ABSTRACT
<p><b>OBJECTIVE</b>To test the effect of CDC42 (a member of Rho family of small GTPases) knockdown mediated by a CDC42 short-hairpin RNA (shRNA) on the morphology of colorectal cancer SW480 cells in vitro.</p><p><b>METHODS</b>Four CDC42 siRNA fragments targeting CDC42 were designed and the most efficient siRNA for CDC42 knockdown was selected to construct the shRNA vector for transfection of colorectal cancer SW480 cells. The interference efficiency in the stably transfected cells (sw480.shCDC) was detected using real-time PCR and Western blotting, and the morphological changes of the transfected cells were observed.</p><p><b>RESULTS</b>Western blotting result showed that siCDC42-3 was the most efficient fragment for CDC42 knockdown, which caused CDC42 knockdown by over 50%. DNA sequencing confirmed successful construction of the CDC42 shRNA vector. Transfection of the cells with the vector significantly reduced CDC42 expressions at both the mRNA and protein levels. The transfected cells exhibited reduced filopodia and cell size with smooth cell margins.</p><p><b>CONCLUSION</b>shRNA-mediated CDC42 knockdown can reduce the cytoskeleton dynamics of colorectal cancer cells to lower their invasiveness. This shRNA construct facilitates further study of the role of CDC42 in the tumorigenesis and progression of colorectal cancer.</p>
Subject(s)
Humans , Cell Line, Tumor , Colorectal Neoplasms , Genetics , Gene Knockdown Techniques , RNA Interference , RNA, Messenger , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Transfection , cdc42 GTP-Binding Protein , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Cdc42-shRNA plasmid to proliferation, migration, invasion and other malignant biological behavior in hepatoma SMMC-7721 cells.</p><p><b>METHODS</b>Cdc42-shRNA interfering vector transfected to SMMC-7721 cells with liposome method. The growth curve of transfected cells SMMC-7721, U6-control, Cdc42-shRNA2 was detected by MTT. The cells mobility was detected by wound healing experiment. Transwell chamber experiments to observe the cell migration and invasion. Detected AFP and PCNA expression level by Western blot.Human hepatoma SMMC-7721 transplanted subcutaneously in nude mouse, detected the expression of Cdc42 of the tumor by immunohistochemistry.t test was used to analyze the data between two groups.</p><p><b>RESULTS</b>The doubling time of Cdc42-shRNA2, U6-control and SMMC7721 was 42.7 h, 34.9 h and 35.1 h. The relative migration distance of Cdc42-shRNA2 and U6-control on 36 h was (47.1 ± 4.1)% and (86.6 ± 5.3)% (t=-10.21, P<0.05). Transwell chamber experimental methods showed the numbers of permeating cells were 18.2 ± 2.1(Cdc42-shRNA2) and 41.0 ± 3.5 (U6-control) (t=-9.67, P<0.05) on 24 h. The AFP and PCNA expression of hepatoma cells is significantly inhibited after the Cdc42-shRNA2 was transfected compared with U6-control group.The tumor average weight of group Cdc42-shRNA2 was (335.1 ± 178.2) mg, which was much lighter than that of SMMC-7721 group ((925.3 ± 241.4) mg) and U6-control group ((910.5 ± 225.6) mg) (t=-4.47, -4.39; P<0.05) and the Cdc42 expression was also weak positive.</p><p><b>CONCLUSION</b>Cdc42 interfere with plasmid significant changes in human malignant biological behavior of hepatocellular carcinoma cells, and reduces liver cancer cell growth, invasion and metastasis of capacity.</p>
Subject(s)
Animals , Humans , Mice , Carcinoma, Hepatocellular , Cell Line, Tumor , Cell Movement , Cell Proliferation , Liver Neoplasms , Mice, Nude , Plasmids , RNA, Small Interfering , Transfection , Tumor Burden , cdc42 GTP-Binding ProteinABSTRACT
OBJECTIVE@#To explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons.@*METHODS@#In this study, expression of Slit1 was observed predominantly in the glia, while expression of Robo2 and srGAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion (DRG). Furthermore, upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting.@*RESULTS@#It was observed that inhibition of neurite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively (1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody. It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-GTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3. These data demonstrated a role for srGAP3 in neurite outgrowth of DRG sensory neurons.@*CONCLUSIONS@#Our observations suggest that srGAP3 promotes neurite outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.
Subject(s)
Animals , Rats , GTPase-Activating Proteins , Metabolism , Ganglia, Spinal , Cell Biology , Wounds and Injuries , Metabolism , Neurites , Metabolism , Neurons , Metabolism , Rats, Sprague-Dawley , Signal Transduction , Physiology , cdc42 GTP-Binding Protein , Metabolism , rac1 GTP-Binding Protein , MetabolismABSTRACT
BACKGROUND: Liver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism. RESULTS: Protein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR. CONCLUSIONS: For the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.
Subject(s)
Animals , Rats , Proteomics , Hepatectomy , Liver/metabolism , Liver Regeneration/physiology , Time Factors , Protein Biosynthesis/physiology , Body Weight/physiology , Electrophoresis, Gel, Two-Dimensional , Signal Transduction/physiology , Random Allocation , Blotting, Western , Rats, Sprague-Dawley , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , cdc42 GTP-Binding Protein/metabolism , 14-3-3 Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Carbohydrate Metabolism/physiology , Lipid Metabolism/physiology , Liver/anatomy & histologyABSTRACT
The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain. Cdc42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture. Previous studies have shown that Cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage (before E12.5). However, the functions of Cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear. Thus, in order to address the role of Cdc42 in the whole brain in later embryo developmental stage or in adulthood, we used Cre/loxP technology to generate two lines of tissue-specific Cdc42-knock-out mice. Inactivation of Cdc42 was achieved in neuroepithelial cells by crossing Cdc42/ flox mice with Nestin-Cre mice and resulted in hydrocephalus, causing death to occur within the postnatal stage. Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted, resulting in aqueductal stenosis. Deletion of Cdc42 in the cerebral cortex also induced obvious defects in interkinetic nuclear migration and hypoplasia. To further explore the role of Cdc42 in adult mice brain, we examined the effects of knocking-out Cdc42 in radial glial cells by crossing Cdc42/flox mice with human glial fibrillary acidic protein (GFAP)-Cre mice. Inactivation of Cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation. Taken together, these results highlight the importance of Cdc42 for ependymal cell differentiation and maintaining, and suggest that these functions likely contribute to the essential roles played by Cdc42 in the development of the brain.
Subject(s)
Animals , Humans , Mice , Brain , Metabolism , Pathology , Cell Differentiation , Cell Polarity , Cerebral Cortex , Cell Biology , Metabolism , Constriction, Pathologic , Embryo, Mammalian , Metabolism , Embryonic Development , Ependyma , Cell Biology , Metabolism , Glial Fibrillary Acidic Protein , Genetics , Metabolism , Hydrocephalus , Metabolism , Pathology , Integrases , Genetics , Metabolism , Mice, Knockout , cdc42 GTP-Binding Protein , Genetics , MetabolismABSTRACT
Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Subject(s)
Animals , Humans , Rats , Albumins/metabolism , Androstadienes/administration & dosage , Creatinine/blood , Desmin/genetics , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Disease Models, Animal , Intracellular Signaling Peptides and Proteins/genetics , Kidney/pathology , Membrane Proteins/genetics , Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Podocytes/drug effects , Rats, Inbred Strains , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
Influenza virus assembly requires the completion of viral protein and vRNP transport to the assembly site at the plasma membrane. Therefore, efficient regulation of intracellular transport of the viral proteins and vRNPs to the surface of the host cell is especially important for virus morphogenesis. Influenza A virus uses the machineries of host cells to transport its own components including ribonucleoproteins (vRNPs) and three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and matrix 2 protein (M2). It has been shown that newly synthesized vRNPs are associated with active form of Rab11 and accumulate at recycling endosomes adjacent to the microtubule organizing center (MTOC) following nuclear export. Subsequently, they are transported along the microtubule network toward the plasma membranes in cargo vesicles. The viral transmembrane proteins are translated on the rough endoplasmic reticulum and transported to the virus assembly site at the plasma membrane. It has been found that several host factors such as ARHGAP21 and GTPase Cdc42 are involved in regulation of intracellular trafficking of influenza A virus transmembrane proteins including NA. In this review, we will highlight the current knowledge about anterograde transport and its regulation of the influenza A virus transmembrane proteins and genome in the host cytoplasm.
Subject(s)
Humans , Cytoplasm , Metabolism , GTP Phosphohydrolases , Metabolism , GTPase-Activating Proteins , Metabolism , Genome, Viral , Hemagglutinin Glycoproteins, Influenza Virus , Metabolism , Influenza A virus , Genetics , Virulence , Physiology , Neuraminidase , Metabolism , Protein Transport , Ribonucleoproteins , Metabolism , Viral Matrix Proteins , Metabolism , cdc42 GTP-Binding Protein , MetabolismABSTRACT
<p><b>BACKGROUND</b>High microvascular permeability plays an essential role in pathological process of multiple diseases such as septic shock, acute lung injury and acute respiratory distress syndrome, and burns. Inhibiting hyperpermeability is significant for controlling these conditions. Cdc42, as a main member of the small Rho GTPase family, plays a critical role in controlling and regulating the endothelial junctional permeability. We aimed to generate and identify endothelial specific cdc42-deficient mice by the Cre/loxp recombination approach, for examination in an animal model of the contribution of the cdc42 gene in the microvascular barrier function.</p><p><b>METHODS</b>We crossed cdc42(Flox/Flox) mice with mice expressing endothelial cell-specific Cre recombinase, and the offspring with the genotype cdc42(Flox/+)Tie2Cre(+/-) were back-crossed with the cdc42(Flox/Flox) mice. The cdc42(Flox/Flox)Tie2Cre(+/-) mice in the F2 generation were the target mice. If the cdc42 deficient mice did not survive, we would observe the cdc42 deficient mice embryos, and compare them with wild-type mice embryos.</p><p><b>RESULTS</b>Cdc42(flox/+)Cre(+/-) mice were mated with the cdc42(Flox/Flox) mice and among the living offspring there were no cdc42(Flox/Flox)Cre(+/-) target mice. We found the endothelial special cdc42 deficient embryos at the E7.5-E16.5 stage. We observed that cdc42 deficient embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos.</p><p><b>CONCLUSIONS</b>Endothelial specific knockout of cdc42 caused embryonic lethality and the mice did not survive to birth. The target embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. These results demonstrated that the cdc42 plays an important role in development of embryos and in development of microvessels as well as microvascular permeability.</p>
Subject(s)
Animals , Female , Male , Mice , Embryo, Mammalian , Metabolism , Endothelium, Vascular , Embryology , Metabolism , Immunohistochemistry , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Physiologic , Genetics , Physiology , cdc42 GTP-Binding Protein , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To compare the change of lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice in acute lung injury.</p><p><b>METHODS</b>The mice with vascular endothelial cell-specific expression of cre recombinase were crossed with cdc42(flox/flox) mice. The cdc42(flox/+)Cre(+/-) F1 offspring mice were crossed back with cdc42(flox/flox) mice, resulting in the F2 generation mice with three genotypes, namely cdc42(flox/+)Cre(+/-), cdc42(flox/flox)Cre(-/-) and cdc42(flox/+)Cre(+/-). The heterozygous mice with cdc42(flox/+)Cre(+/-) genotype were selected as the model mice, with the other two genotype groups as the control. After intratracheal instillation of 2 mg/kg LPS to induce acute lung injury, the mice were sacrificed to examine the lung pathologies, lung wet/dry ratio and lung microvascular permeability.</p><p><b>RESULTS</b>The heterozygous mice with cdc42 gene knockout (cdc42(flox/+)Cre(+/-)) showed no significant differences from the two control groups in the lung pathological score, lung wet/dry ratio or the lung microvascular permeability coefficient.</p><p><b>CONCLUSION</b>There were no significant difference on lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice.</p>
Subject(s)
Animals , Mice , Acute Lung Injury , Pathology , Capillary Permeability , Endothelial Cells , Pathology , Integrases , Genetics , Lung , Pathology , Mice, Knockout , cdc42 GTP-Binding Protein , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of Cdc42 expression under estrogen stimulation, and to explore the signaling pathway of intracellular material transportation caused by estrogen.</p><p><b>METHODS</b>MTT was used to test the drug sensitivity of cells. Real-time PCR was used to evaluate the expression of Cdc42 mRNA. The amount of ADM accumulated in MCF-7 cells was detected by flow cytometry. The protein levels of active-Cdc42 and Total-Cdc42 were measured by Western blot.</p><p><b>RESULTS</b>IC(50) of ADM in MCF-7 cells was increased from (0.098 ± 0.011) µg/ml to (0.134 ± 0.130) µg/ml (P < 0.05) after estrogen stimulation. The amount of ADM accumulated in MCF-7 cells was reduced from 7.253 ± 0.310 to 3.233 ± 0.313 (P < 0.05). All of Cdc42 mRNA, active-Cdc42 protein and total-Cdc42 protein were increased (P < 0.05). After the treatment with siRNA, the IC(50) of ADM in siRNA group was decreased to (0.057 ± 0.017) µg/ml (P < 0.05) compared with that in the control group. The amount of accumulated ADM was significantly increased in the siRNA group, and all the expression levels of Cdc42 mRNA, active-Cdc42 protein and total-Cdc42 protein were decreased in the siRNA group (P < 0.05).</p><p><b>CONCLUSIONS</b>Estrogen enhances the drug resistance in breast cancer cells. The mechanism of this effect may be via the enhancing Cdc42 expression and decreasing the accumulation of chemotherapeutic drugs in the cancer cells.</p>
Subject(s)
Female , Humans , Antibiotics, Antineoplastic , Metabolism , Pharmacology , Breast Neoplasms , Genetics , Metabolism , Pathology , Cell Line, Tumor , Doxorubicin , Metabolism , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Estrogens , Pharmacology , Inhibitory Concentration 50 , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Transfection , cdc42 GTP-Binding Protein , Genetics , MetabolismABSTRACT
<p><b>BACKGROUND AND OBJECTIVE</b>MicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells.</p><p><b>METHODS</b>We detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion.</p><p><b>RESULTS</b>Similar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05).</p><p><b>CONCLUSIONS</b>Members of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.</p>
Subject(s)
Animals , Humans , Mice , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , MicroRNAs , Genetics , Metabolism , NIH 3T3 Cells , Neoplasm Invasiveness , Phosphorylation , Stomach Neoplasms , Genetics , Metabolism , Pathology , Transfection , cdc42 GTP-Binding Protein , Metabolism , p21-Activated Kinases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques.</p><p><b>METHODS</b>Using isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins.</p><p><b>RESULTS</b>Significant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP_040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis.</p><p><b>CONCLUSION</b>Loss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.</p>
Subject(s)
Female , Humans , Male , Calmodulin , Metabolism , Colorectal Neoplasms , Metabolism , Pathology , Electrophoresis, Gel, Two-Dimensional , Globins , Metabolism , Isoelectric Focusing , Liver Neoplasms , Metabolism , Peptide Mapping , Proteome , Metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , cdc42 GTP-Binding Protein , MetabolismABSTRACT
Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively
Subject(s)
Humans , Animals , Cadherins , Cell-Matrix Junctions , Integrins , rho GTP-Binding Proteins , cdc42 GTP-Binding Protein , Cell Adhesion , Cytoskeleton , rac1 GTP-Binding Protein , rho GTP-Binding Proteins , rhoA GTP-Binding Protein , Signal TransductionABSTRACT
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
Subject(s)
Cattle , Rats , Aging , Animals , Brain/metabolism , Calcium/pharmacology , Comparative Study , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Molecular Weight , Phosphorylation/drug effects , Rats, Sprague-Dawley , Synaptic Membranes/metabolism , Synaptosomes/metabolism , cdc42 GTP-Binding Protein/biosynthesis , rab3A GTP-Binding Protein/metabolism , rab5 GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/biosynthesisABSTRACT
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
Subject(s)
Cattle , Rats , Aging , Animals , Brain/metabolism , Calcium/pharmacology , Comparative Study , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Molecular Weight , Phosphorylation/drug effects , Rats, Sprague-Dawley , Synaptic Membranes/metabolism , Synaptosomes/metabolism , cdc42 GTP-Binding Protein/biosynthesis , rab3A GTP-Binding Protein/metabolism , rab5 GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/biosynthesisABSTRACT
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.