The role of Rho GTPases in the regulation of the rearrangement of actin cytoskeleton and cell movement

The rearrangement of the actin cytoskeleton has been shown to play a critical role in the development of transformation and malignant phenotype of cancer cells. Rho family GTPases regulate the arrangement of the actin cytoskeleton. By wound-healing assay, we have found that NIH 3T3 fibroblast cells move towards the wound- gaps by extending filopodial and lamellipdial structures at the leading edge of the moving cells. We have inactivated the function of Rho GTPases of v-Ras transformed NIH 3T3 cells by overexpressing Rho GTPase-activating (RhoGAP) domain of RhoGAP of p190. We have observed that inactivation of Rho, Rac and Cdc42 GTPases by overexpressing RHG causes inhibition of: (i) polymerization of actin to form filaments, (ii) formation of lamellipodia, filopodia and stress fibres, (iii) cell motility, (iv) cell spreading and (v) cell-to-cell adhesions. These results further strengthen the current knowledge on the role of Rho, Rac and Cdc42 GTPases in the regulation of the rearrangement of actin cytoskeleton. Our results, for the first time, demonstrate that RhoGAP domain of RhoGAP could be used to study the molecular mechanism of Ras-mediated signalling in growth, differentiation and carcinogenesis.     

bPAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin

p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.     

Role of mTOR signaling in tumor cell motility, invasion and metastasis

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.     

Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components

Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.     

Phagocytosis induces superoxide formation and apoptosis in macrophages

Phagocytosis by inflammatory cells is an essential step and a part of innate immunity for protection against foreign pathogens, microorganism or dead cells. Phagocytosis, endocytotic events sequel to binding particle ligands to the specific receptors on phagocyte cell surface such as Fcgamma recptor (FcgammaR), complement receptor (CR), beta-glucan receptor, and phosphatidylserine (PS) receptor, require actin assembly, pseudopod extension and phagosome closure. Rho GTPases (RhoA, Cdc42, and Rac1) are critically involved in these processes. Abrupt superoxide formation, called as oxidative burst, occurs through NADPH oxidase complex in leukocytes following phagocytosis. NADPH oxidase complex is composed of membrane proteins, p22PHOX and gp91PHOX, and cytosolic proteins, p40PHOX, p47PHOX and p67PHOX. The cytosolic subunits and Rac-GTP are translocated to the membrane, forming complete NADPH oxidase complex with membrane part subunits. Binding of imunoglobulin G (IgG)- and complement-opsonized particles to FcgammaR and CR of leukocytes induces apoptosis of the cells, which may be due to oxidative burst and accompanying cytochrome c release and casapase-3 activation.     

Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells

AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases K_ATP currents by increasing the surface levels of K_ATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced K_ATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of K_ATP channel activity and membrane potentials revealed that AMPK-dependent K_ATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of K_ATP channels to the plasma membrane in pancreatic β-cells.     

The concentration of oleocanthal in olive oil waste

The aim of this study was to determine the concentration of oleocanthal in olive pomace waste and compare this to its concentration in extra-virgin olive oil (EVOO). The concentration of oleocanthal in freshly pressed EVOO and its subsequent waste was analysed at early, mid and late season harvests. Oleocanthal concentrations were quantified using high-performance liquid chromatography-mass spectrometry. In oil, oleocanthal concentration was as follows: 123.24 ± 6.48 mg kg(-1) in early harvest, 114.20 ± 17.42 mg kg(-1) in mid harvest and 152.22 ± 10.54 mg kg(-1) in late harvest. Its concentration in waste was determined to be: 128.25 ± 11.33 mg kg(-1) in early harvest, 112.15 ± 1.51 mg kg(-1) in mid harvest and 62.35 ± 8.00 mg kg(-1) in late harvest. Overall, olive pomace waste is a valuable source of oleocanthal.     

Fungus-Derived 3-Hydroxyterphenyllin and Candidusin A Ameliorate Palmitic Acid-Induced Human Podocyte Injury via Anti-Oxidative and Anti-Apoptotic Mechanisms

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. An elevated fatty acid plasma concentration leads to podocyte injury and DN progression. This study aimed to identify and characterize cellular mechanisms of natural compounds that inhibit palmitic acid (PA)–induced human podocyte injury. By screening 355 natural compounds using a cell viability assay, 3-hydroxyterphenyllin (3-HT) and candidusin A (CDA), isolated from the marine-derived fungus Aspergillus candidus PSU-AMF169, were found to protect against PA-induced podocyte injury, with half-maximal inhibitory concentrations (IC₅₀) of ~16 and ~18 µM, respectively. Flow cytometry revealed that 3-HT and CDA suppressed PA-induced podocyte apoptosis. Importantly, CDA significantly prevented PA-induced podocyte barrier impairment as determined by 70 kDa dextran flux. Reactive oxygen species (ROS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) direct scavenging assays indicated that both compounds exerted an anti-oxidative effect via direct free radical–scavenging activity. Moreover, 3-HT and CDA upregulated the anti-apoptotic Bcl2 protein. In conclusion, 3-HT and CDA represent fungus-derived bioactive compounds that have a novel protective effect on PA-induced human podocyte apoptosis via mechanisms involving free radical scavenging and Bcl2 upregulation.     

Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection.

The molecular link between the signalling pathway regulating the formation of filopodia and the initiation of local actin polymerization has been elucidated: N-WASP, a close homologue of WASP, which is the product of the gene responsible for the Wiskott-Aldrich syndrome, mediates a direct connection between the small G-protein Cdc42 and the Arp2/3 complex.     

Blue light-induced chloroplast reorientations in Lemna trisulca L. (duckweed) are controlled by two separable cellular mechanisms as suggested by different sensitivity to wortmannin

Chloroplast reorientations within mesophyll cells are among the most rapid physiological responses of higher plants to blue light. At light intensities below the saturation point of photosynthesis, chloroplasts move to the cell walls perpendicular to the direction of light and maximize light absorption (low-fluence rate response [LFR]). At light intensities above the saturation point of photosynthesis, chloroplasts redistribute to cell walls parallel to the direction of light (high-fluence rate response [HFR]). The actin-based mechanism is responsible for the light-induced chloroplast movements. We have found that an inhibitor of phosphoinositide-3-kinases, wortmannin, potently and irreversibly inhibited LFR and HFR chloroplast responses to blue light in Lemna trisulca L. mesophyll cells. Microscopic observations and photometric measurement indicated that 100 nM wortmannin specifically inhibited LFR in Lemna, whereas HFR displayed no sensitivity to the inhibitor at this concentration. A complete inhibition of the HFR could be obtained by 1 microM wortmannin. These data indicate that LFR is more sensitive to wortmannin than HFR and suggest that these two responses may be under the control of different cellular mechanisms. Our results suggest that phosphoinositide kinases and other phosphoinositide cycle enzymes may play a role in the transduction of the light signal to the actin cytoskeleton in Lemna as factors specifying the direction of chloroplast movements. A hypothetical model assuming three signaling pathways regulating light-induced chloroplast reorientations in mesophyll cells is proposed.     

Molecular cloning and sequencing of rat Cdc42 GTPase cDNA

Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.     

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin- damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced- tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA- PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.     

Ezrin Modulates the Cell Surface Expression of Programmed Cell Death Ligand-1 in Human Cervical Adenocarcinoma Cells

Cancer cells employ programmed cell death ligand-1 (PD-L1), an immune checkpoint protein that binds to programmed cell death-1 (PD-1) and is highly expressed in various cancers, including cervical carcinoma, to abolish T-cell-mediated immunosurveillance. Despite a key role of PD-L1 in various cancer cell types, the regulatory mechanism for PD-L1 expression is largely unknown. Understanding this mechanism could provide a novel strategy for cervical cancer therapy. Here, we investigated the influence of ezrin/radixin/moesin (ERM) family scaffold proteins, crosslinking the actin cytoskeleton and certain plasma membrane proteins, on the expression of PD-L1 in HeLa cells. Our results showed that all proteins were expressed at mRNA and protein levels and that all ERM proteins were highly colocalized with PD-L1 in the plasma membrane. Interestingly, immunoprecipitation assay results demonstrated that PD-L1 interacted with ERM as well as actin cytoskeleton proteins. Furthermore, gene silencing of ezrin, but not radixin and moesin, remarkably decreased the protein expression of PD-L1 without affecting its mRNA expression. In conclusion, ezrin may function as a scaffold protein for PD-L1; regulate PD-L1 protein expression, possibly via post-translational modification in HeLa cells; and serve as a potential therapeutic target for cervical cancer, improving the current immune checkpoint blockade therapy.     

Involvement of ��PIX in angiotensin II-induced migration of vascular smooth muscle cells

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a βPIX GEF-dependent manner. βPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of βPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of βPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of βPIX-Rac1. Taken together, our results suggest that βPIX is involved in Ang II-induced VSMC migration.     

3,6-二取代三唑并噻二唑衍生物的合成及其对细胞分裂周期25B磷酸酶和蛋白酪氨酸磷酸酶1B抑制活性评价

合成出了一系列含苯并咪唑/芳氧甲基骨架的3,6-二取代三唑并噻二唑衍生物3a~3l,其结构经傅里叶变换红外光谱仪(FT-IR)、核磁共振波谱仪(NMR)和元素分析得以确认。 评价了它们对细胞分裂周期25B磷酸酶(Cdc25B)/蛋白酪氨酸磷酸酶1B(PTP1B)的抑制活性,讨论了构效关系。 生物活性测试结果显示,化合物3a对Cdc25B和PTP1B的抑制活性最高,其半数抑制浓度(IC₅₀)值分别为(0.46±0.02) μg/mL和(1.77±0.40) μg/mL。 所得研究结果为开发新型Cdc25B/PTP1B抑制剂提供了参考依据。     

糖尿病肾病患者AR基因表达量测定

对50例正常人和80名糖尿病肾病不同分期的患者进行醛糖还原酶(Aldose Reductase, AR)基因的表达量测定, 提示AR基因有望成为糖尿病肾病(DN)早期诊断的生物标志物以及DN治疗上潜在的药物靶点, 并且利用AR基因对DN的中医诊断进行了分子生物学验证.     

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.     

Steering cell migration using microarray amplification of natural directional persistence

Cell locomotion plays a key role in embryonic morphogenesis, wound healing, and cancer metastasis. Here we show that intermittent control of cell shape using microarrays can be used to amplify the natural directional persistence of cells and guide their continuous migration along preset paths and directions. The key to this geometry-based, gradient-free approach for directing cell migration is the finding that cell polarization, induced by the asymmetric shape of individual microarray islands, is retained as cells traverse between islands. Altering the intracellular signals involved in lamellipodia extension (Rac1), contractility (RhoA), and cell polarity (Cdc42) alters the speed of fibroblast migration on these micropatterns but does not affect their directional bias significantly. These results provide insights into the role of cell morphology in directional movement and the design of micropatterned materials for steering cellular traffic.     

基于咔唑的单-/双-硫代碳酰腙衍生物的合成及Cdc25B/PTP1B抑制活性评价

合成了一系列新型的基于咔唑的单-/双-硫代碳酰腙衍生物.利用IR、1H NMR、13C NMR和元素分析对其进行了结构表征.评价了目标化合物对Cdc25B和PTP1B的抑制活性,讨论了其结构与活性的关系.实验结果显示,大部分目标化合物对Cdc25B和PTP1B表现出良好的抑制活性.其中,1,5-双[(9-戊基-3-咔唑基)亚甲基]硫代碳酰腙(4d)对Cdc25B的抑制活性最高,IC50为(0.23±0.02)μg/m L.1,5-双[(9-乙基-3-咔唑基)亚甲基]硫代碳酰腙(4a)对PTP1B的抑制活性最高, IC50为(1.00±0.16)μg/m L.对目标化合物4a和4d进行分子对接研究和密度泛函理论(DFT)计算,结果表明,目标化合物4d和4a分别进入到了Cdc25B和PTP1B酶的活性位点区域,有活性作用的主要是硫代碳酰腙和咔唑基团.