STIM1调控细胞运动促进骨肉瘤转移的研究

目的:明确STIM1是否参与调控细胞运动促进骨肉瘤的转移。方法:应用靶向STIM1的si RNA沉默MG-63骨肉瘤细胞中STIM1的表达,然后用侵袭实验、迁移实验以及黏附实验检测骨肉瘤细胞侵袭、迁移与黏附能力的变化,采用Western Blot检测细胞FAK和paxillin的表达及Rac1和RhoA信号通路的活性。结果:转染靶向STIM1的si RNA后,MG-63骨肉瘤细胞中STIM1的蛋白表达和m RNA表达均明显降低(P0.05),细胞的侵袭、迁移与黏附能力均显著下降(P0.05),细胞伪足与细胞骨架的重要组分FAK和paxillin的表达及调控细胞运动的Rac1和RhoA信号通路活性均显著降低(P0.05)。结论:STIM1可能通过激活RhoA和Rac1的信号通路,增加FAK和paxillin的表达,从而调控骨肉瘤细胞运动,促进骨肉瘤转移。     

Regulation of ezrin localization by Rac1 and PIPK in human epithelial cells

Regulation of ezrin and other ERM proteins is not completely understood, but the involvement of Rho GTPases seems crucial. In this work, expression plasmids encoding full-length, deleted or truncated ezrin were constructed and coexpressed with Rac1 GTPase in HeLa human epithelial cells in order to elucidate the mechanisms of ezrin activation and function. We observed induction of actin stress fiber formation by ezrin constructs harboring the F-actin binding site but devoid of sequences required for intra- or intermolecular binding. Stress fiber-inducing ezrin mutants were localized in adherens junctions containing N-cadherin but no E-cadherin, and also colocalized with F-actin in stress fibers. This localization required the activity of Rac1 and phosphatidylinositol-4-phosphate 5-kinase and involved RhoA. We suggest that localization of ezrin in adherens junctions is regulated by Rac in a manner involving PIPK.     

T1alpha/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.     

Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix. While measuring the activity of Rho family GTPases, we found that Rac1, but not RhoA or Cdc 42, is activated in ezrin T567D-expressing cells, compared with cells expressing wild-type ezrin. Together with Rac1 activation, we observed an accumulation of E-cadherin in intracellular compartments and a concomitant decrease in the level of E-cadherin present at the plasma membrane. This effect could be reversed with a dominant negative form of Rac1, N17Rac1. We show that after a calcium switch, the delivery of E-cadherin from an internalized pool to the plasma membrane is greatly delayed in ezrin T567D-producing cells. In confluent cells, ezrin T567D production decreases the rate of E-cadherin internalization. Our results identify a new role for ezrin in cell adhesion through the activation of the GTPase Rac1 and the trafficking of E-cadherin to the plasma membrane.     

Cloning, tissue distribution and sub-cellular localisation of phospholipase C X-domain containing protein (PLCXD) isoforms

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes comprise a small family of receptor-regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. To date, six distinct classes of PI-PLC are known to exist in mammals. Here we characterise a seventh class of PI-PLC, which contains only the catalytic X domain in its structure, termed phospholipase C X-domain containing protein (PLCXD). At least three tissue-specific PLCXD isoforms exist in humans, comprising hPLCXD-1, hPLCXD-2 and hPLCXD-3, with hPLCXD-2 exhibiting three C-terminal spliceforms (2.1, 2.2 and 2.3). Specific amino acids known to be essential for the catalytic function of PI-PLCs were found to be conserved in all three human PLCXDs and over-expression of hPLCXD-1, 2.1 and 3 in the HeLa cell line increased endogenous PI-PLC activity. Human PLCXD isoforms exhibited tissue-specific expression profiles in mice and humans and immunocytochemistry revealed distinct sub-cellular localisations when over-expressed in human cultured cell lines. These novel proteins may therefore possess fundamental, and as yet uncharacterised roles in cell physiology.     

Lysophosphatidic acid acyltransferase β (LPAATβ) promotes the tumor growth of human osteosarcoma

Background

Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated.

Methodology/Principal Findings

Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma.

Conclusions/Significance

Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors.     

ErbB3 silencing reduces osteosarcoma cell proliferation and tumor growth in vivo

Osteosarcoma is the most common primary bone tumor in children and adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of the molecular signals that contribute to the aberrant osteosarcoma cell growth may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here we show that the expression of ErbB3 is increased in human osteosarcoma cells in vitro. Tissue microarray analysis of tissue cores from osteosarcoma patients further showed that the ErbB3 protein expression is higher in bone tumors compared to normal bone tissue, and is further increased in patients with recurrent disease or soft tissue metastasis. In murine osteosarcoma cells, silencing ErbB3 using shRNA decreased cell replication, cell migration and invasion, indicating that ErbB3 contributes to tumor cell growth and invasiveness. Furthermore, ErbB3 silencing markedly reduced tumor growth in a murine allograft model in vivo. Immunohistochemal analysis showed that the reduced tumor growth induced by ErbB3 silencing in this model resulted from decreased cell osteosarcoma cell proliferation, supporting a role of ErbB3 in bone tumor growth in vivo. Taken together, the results reveal that ErbB3 expression in human osteosarcoma correlates with tumor grade. Furthermore, silencing ErbB3 in a murine osteosarcoma model results in decreased cell growth and invasiveness in vitro, and reduced tumor growth in vivo, which supports the potential therapeutic interest of targeting ErbB3 in osteosarcoma.     

Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

Objective

Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism.

Methods

The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed.

Results

It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively.

Conclusion

Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.     

Regulator of G-protein signalling 3 redirects prototypical Gi-coupled receptors from Rac1 to RhoA activation

The small GTPases, Rac1 and RhoA, are pivotal regulators of several essential, but distinct cellular processes. Numerous G-protein-coupled receptors signal to these GTPases, but with different specificities. Specifically, Gi-coupled receptors (GiPCRs) are generally believed to activate Rac1, but not RhoA, a process involving Gbetagamma-dimers and phosphatidylinositol 3-kinase (PI3K). Here we show that, depending on the expression level of the 519 amino acid isoform of regulator of G-protein signalling 3 (RGS3L), prototypical GiPCRs, like M2 muscarinic, A1 adenosine, and alpha2-adrenergic receptors, activate either Rac1 or RhoA in human embryonic kidney cells and neonatal rat cardiomyocyte-derived H10 cells. The switch from Rac1 to RhoA activation in H10 cells was controlled by fibroblast growth factor-2 (FGF-2), lowering the expression of RGS3L. Activation of both, Rac1 and RhoA, seen at low and high expression levels of RGS3L, respectively, was sensitive to pertussis toxin and the PI3K inhibitor LY294002 and mediated by Gbetagamma-dimers. We conclude that RGS3L functions as a molecular switch, redirecting GiPCRs via Gbetagamma-dimers and PI3K from Rac1 to RhoA activation. Considering the essential roles of Rac1 and RhoA in many signalling pathways, this additional function of RGS3L indicates a specific role of this protein in cellular signalling networks.     

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.     

Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling

The Rho family of small GTPases (RhoA, Rac1 and Cdc42) controls signal-transduction pathways that influence many aspects of cell behaviour, including cytoskeletal dynamics. At the leading edge, Rac1 and Cdc42 promote cell motility through the formation of lamellipodia and filopodia, respectively. On the contrary, RhoA promotes the formation of contractile actin-myosin-containing stress fibres in the cell body and at the rear. Here, we identify synaptopodin, an actin-associated protein, as a novel regulator of RhoA signalling and cell migration in kidney podocytes. We show that synaptopodin induces stress fibres by competitive blocking of Smurf1-mediated ubiquitination of RhoA, thereby preventing the targeting of RhoA for proteasomal degradation. Gene silencing of synaptopodin in kidney podocytes causes the loss of stress fibres and the formation of aberrant non-polarized filopodia and impairment of cell migration. Together, these data show that synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration.     

Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMβ causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.     

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (¹⁸⁸S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (¹⁰⁸T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on ⁸⁸S and ¹⁰⁰T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on ⁸⁸S and ¹⁰⁰T in response to EGF, which upregulates RhoA activity.     

Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK₁₀ and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations. Rac; Cdc42; actin; ezrin; adenosine 5'-triphosphate; guanosine 5'-triphosphate     

MicroRNA‑224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1

Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR‐224 in the development and progression of osteosarcoma. We demonstrated that miR‐224 was down‐regulated in osteosarcoma cell lines and tissues. Lower miR‐224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR‐224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG‐63 cells to cisplatin. We identified Rac1 as a direct target gene of miR‐224 in osteosarcoma. Rac1 expression was up‐regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR‐224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR‐224‐overexpressing MG‐63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR‐224‐overexpressing MG‐63 cells. These data suggest that miR‐224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin.     

Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2^kd/kd) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2^+/+) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2^kd/kd mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2^kd/kd neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2^kd/kd neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.