RhoGDIβ-induced hypertrophic growth in H9c2 cells is negatively regulated by ZAK

We found that overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. We further demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions. Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical interaction. Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ.     

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase–related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.     

Glucocorticoid induces mesenchymal-to-epithelial transition and inhibits TGF-β1-induced epithelial-to-mesenchymal transition and cell migration

Epithelial-to-mesenchymal transition (EMT) has been implicated in various physiological and pathological events. In this study, we found that the synthetic glucocorticoid dexamethasone (Dex) can inhibit transforming growth factor-beta1-induced EMT and cell migration. We also demonstrated that Dex inhibits EMT through a mechanism involving the suppression of ROS generation. Surprisingly, Dex alone induced mesenchymal-to-epithelial transition (MET). Dexamethasone treatment abolished Snail1 binding to the E-cadherin promoter, suggesting that suppression of Snail1 contributes to the above roles of Dex. Our findings demonstrate that Dex functions as both a suppressor of EMT and as an inducer of MET and therefore may be implicated in certain pathophysiological events.     

β2-Chimaerin binds to EphA receptors and regulates cell migration

Ephrins and Eph receptors have key roles in regulation of cell migration during development. We found that the RacGAP β2-chimaerin (chimerin) bound to EphA2 and EphA4 and inactivated Rac1 in response to ephrinA1 stimulation. EphA4 bound to β2-chimaerin through its kinase domain and promoted binding of Rac1 to β2-chimaerin. In addition, knockdown of endogenous β2-chimaerin blocked ephrinA1-induced suppression of cell migration. These results suggest that β2-chimaerin is activated by EphA receptors and mediates the EphA receptor-dependent regulation of cell migration.

Structured summary

MINT-7013428: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with Chimaerin beta 2 (uniprotkb:Q80XD1-2) and EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013515: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by anti tag coimmunoprecipitation (MI:0007)MINT-7013410: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with Chimaerin beta 1 (uniprotkb:Q80XD1-1) and EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013503: Chimaerin beta 1 (uniprotkb:Q80XD1-1) physically interacts (MI:0218) with EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)MINT-7013472: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with EphA2 (uniprotkb:O43921) by anti tag coimmunoprecipitation (MI:0007)MINT-7013450: EphA1 (uniprotkb:Q60750) physically interacts (MI:0218) with EphA2 (uniprotkb:O43921) and Chimaerin beta 2 (uniprotkb:P52757-1) by anti tag coimmunoprecipitation (MI:0007)MINT-7013491: Chimaerin beta 2 (uniprotkb:Q80XD1-2) physically interacts (MI:0218) with EphA4 (uniprotkb:O08542) by anti tag coimmunoprecipitation (MI:0007)     

Type I PIPK-α regulates directed cell migration by modulating Rac1 plasma membrane targeting and activation

Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) is a critical regulator of cell migration, but the roles of the type I phosphatidylinositol-4-phosphate 5-kinases (PIPKIs), which synthesize PI4,5P₂, have yet to be fully defined in this process. In this study, we report that one kinase, PIPKI-α, is a novel upstream regulator of Rac1 that links activated integrins to the regulation of cell migration. We show that PIPKI-α controls integrin-induced translocation of Rac1 to the plasma membrane and thereby regulates Rac1 activation. Strikingly, this function is not shared with other PIPKI isoforms, is independent of catalytic activity, and requires physical interaction of PIPKI-α with the Rac1 polybasic domain. Consistent with its role in Rac1 activation, depletion of PIPKI-α causes pronounced defects in membrane ruffling, actin organization, and focal adhesion formation, and ultimately affects the directional persistence of migration. Thus, our study defines the role of PIPKI-α in cell migration and describes a new mechanism for the spatial regulation of Rac1 activity that is critical for cell migration.     

Rac1 modulates TGF-β1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-β1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-β1, while N17TRac1 was inhibitory. TGF-β1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-β1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-β1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.     

αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion

The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin–RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvβ8 integrin–RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion.     

FERM domain-containing unconventional myosin VIIA interacts with integrin β5 subunit and regulates αvβ5-mediated cell adhesion and migration

Unconventional myosin VIIA (Myo7a) has been known to associate with hereditary deafness. Here we present a novel function of Myo7a by identifying that Myo7a directly interacts with integrin β5 subunit and regulates cell adhesion and motility in an integrin-dependent manner. We found that Myo7a bound to the cytoplasmic tail of integrin β5. Further, we pinpointed an integrin-binding domain at F3 of the first FERM domain and F1 of the second FERM domain. Functionally, Myo7a-induced cell adhesion and migration were mediated by integrin αvβ5. These findings indicated that Myo7a interacts with integrin β5 and selectively promotes integrin αvβ5-mediated cell migration.     

LRP6 regulates Rab7-mediated autophagy through the Wnt/β-catenin pathway to modulate trophoblast cell migration and invasion

Pre-eclampsia is a common complication during pregnancy; however, the underlying mechanisms of the crosstalk between low-density lipoprotein receptor-related protein 6 (LRP6) and autophagy in trophoblast cells are still not fully explored. Messenger RNA (mRNA) and protein levels of LRP6, beclin 1, Unc-51-like autophagy activating kinase 1 (ULK1), p62, vimentin, matrix metallopeptidase-9 (MMP-9), β-catenin, c-Myc, and Rab7, as well as the ratio of LC3-II/LC3-I, were analysed by quantitative real-time polymerase chain reaction or Western blot analysis, respectively. An MTT assay was used to measure cell growth, and transwell and wound healing assays were carried out to evaluate the invasion and migration abilities of the trophoblasts used. An immunofluorescence assay was used to measure LC3. The mRFP-GFP-LC3 tandem fluorescence assay was applied to detect autophagic flow. LRP6 overexpression was achieved by constructing pcDNA3.1-LRP6 vectors. LRP6 was expressed at low levels in HTR-8/SVneo cells under hypoxia/reoxygenation (H/R) conditions. H/R inhibited the activation of autophagy. LRP6 overexpression promoted cell proliferation and activated autophagy, which led to the upregulation of beclin 1 and ULK1, as well as the ratio of LC3-II/LC3-I and the downregulation of p62. Furthermore, LRP6 overexpression elevated the migration and invasion abilities of the indicated cells and increased vimentin and MMP-9 expression levels. Furthermore, LRP6 upregulated Rab7 and activated autophagy through the Wnt/β-catenin pathway. The late autophagy inhibitor bafilomycin A1 (Baf-A1) and the Wnt/β-catenin pathway inhibitor PKF115-584 reversed the effects of LRP6 on trophoblast autophagy, migration and invasion. LRP6 promotes Rab7-mediated autophagy by activating the Wnt/β-catenin pathway, which leads to increasing migration and invasion of trophoblast cells. Our study paves a new avenue for clinical treatment, and LRP6 may serve as an essential target in pre-eclampsia.     

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function.     

β-Actin specifically controls cell growth, migration, and the G-actin pool

Ubiquitously expressed β-actin and γ-actin isoforms play critical roles in most cellular processes; however, their unique contributions are not well understood. We generated whole-body β-actin-knockout (Actb(-/-)) mice and demonstrated that β-actin is required for early embryonic development. Lethality of Actb(-/-) embryos correlated with severe growth impairment and migration defects in β-actin-knockout primary mouse embryonic fibroblasts (MEFs) that were not observed in γ-actin-null MEFs. Migration defects were associated with reduced membrane protrusion dynamics and increased focal adhesions. We also identified migration defects upon conditional ablation of β-actin in highly motile T cells. Of great interest, ablation of β-actin altered the ratio of globular actin (G-actin) to filamentous actin in MEFs, with corresponding changes in expression of genes that regulate the cell cycle and motility. These data support an essential role for β-actin in regulating cell migration and gene expression through control of the cellular G-actin pool.     

Ligularia fischeri regulates lung cancer cell proliferation and migration through down-regulation of epidermal growth factor receptor and integrin β1 expression

In the present study, we report the effect and molecular mechanism of Ligularia fischeri (LF) on proliferation and migration in human lung cancer cells. LF-mediated inhibition of cell proliferation in p53 wild-type A549 and p53-deficient H1299 cells is accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G₁ phase cell cycle arrest. In contrast, LF inhibits cell migration in A549 cells, but not in H1299 cells. These regulatory effects of LF on cell proliferation and migration are associated with inactivation of mitogenic signaling pathways such as ERK, Akt and p70^S6K, and down-regulation of epidermal growth factor receptor and integrin β1 expression. Collectively, these findings suggest further development and evaluation of LF for the prevention and treatment of lung cancer with mutated p53 as well as wild-type p53.     

MicroRNA-34a regulates migration of chondroblast and IL-1β-induced degeneration of chondrocytes by targeting EphA5

MicroRNAs function as an endogenous mode of fine gene regulation and have been implicated in multiple differentiation and developmental processes. In the present study, we investigated the role of miRNA-34 during chondrogenic differentiation of chick limb mesenchymal cells. We found that the expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. Furthermore, we determined that EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory actions of miR-34 inhibition on cell migration and condensation of chick limb mesenchymal cells on collagen substrate. Taken together, our data suggest that miR-34a is a negative modulator of chondrogenesis, particularly in migration of chondroblasts, by targeting EphA5 and resulting inhibition of cellular condensation during chondrogenesis of chick limb mesenchymal cells.     

A c-Myc–SIRT1 feedback loop regulates cell growth and transformation

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1''s role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc''s transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression.     

Integrin α9β1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway

The α9β1 integrin accelerates cell migration through binding of the α9 cytoplasmic domain to SSAT, which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. SSAT levels were downregulated at both the mRNA and protein levels by shRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Further, SSAT knockdown in glioma xenograft cells significantly reduced their migration potential. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. The migratory potential of MMP-9/uPAR/cathepsin B-overexpressed 4910 and 5310 cells was not affected by either glybenclamide (Kir 6.x inhibitor) or tertiapin-Q (Kir 1.1 and 3.x inhibitor) but instead was significantly inhibited by either barium or Kir4.2 siRNA treatments. Co-localization of α9 integrin with Kir4.2 was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with Kir4.2. Taken together, our results clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and the Kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with Kir4.2 inward rectifier potassium channels.     

TGFβ-induced GRK2 expression attenuates AngII-regulated vascular smooth muscle cell proliferation and migration

Through diametric actions, the transforming growth factor β (TGFβ) and Angiotensin II (AngII) play important roles in regulating various biological responses such as cell proliferation and migration. Signaling initiated by TGFβ and AngII occurs through two structurally and functionally distinct receptor super families, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Previously, we identified the G protein-coupled receptor kinase-2 (GRK2), a key regulatory factor in the desensitization of GPCRs, as a direct downstream target of the TGFβ signaling cascade. GRK2 acts through a negative feed-back loop mechanism to terminate TGFβ-induced smad signaling. To investigate the impact of TGFβ-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). In this study, we show that activation of the TGFβ signaling cascade in VSMCs results in increased GRK2 expression levels, which consequently inhibits AngII-induced ERK phosphorylation and antagonizes AngII-induced VSMC proliferation and migration. Moreover, the inhibitory effect of TGFβ on AngII signaling occurs at the Mek-Erk interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFβ signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation and that GRK2 is a key factor mediating the cross-talk between these two receptor super families.     

TRIM45 negatively regulates NF-κB-mediated transcription and suppresses cell proliferation

The signaling adapter protein CRK is an indispensable molecule involved in regulating the malignant potential of human cancers. CRK-like (CRKL) is a hematopoietic cell-dominant homologue of CRK that is reported to be phosphorylated by BCR-ABL tyrosine kinase in chronic myelogenous leukemia patients, but its biological function in non-hematopoietic tumors remains unclear. In this study, we explored the tumorigenic role of CRKL in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Immunoprecipitation analysis of HNSCC cell line, HSC-3 cells, showed that the dominant binding partner for C3G was CRKL, not CRK. To clarify the molecular function of CRKL, we established lentiviral shRNA-mediated CRKL-knockdown HNSCC cell lines. In CRKL-knockdown HSC-3 and HSC-4 cells, cell growth and motility were diminished compared to control cells. Cell adhesion assays showed that cell attachment onto both fibronectin- and collagen-coated dishes was significantly suppressed in CRKL-knockdown HSC-3 cells, while no significant change was observed for poly-l-lysine-coated dishes. Immunofluorescence staining revealed that focal adhesion was reduced in CRKL-knockdown HSC-3 cells. With a pulldown assay, CRKL-knockdown HSC-3 cells showed decreased amounts of active Rap1 compared to control cells. Moreover, in an in vivo assay, tumor formation of CRKL-knockdown HSC-3 cells in nude mice was significantly abrogated. Our results indicate that CRKL regulates HNSCC-cell growth, motility, and integrin-dependent cell adhesion, suggesting that CRKL plays a principal role in HNSCC tumorigenicity.     

PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein–coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin–WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration.