted the growth and motility of HCC cells, and up-regulated the target genes (Axin2 and cyclin D1). The down-regulation of TM4SF1 impaired the capability of HCC cells for growth, migration and metastasis. In addition, the down-regulation of TM4SF1 promoted the ubiquitination of beta-catenin. In summary, these results reveal the oncogenic functions of TM4SF1 in HCC progression and suggest that TM4SF1 might be a target for treatment.     

Pharmacological profile of hemokinin 1: a novel member of the tachykinin family

Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK(1), the rabbit pulmonary artery (rbPA) for NK(2), and rat portal vein (rPV) for NK(3) receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB > SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK(1) receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pK(B) 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK(1) antagonist, MEN 11467 (pK(B) 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK(1) preferring receptor agonist.     

Focal adhesion and actin organization by a cross-talk of TM4SF5 with integrin alpha2 are regulated by serum treatment

The biological functions of transmembrane 4 L6 family member 5 (TM4SF5) homologues to a tumor-associated antigen L6 are unknown, although it is over-expressed in certain forms of cancer. In the present study, the ectopic expression of TM4SF5 in Cos7 cells reduced integrin signaling under serum-containing conditions, but increased integrin signaling upon serum-free replating on substrates. TM4SF5 regulated actin organization and focal contact dynamics via the serum treatment-dependent differential regulation of FAK Tyr925 and paxillin Tyr118 phosphorylations and their localizations on peripheral cell boundaries. Y925F FAK mutation abolished the TM4SF5 effects. TM4SF5 associated with integrin alpha2 subunit, and this association was abolished by serum treatment. Furthermore, functional blocking anti-integrin alpha2 antibody abolished TM4SF5-enhanced signaling activity and caused membrane blebbing with abnormal actin organization. TM4SF5 increased chemotactic but decreased haptotactic migration. Altogether, this study reveals the functions of TM4SF5 collaborative with integrin signaling to alter focal contact dynamics, actin reorganization, and migration. Furthermore, this study suggests a mechanism of cross-talk between TM4SF5 and integrin which is further regulated by growth factor signaling.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

KCNQ1OT1 regulates the retinoblastoma cell proliferation,migration and SIRT1/JNK signaling pathway by targeting miR-124/SP1 axis

Objective: Long non-coding RNA (lncRNA) KCNQ1OT1 was reported to be tightly associated with tumorigenesis and progression of multiple cancers. However, the expression and biological functions of KCNQ1OT1 in retinoblastoma (RB) are still unknown. We aim to elucidate the potential function and underlying mechanism of KCNQ1OT1 in regulating the progression of RB. Methods: The levels of KCNQ1OT1 were assayed by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) analysis. The cell proliferation of RB cells (Y79 and WERI-Rb-1) were evaluated through Cell Counting Kit 8 (CCK-8) assay. Meanwhile, Y79 and WERI-Rb-1 cell apoptosis and cell cycle were assessed by Flow Cytometry analysis. Dual luciferase reporter assay were performed to illustrate the interaction between KCNQ1OT1, miR-124, and SP1. Results: We found that KCNQ1OT1 was up-regulated and miR-124 was down-regulated in RB tissues and cells. Moreover, knockdown of KCNQ1OT1 reduced the proliferation, migration, and cell cycle, as well as promoted cell apoptosis of Y79 and WERI-Rb-1 cells. Western blot analysis consistently proved cell cycle and apoptosis related protein expression levels. More importantly, KCNQ1OT1 was a sponge of microRNA (miR)-124. MiR-124 inhibition strongly reversed the effect on cell proliferation, cycle arrest, and apoptosis by KCNQ1OT1 knockdown mediation. In addition, KCNQ1OT1 regulated expression of SP1, a direct target of miR-124 in RB. On the other hand, miR-124 inhibitor abrogated the active effect of KCNQ1OT1 silencing on silent information regulator 1 (SIRT1)/c-Jun N-terminal kinase (JNK) signaling pathway. The function of KCNQ1OT1 was verified in vivo. Conclusions: These findings implied that KCNQ1OT1 silencing inhibited RB progression and activated SIRT1/JNK signaling pathway partially by modulating the miR-124/SP1 axis.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.     

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1gene (Nramp1/Slc11a1) is a gene that controls the susceptibility ofinbred mice to intracellular pathogens. Polymorphisms in the humanSlc11a1/Nramp1 gene have been associated with host susceptibilityto leprosy. This study has evaluated nine polymorphisms of theSlc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T,1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19patients had the multibacillary and the paucibacillary clinical forms of the disease,respectively), and 239 healthy controls matched by age, gender, and ethnicity. Thefrequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p =0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in thecontrol group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p= 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), suchas the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/Cpolymorphisms, respectively, were more frequent in the leprosy group. The leprosy andcontrol groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A,1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy,while the allele 2 and 3 of the (GT)n polymorphism in the promoter region wereassociated with susceptibility and protection to leprosy, respectively.     

Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes

Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin‐mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP‐1, AP‐2, and AP‐3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ‐type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP‐3 suggesting that calcyon could regulate membrane‐bound pools of AP‐3 and AP‐3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP‐3, and AP‐3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol‐4‐kinase type II alpha (PI4KIIα), two well‐defined AP‐3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock‐out brain, a phenotype previously described in AP‐3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP‐3 and the targeting of AP‐3 cargoes.     

Cutting edge: a novel function for the SLAP-130/FYB adapter protein in beta 1 integrin signaling and T lymphocyte migration

The role of integrin-mediated signaling events in T cell function remains incompletely characterized. We report here that alpha4beta1 integrin stimulation of H9 T cells and normal human T cell blasts results in rapid and transient tyrosine phosphorylation of the adapter protein, SH2 domain-containing 76-kDa protein (SLP-76)-associated phosphoprotein of 130 kDa (SLAP-130)/FYB at levels comparable to those observed following TCR stimulation. Stimulation of T cells via the alpha4beta1 integrin enhances the association of tyrosine phosphorylated SLAP-130/FYB with the SH2 domain of the src tyrosine kinase p59fyn. Activation of normal T cells, but not H9 T cells, via alpha4beta1 leads to tyrosine phosphorylation of SLP-76 as well as SLAP-130/FYB. Overexpression of SLAP-130/FYB in normal T cells enhances T cell migration through fibronectin-coated filters in response to the chemokine stromal cell-derived factor (SDF)-1alpha. These results identify SLAP-130/FYB as a new tyrosine phosphorylated substrate in beta1 integrin signaling and suggest a novel function for SLAP-130/FYB in regulating T lymphocyte motility.