Cellular Microenvironment ; Humans ; Myocardial Infarction ; metabolism ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the mechanisms of lysophosphatidic acid (LPA) in stimulating invasion and metastatic colonization of ovarian cancer cells.

METHODSThe metastatic ability in vivo of ovarian cancer SK-OV3, HEY, OVCAR3, and IGROV1 cells was determined in tumor-bearing nude mouse models. Matrigel assay was used to detect the changes of response in vitro of ovarian cancer cells to LPA after Rac(-) or Rac(+) adenovirus treatment. LPA-induced Rho GTPase activation was detected by GST-fusion protein binding assay.

RESULTSThe peritoneal metastatic colonization assay showed overt metastatic colonization in mice receiving SK-OV3 and HEY cell inoculation, indicating that they are invasive cells. Metastatic colonization was not detected in animals receiving OVCAR3 and IGROV1 cells, indicating that these cells are non-invasive cells. In the matrigel invasion assay, exposure to LPA led to a notably greater migratory response in metastatic SK-OV3 and HEY cells (Optical density: SK-OV3 cells: 0.594±0.023 vs. 1.697±0.049, P<0.01; HEY cells: 0.804±0.070 vs. 1.851±0.095, P<0.01). But LPA did little in the non-metastatic OVCAR3 and IGROV1 cells (Optical density A: OVCAR3 cells: 0.336±0.017 vs. 0.374±0.007, P>0.05; IGROV1 cells: 0.491±0.036 vs. 0.479±0.061, P>0.05). LPA migratory responses of ovarian cancer cells were closely related to their metastatic colonization capabilities (r = 0.983, P<0.05). Rac(-) blocked the LPA response of invasive SK-OV3 and HEY cells (LPA-induced fold increase of cell migration: SK-OV3 cells: 2.988±0.095 vs. 0.997±0.100,P=0.01; HEY cells: 2.404±0.059 vs. 0.901±0.072, P=0.01). But Rac(+) confered the non-invasive cells with LPA response and invasion capability (LPA-induced fold increase of cell migration: OVCAR3 cells: 1.072±0.080 vs. 1.898±0.078, P<0.01; IGROV1 cells: 1.002±0.044 vs. 2.141±0.057, P<0.05). Among Rho GTPases, only Rac activation was different between ovarian cancer cell lines with different metastatic capability after LPA stimulation: Cdc42 could not be activated in both the invasive and non-invasive cell lines. RhoA could be activated in both the invasive and non-invasive cell lines. Rac could be activated by LPA in the invasive ovarian cancer cell lines. However, Rac could not be activated in the non-invasive cell lines.

CONCLUSIONLysophosphatidic acid stimulates invasion and metastasis of ovarian cancer cells through Rac activation.

Animals ; Cell Movement ; Female ; Humans ; Lysophospholipids ; metabolism ; Mice ; Ovarian Neoplasms ; metabolism ; Tumor Cells, Cultured ; rho GTP-Binding Proteins ; rhoA GTP-Binding Protein

Erectile dysfunction (ED) has been plaguing men for a long time and the incidence of this disease is as high as 52% among males aged between 40 and 70. Recent discovery has shown a connection between the RhoA/Rho-kinase signaling system and ED. This paper reviews the progress in the study of RhoA/Rho-kinase signaling pathway, expounds its mechanism in penile erection and provides a base for further research on the role of RhoA/Rho-kinase signaling pathway in penile erection.
Animals ; Humans ; Male ; Penile Erection ; physiology ; Rabbits ; Signal Transduction ; physiology ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) points combined with nerve mobilization on protein and mRNA expression of RhoA in rabbits with sciatic nerve injury, and to provide theoretical basis for the treatment of peripheral nerve injury by EA at "Jiaji" (EX-B 2) points combined with nerve mobilization. METHODS: A total of 180 New Zealand rabbits were randomly divided into a normal control group, a model control group, a nerve mobilization group, an EA group, an EA plus nerve mobilization group, 36 rabbits in each group. Each group was further divided into a 1-week subgroup, 2-week subgroup and 4-week subgroup, 12 rabbits in each subgroup. The sciatic nerve injury model was made by clamping method. The rabbits in the normal control group did not receive any intervention. The rabbits in the model control group was normally fed after operation. The rabbits in the nerve mobilization group were treated with nerve mobilization; the manipulation lasted for 1 s and relaxed for 5 s, 10 times per day, 6 days per week. The rabbits in the EA group were treated with EA at "Jiaji" (EX-B 2) points (L-L), once a day, 30 min each time, 6 times per week. The rabbits in the EA plus nerve mobilization group were treated with EA at "Jiaji" (EX-B 2) points, followed by nerve mobilization. The function of sciatic nerve on the injured side was evaluated by toe tension reflex and modified Tarlov score; the tissues of corresponding segments of spinal cord L-L and sciatic nerve were taken; the expression of RhoA gene was detected by real-time PCR and the expression of RhoA protein was detected by Western Blot. RESULTS: ① Toe tension reflex and modified Tarlov score: at 1, 2 and 4 weeks, the scores in the model control group were lower than those in the normal control group (all <0.01). The scores in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group were higher than those in the model control group (all <0.01), and the scores in the subgroup of EA plus nerve mobilization group were higher than those in the nerve mobilization group and the EA group (all <0.01); the recovery was the best at 4 weeks. ② The mRNA and protein expression of RhoA: in segment of spinal cord, at 1, 2 and 4 weeks, the expression in the model control group was higher than that in the normal control group (all <0.01). The expression in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group was lower than that in the model control group (all <0.01), and the expression in the subgroup of EA plus nerve mobilization group was lower than that in the nerve mobilization group and the EA group (all <0.01); at 1 week and 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all <0.01); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all <0.01). In the sciatic nerve, at 1, 2 and 4 weeks, the expression in the model control group was higher than that in the normal control group (all <0.01). The expression in the subgroup of nerve mobilization group, EA group and EA plus nerve mobilization group was lower than that in the model control group (all <0.01); at 2 weeks and 4 weeks, the expression in the EA plus nerve mobilization group was lower than that in the nerve mobilization group and EA group (all <0.01); at 1 week, the expression in the nerve mobilization group was lower than that in the EA group and EA plus nerve mobilization group (all <0.01), but the differences between the EA group and the EA plus nerve mobilization group were not significant (>0.05); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all <0.01); at 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all <0.01). CONCLUSION The nerve mobilization and EA at "Jiaji" (EX-B 2) points could both promote the repair of injured sciatic nerve, which may be related to the down-regulation of RhoA expression, and the combination of the two methods has better effects.
Acupuncture Points ; Animals ; Chlorophenols ; Electroacupuncture ; Peripheral Nerve Injuries ; RNA, Messenger ; metabolism ; Rabbits ; Sciatic Nerve ; injuries ; rhoA GTP-Binding Protein

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.
*Aging ; Animal ; Brain/metabolism ; Calcium/pharmacology ; Cattle ; Comparative Study ; GTP-Binding Proteins/*metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Phosphorylation/drug effects ; Rats ; Rats, Sprague-Dawley ; Synaptic Membranes/*metabolism ; Synaptosomes/*metabolism ; cdc42 GTP-Binding Protein/biosynthesis/metabolism ; rab3A GTP-Binding Protein/metabolism ; rab5 GTP-Binding Proteins/metabolism ; rhoA GTP-Binding Protein/biosynthesis/metabolism

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.
*Aging ; Animal ; Brain/metabolism ; Calcium/pharmacology ; Cattle ; Comparative Study ; GTP-Binding Proteins/*metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Phosphorylation/drug effects ; Rats ; Rats, Sprague-Dawley ; Synaptic Membranes/*metabolism ; Synaptosomes/*metabolism ; cdc42 GTP-Binding Protein/biosynthesis/metabolism ; rab3A GTP-Binding Protein/metabolism ; rab5 GTP-Binding Proteins/metabolism ; rhoA GTP-Binding Protein/biosynthesis/metabolism

RhoA, a small GTPase, is involved in a wide array of cellular functions in the central nervous system, such as cell motility, cytoskeleton rearrangement, transcriptional regulation, phagocytosis and cell growth. It is not known how spinal cord injury (SCI) affects the expression of RhoA in different nerve cells. In the present study, we investigated the changes of RhoA expression in remote areas of the injury at the 3rd, 7th and 30th day after SCI, which was established by T10 contusion method. Moreover, we examine its expression profile in neurons, astrocytes and microglia. RhoA was found to be weakly expressed in these nerve cells in normal spinal cord. Western blotting showed that, after SCI, the total RhoA expression was up-regulated, and the RhoA expression was increased and peaked at the 7th day. Double immunostaining revealed specific and temporal expression patterns of RhoA in different nerve cells. The expression of RhoA in neurons started to increase at day 3, peaked at day 7 and then decreased slightly at day 30. Expression of RhoA in astrocytes increased moderately after SCI and peaked at day 7. There was no obvious change in RhoA expression in microglia after SCI in remote areas. This study demonstrated that, after SCI, RhoA expression exhibited different patterns with different nerve cells of spinal cord. RhoA expression patterns also changed with time after SCI, and among different nerve cells in the injured spinal cord. These findings can help us better understand the roles of RhoA in SCI.
Animals ; Astrocytes ; metabolism ; Blotting, Western ; Immunohistochemistry ; Male ; Microglia ; metabolism ; Microscopy, Confocal ; Neurons ; metabolism ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; Time Factors ; rhoA GTP-Binding Protein ; metabolism

SPRED1 protein coded by SPRED1 gene, a kind of tumors suppressor, belongs to Sprouty related protein family and mainly distributes in human brain. The activity of SPRED1 is mainly regulated by the tyrosine phosphorylation, which is stimulated by the hemopoietic factors. As an inhibitor of Ras-MAPK and RhoA cell signaling pathways, SPRED1 plays an important role in tumorigenesis and metastasis of solid tumor. Recently, the inactivation of SPRED1 is reported to result in proliferation, survival time extension and induction angiogenesis of AML cells. There is a clue that SPRED1 is highly related to leukemia genesis. Recently our study proved that the expression level of SPRED1 decreased in patients with acute myeloid leukemia (AML). This review summarizes the recent progress of study on the relationship between SPRED1 and AML, so as to explore the pathogenesis of leukemia and provide a new approach for clinical diagnosis.
Animals ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Membrane Proteins ; metabolism ; Repressor Proteins ; metabolism ; Signal Transduction ; rhoA GTP-Binding Protein ; metabolism

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.
Actins ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; immunology ; metabolism ; Blood Platelets ; metabolism ; Cells, Cultured ; Humans ; Phosphatidylinositol 3-Kinases ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism ; pharmacology

OBJECTIVEWhite matter damage (WMD) in preterm infants is a well-recognized serious complication of prematurity. The collapse of cell skeleton of growth cone after hypoxia-ischemia (HI) is considered as the basic neuropathologic change of the long-term residuals of premature white matter damage. F-actin is the major component of cell skeleton and maintains the normal form of cells, its function and potential mechanism of WMD have not been reported. In this study, changes of F-actin and its influencing factor RhoA were investigated.

METHODSTotally 184 Sprague-Dawley (SD) rats (age 2 days, body weight 6 to 8 grams) were randomly divided into 14 groups: 7 different time WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, 28 d) and 7 corresponding control groups. The 2 day-old SD rats were subjected to ligation of right carotid artery (ischemia), and then they were put into a box full with 6% oxygen and 94% nitrogen for 4 hours (hypoxia). The light microscopy was used to observe the brain pathological changes and the electron microscopy was used to detect the brain ultrastructural changes after hypoxia and ischemia. Eighty SD rats were used for flurescent-immunohistochemical method to detect the distribution of F-actin in cell membrane and cytoplasm of both WMD groups and the control groups at 12 h, 24 h, 48 h, 72 h, 7 d after HI respectively. The distribution of F-actin was reflected by the percentage of non-integrity cells. Another 80 SD rats were used for real time RT-PCR to detect the expression of RhoAmRNA in the white matter tissue of both WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d) and the control groups.

RESULTS(1) Necrosis of lateral ventricle tissue was observed by 72 h after HI. Dilatation of ventricle and formation of capsular space beneath white matter had been observed by 14 d after HI. (2) Disregulation, pyknosis, mitochondrion swelling and chromatin agglutination were observed in WMD groups. The maldevelopment of myelins in WMD groups was detected at 1 h after HI. (3) The fluorescent stains decreased on cellular membrane, but increased in cytoplasm with time. The percentage of non-integrity cells was significantly higher (P < 0.05) in HI groups (0.32 +/- 0.04, 0.43 +/- 0.04, 0.56 +/- 0.03, 0.65 +/- 0.04, 0.87 +/- 0.03) than the controls (0.02 +/- 0.01, 0.02 +/- 0.01, 0.01 +/- 0.01, 0.02 +/- 0.01, 0.02 +/- 0.01). (4) The expression of RhoA mRNA was significantly increased (P < 0.05) in HI groups (1.205, 2.415, 4.830, 1.500) in the white matter tissue compared with the controls (0.300, 0.375, 0.375, 0.530) at 12 h, 24 h, 48 h, 72 h after HI. The expression of RhoA mRNA reached the peak value at HI 48 h, and then gradually decreased. The expression of RhoA mRNA at HI 7 d in WMD group (0.500) was not significantly different from the control (P > 0.05).

CONCLUSION(1) The pathological and ultrastructural changes of white matter in WMD groups after HI suggest that the WMD model was successfully set up in premature 2 days SD rats. (2) F-actin is redistributed within cells after HI: expression in membrane is decreased and expression in plasma was increased. The redistribution possibly results in the collapse and retraction of cells. (3) The expression of RhoA mRNA is increased significantly after HI, which may lead to the redistribution of F-actin. (4) The increase of the expression of RhoA mRNA is not persistent, but the redistribution of F-actin is continued, which suggests that RhoA may not be the only factor affecting the redistribution of F-actin.

Actins ; genetics ; metabolism ; Animals ; Humans ; Hypoxia-Ischemia, Brain ; metabolism ; Infant ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; rhoA GTP-Binding Protein ; genetics ; metabolism