HCF-1 promotes cell cycle progression by regulating the expression of CDC42.

The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the -881 to -575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.

The bacterial protein CNF1 as a new strategy against Plasmodium falciparum cytoadherence.

Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.

Cdc42 Is Essential for Both Articular Cartilage Degeneration and Subchondral Bone Deterioration in Experimental Osteoarthritis.

Cdc42, a member of Rho family small guanosine triphosphatases (GTPases), is critical for cartilage development. We investigated the roles of Cdc42 in osteoarthritis and explored the potential mechanism underlying Cdc42-mediated articular cartilage degeneration and subchondral bone deterioration. Cdc42 is highly expressed in both articular cartilage and subchondral bone in a mouse osteoarthritis model with surgical destabilization of the medial meniscus (DMM) in the knee joints. Specifically, genetic disruption of Cdc42, knockdown of Cdc42 expression, or inhibition of Cdc42 activity robustly attenuates the DMM-induced destruction, hypertrophy, high expression of matrix metallopeptidase-13 and collagen X, and activation of Stat3 in articular cartilages. Notably, genetic disruption of Cdc42, knockdown of Cdc42 expression or inhibition of Cdc42 activity significantly restored the increased numbers of mesenchymal stem cells, osteoprogenitors, osteoblasts, osteoclasts, and neovascularized vessels, the increased bone mass, and the activated Erk1/2, Smad1/5 and Smad2 in subchondral bone of DMM-operated mice. Mechanistically, Cdc42 mediates interleukin-1ß-induced interleukin-6 production and subsequent Jak/Stat3 activation to regulate chondrocytic inflammation, and also lies upstream of Erk/Smads to regulate subchondral bone remodeling during transform growth factor-ß1 signaling. Cdc42 is apparently required for both articular cartilage degeneration and subchondral bone deterioration of osteoarthritis, thus, interventions targeting Cdc42 have potential in osteoarthritic therapy. © 2018 American Society for Bone and Mineral Research.

High Expression of Cell Division Cycle 42 Promotes Pancreatic Cancer Growth and Predicts Poor Outcome of Pancreatic Cancer Patients.

BACKGROUND: Cell division cycle 42 (CDC42), an important member of the Rho family, is overexpressed in various human cancers. However, its expression and role in pancreatic cancer (PC) are not well understood. AIM: The present study was designed to investigate the expression patterns and underlying cellular mechanisms of CDC42 in PC. METHODS: First, immunohistochemical analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect CDC42 expression in clinical pancreatic carcinoma and adjacent tissues. Second, differential expression of CDC42 between PC cells and normal cells was evaluated by qRT-PCR and Western blotting. Third, the correlation between CDC42 expression as well as clinicopathological characteristics and patient survival was analyzed. Finally, CDC42 was knocked down to examine its role both in vivo and in vitro. RESULTS: The results showed significantly increased CDC42 expression in pancreatic tumor tissues compared with adjacent normal tissues, as revealed by qRT-PCR, Western blotting and immunostaining. Compared to PanC-1 cells, CDC42 expression was downregulated in HPDE6-C7 cells as shown by qRT-PCR and Western blotting. High CDC42 expression was observed in 69.2% (83/120) of pancreatic adenocarcinoma patients and was significantly associated with tumor differentiation (p = 0.013), median tumor size (p = 0.005), tumor infiltration (pT stage, p = 0.04), lymph nodal status (pN stage, p = 0.044) and TNM staging (p = 0.003). Multivariate Cox regression analysis revealed CDC42 expression to be an independent predictor of survival of PC patients (HR 3.0, 95% CI 1.60-5.61, p = 0.001). Finally, we found that CDC42 promoted the proliferation of PanC-1 cells both in vivo and in vitro. CONCLUSIONS: Our findings reveal that CDC42 might play an important role in promoting PC development, and the findings suggest that CDC42 might serve as a potential prognostic indicator of PC.

Cdc42 Promotes Schwann Cell Proliferation and Migration Through Wnt/ß-Catenin and p38 MAPK Signaling Pathway After Sciatic Nerve Injury.

Schwann cells (SCs) are unique glial cells in the peripheral nerve and may secrete multiple neurotrophic factors, adhesion molecules, extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration, guiding and supporting nerve proliferation and migration. Cdc42 plays an important regulatory role in dynamic changes of the cytoskeleton. However, there is a little study referred to regulation and mechanism of Cdc42 on glial cells after peripheral nerve injury. The present study investigated the role of Cdc42 in the proliferation and migration of SCs after sciatic nerve injury. Cdc42 expression was tested, showing that the mRNA and protein expression levels of Cdc42 were significantly up-regulated after sciatic nerve injury. Then, we isolated and purified SCs from injuried sciatic nerve at day 7. The purified SCs were transfected with Cdc42 siRNA and pcDNA3.1-Cdc42, and the cell proliferation, cell cycle and migration were assessed. The results implied that Cdc42 siRNA remarkably inhibited Schwann cell proliferation and migration, and resulted in S phase arrest. While pcDNA3.1-Cdc42 showed a contrary effect. Besides, we also observed that Cdc42 siRNA down-regulated the protein expression of ß-catenin, Cyclin D1, c-myc and p-p38, which were up-regulated by pcDNA3.1-Cdc42. Meanwhile, the inhibitor of Wnt/ß-catenin and p38 MAPK signaling pathway IWP-2 and SB203580 significantly inhibited the effect of pcDNA3.1-Cdc42 on cell proliferation and migration. Overall, our data indicate that Cdc42 regulates Schwann cell proliferation and migration through Wnt/ß-catenin and p38 MAPK signaling pathway after sciatic nerve injury, which provides further insights into the therapy of the sciatic nerve injury.

Bif-1 promotes tumor cell migration and metastasis via Cdc42 expression and activity.

Tumor metastasis is the process by which tumor cells disseminate from tumors and enter nearby and distant microenvironments for new colonization. Bif-1 (BAX-interacting factor 1), which has a BAR domain and an SH3 domain, has been reported to be involved in cell growth, apoptosis and autophagy. However, the influence of Bif-1 on metastasis has been less studied. To understand the role of Bif-1 in metastasis, we studied the expression levels of Bif-1 in human HCC specimens using immunohistochemistry, a tissue microarray and quantitative PCR. The function of Bif-1 was assessed in migration and translocation assays and the pulmonary metastatic animal model. The relationship between Bif-1 and the Rho family was determined using immunoblot analyses and chromatin immunoprecipitation. The results showed that the expression of Bif-1 was higher in hepatocellular carcinoma (HCC) than matched adjacent non-tumor liver tissues. Increased Bif-1 expression was associated with tumor size and the intercellular spread and metastasis of HCC. Analysis of the relationship between Bif-1 expression and patients' clinical characteristics revealed that patients with higher levels of Bif-1 had shorter disease-free and overall survival rates. Knockdown of Bif-1 with RNAi suppressed the migration of HCC cells and pulmonary metastasis and decreased the expression of Cdc42, a member of the Rho family. Bif-1 localized to the cytosol and nucleus and interacted with the promoter transcription region of Cdc42, which may regulate Cdc42 expression. Our results demonstrate a novel role of Bif-1 in HCC, in which Bif-1 promotes cell metastasis by regulating Cdc42 expression and activity.

Transcript levels of the Aspergillus fumigatus Cdc42 module, polarisome, and septin genes show little change from dormancy to polarity establishment.

Aspergillus fumigatus is the most common airborne pathogen causing fatal mycoses in immunocompromised patients. During the first 8 hours of development A. fumigatus conidia break dormancy, expand isotopically, establish an axis of polarity, and begin to extend germ tubes in a polar manner. The transition from isotropic to polar growth is critical for tissue invasion and pathogenesis. In the current work, we used two-color microarrays to examine the A. fumigatus transcriptome during early development, focusing on the isotropic to polar switch. The most highly regulated transcripts in the isotropic to polar switch did not include known polarity genes. Transcripts encoding the Cdc42 module, polarisome components, and septins, known to be critical players in polarity, showed relatively steady levels during the isotropic to polar switch. Indeed, these transcripts were present in dormant conidia, and their levels changed little from dormancy through germ tube emergence. Not only did the isotropic to polar switch show little change in the expression of key polarity genes of the Cdc42 module, polarisome, and septins, it also showed the lowest overall levels of both up- and downregulation in early development.

Heme oxygenase-1 protects spinal cord neurons from hydrogen peroxide-induced apoptosis via suppression of Cdc42/MLK3/MKK7/JNK3 signaling.

The mechanisms by which oxidative stress induces spinal cord neuron death has not been completely understood. Investigation on the molecular signal pathways involved in oxidative stress-mediated neuronal death is important for development of new therapeutics for oxidative stress-associated spinal cord disorders. In current study we examined the role of heme oxygenase-1 (HO-1) in the modulation of MLK3/MKK7/JNK3 signaling, which is a pro-apoptotic pathway, after treating primary spinal cord neurons with H2O2. We found that MLK3/MKK7/JNK3 signaling was substantially activated by H2O2 in a time-dependent manner, demonstrated by increase of activating phosphorylation of MLK3, MKK7 and JNK3. H2O2 also induced expression of HO-1. Transduction of neurons with HO-1-expressing adeno-associated virus before H2O2 treatment introduced expression of exogenous HO-1 in neurons. Exogenous HO-1 reduced phosphorylation of MLK3, MKK7 and JNK3. Consistent with its inhibitory effect on MLK3/MKK7/JNK3 signaling, exogenous HO-1 decreased H2O2-induced neuronal apoptosis and necrosis. Furthermore, we found that exogenous HO-1 inhibited expression of Cdc42, which is crucial for MLK3 activation. In addition, HO-1-induced down-regulation of MLK3/MKK7/JNK3 signaling might be related to up-regulation of microRNA-137 (mir-137). A mir-137 inhibitor alleviated the inhibitory effect of HO-1 on JNK3 activation. This inhibitor also increased neuronal death even when exogenous HO-1 was expressed. Therefore, our study suggests a novel mechanism by which HO-1 exerted its neuroprotective efficacy on oxidative stress.

Rapamycin inhibits epithelial-to-mesenchymal transition of peritoneal mesothelium cells through regulation of Rho GTPases.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-ß1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).

The serotonin receptor 5-HT7R regulates the morphology and migratory properties of dendritic cells.

Dendritic cells are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of dendritic cell functions is poorly understood. Here, we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7R) as well as its downstream effector Cdc42 is upregulated in dendritic cells upon maturation. Although dendritic cell maturation was independent of 5-HT7R, receptor stimulation affected dendritic cell morphology through Cdc42-mediated signaling. In addition, basal activity of 5-HT7R was required for the proper expression of the chemokine receptor CCR7, which is a key factor that controls dendritic cell migration. Consistent with this, we observed that 5-HT7R enhances chemotactic motility of dendritic cells in vitro by modulating their directionality and migration velocity. Accordingly, migration of dendritic cells in murine colon explants was abolished after pharmacological receptor inhibition. Our results indicate that there is a crucial role for 5-HT7R-Cdc42-mediated signaling in the regulation of dendritic cell morphology and motility, suggesting that 5-HT7R could be a new target for treatment of a variety of inflammatory and immune disorders.

miR-30c Mediates Upregulation of Cdc42 and Pak1 in Diabetic Cardiomyopathy.

AIM: Cardiac hypertrophy and myocardial fibrosis significantly contribute to the pathogenesis of diabetic cardiomyopathy (DCM). Altered expression of several genes and their regulation by microRNAs has been reported in hypertrophied failing hearts. This study aims to examine the role of Cdc42, Pak1, and miR-30c in the pathogenesis of cardiac hypertrophy in DCM. METHODS: DCM was induced in Wistar rats by low-dose streptozotocin-high-fat diet for 12 weeks. Cardiac expression of Cdc42, Pak1 and miR-30c, and hypertrophy markers (ANP and ß-MHC) was studied in DCM vs control rats and in high-glucose (HG)-treated H9c2 cardiomyocytes. RESULTS: Diabetic rats showed cardiomyocyte hypertrophy, increased heart-to-body weight ratio, and an increased expression of ANP and ß-MHC. Cardiac expression of Cdc42 and Pak1 genes was increased in diabetic hearts and in HG-treated cardiomyocytes. miR-30c was identified to target Cdc42 and Pak1 genes, and cardiac miR-30c expression was found to be decreased in DCM rats, patients with DCM, and in HG-treated cardiomyocytes. miR-30c overexpression decreased Cdc42 and Pak1 genes and attenuated HG-induced cardiomyocyte hypertrophy, whereas miR-30c inhibition increased Cdc42 and Pak1 gene expression and myocyte hypertrophy in HG-treated cardiomyocytes. CONCLUSION: Downregulation of miR-30c mediates prohypertrophic effects of hyperglycemia in DCM by upregulation of Cdc42 and Pak1 genes.

Cdc42 expression in cervical cancer and its effects on cervical tumor invasion and migration.

The aim of the present study was to examine Cdc42 expression in cervical cancer and explore its effects on invasion and migration capability of cervical cancer cells. Immunohistochemistry was used to detect Cdc42 expression in normal cervical tissues as well as CIN I or below, CIN II or above, and cervical cancer tissues. Western blot analysis was used to explore Cdc42 expression in normal cervical cell line Crl-2614 and cervical cancer cell line HeLa. Plasmids of constitutively active Cdc42 (Cdc42 CA), wild-type Cdc42 (Cdc42 WT) and dominant negative Cdc42 (Cdc42 DN) were transfected, respectively, into HeLa cells to investigate the impacts of Cdc42 on migration and invasion of cervical cancer cells using Transwell and on cytoskeleton microfilaments using confocal microscopy after immunofluorescence staining. Cdc42 expression was gradually increased in the order of cervical tissues with CIN I or below, CIN II or above and cancer, showing significant difference (P<0.05), and was significantly higher in HeLa cells than in Crl-2614 cells (P<0.05). Migration ability of HeLa cells transfected with Cdc42 CA was significantly higher than that of non-transfected, as well as Cdc42 WT- or Cdc42 DN-transfected HeLa cells (P<0.05). Overexpression of Cdc42 CA can promote filopodia formation in HeLa cells. We concluded that Cdc42 overexpression significantly improved the ability of cervical cancer cells to migrate possibly due to improved pseudopodia formation.

δ-catenin promotes the malignant phenotype in breast cancer.

δ-Catenin is a member of the p120 catenin family. Similar to p120ctn, δ-catenin contains nine central Armadillo repeats and binds to the juxtamembrane domain (JMD) of E-cadherin. We used immunohistochemistry to detect δ-catenin expression in breast carcinoma (128 cases), and δ-catenin mRNA and protein expression was detected by reverse transcription-polymerase chain reaction and Western blotting (45 cases). The effects of δ-catenin on the activity of small GTPases and the biological behavior of breast cancer cells were explored by pulldown, flow cytometry, methyl thiazolyl tetrazolium, and Matrigel invasion assays. The results showed that δ-catenin expression increased in breast cancer tissues and was associated with a higher degree of malignancy (invasive lobular breast cancer, high tumor-node-metastasis stage, lymph node metastasis, and C-erbB-2+) and poor prognosis. Postoperative survival was shorter in patients with δ-catenin-positive expression than in patients with negative expression. δ-Catenin may regulate Cdc42/Rac1 activity, promote proliferation and invasion of breast cancer cells, and alter cell cycle progression. We conclude that δ-catenin tends to overexpress in breast carcinoma and promotes the malignant phenotype.

Upregulation of miR-137 protects anesthesia-induced hippocampal neurodegeneration.

PURPOSE: Ketamine is commonly used in pediatric anesthesia but may cause neurodegeneration in young brains. The aim of the study is to use an animal model to characterize the role of microRNA 137 (miR-137) in ketamine-induced neurodegeneration in neonatal hippocampus. METHODS: Young Sprague-Dawley Rats (1 month old) was systemically administrated with ketamine (75 mg/kg) for 3 days. TUNEL assay was used to assess the ketamine-induced neurodegeneration of hippocampal CA1 neurons, quantitative real-time PCR (qRT-PCR) to assess the expression of miR-137 and Morris water maze test (MWM) to assess the damaged memory function. Alternatively, lentivirus over-expressing miR-137 was injected into hippocampus before ketamine administration, and the subsequent effects of miR-137 upregulation on ketamine-induced hippocampal neurodegeneration and memory dysfunction were investigated. Furthermore, the direct downstream target of miR-137, CDC42, was down-regulated by siRNA injection into hippocampus. The effects of CDC42 inhibition on hippocampal apoptosis and memory function were also investigated. RESULTS: Excessive ketamine treatment resulted in severe apoptosis in hippocampal CA1 neurons, downregulation of miR-137 in hippocampus and significant long-term memory dysfunction. Conversely, pre-treatment of overexpressing miR-137 protected hippocampal neurodegeneration and memory loss. The molecular target of miR-137, CDC42 was down-regulated by ketamine in hippocampus. Knocking down hippocampal CDC42 exerted an apoptotic effect on hippocampal neurons and memory loss, similar to the effect of ketamine treatment. CONCLUSIONS: Our results demonstrated that miR-137 played an important role in regulating ketamine induced hippocampal neurodegeneration, possibly through CDC42.

Podocalyxin-like 1 promotes invadopodia formation and metastasis through activation of Rac1/Cdc42/cortactin signaling in breast cancer cells.

Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXL-knockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network.

miR-137 controls proliferation and differentiation of human adipose tissue stromal cells.

BACKGROUND/AIMS: Demonstrating the molecular mechanisms of human adipose tissue-derived mesenchymal stem cells (hADSCs) differentiation and proliferation could develop hADSCs-based cell therapy. METHODS: The microRNA-137 (miR-137) and cell division control protein 42 homolog (CDC42) levels were regulated by oligonucleotides transfection. The adipogenic differentiation was induced for 10 days in an adipogenic medium and assessed by using an Oil Red O stain. The regulation of miR-137 on CDC42 expression was determined by western blot, real-time PCR and luciferase reporter assay. RESULTS: We confirmed the roles of miR-137 on hADSCs proliferation and adipogenic differentiation. We showed that overexpression of miR-137 inhibited both hADSCs proliferation and adipogenic differentiation. Overexpression of miR-137 also downregulated protein and mRNA levels of CDC42, a predicted target of miR-137. In contrast, inhibition of miR-137 with 2'-O-methyl antisense RNA increased proliferation and adipogenic differentiation in hADSCs. Luciferase reporter activity in the miR-137 target site within the CDC42 3'UTR was lower in miR-137-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDC42 in hADSCs inhibited their proliferation and adipogenic differentiation. CONCLUSION: Our results indicate that miR-137 regulates hADSCs adipogenic differentiation and proliferation by directly targeting CDC42. These findings improve our knowledge of the molecular mechanisms governing hADSCs differentiation and proliferation.

IBP regulates epithelial-to-mesenchymal transition and the motility of breast cancer cells via Rac1, RhoA and Cdc42 signaling pathways.

Epithelial-to-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors. However, the molecular mechanisms underlying this transition are poorly understood. In this study, we demonstrate that interferon regulatory factor 4 binding protein (IBP) regulates EMT and the motility of breast cancer cells through Rac1, RhoA and Cdc42 signaling pathways. We found that increased expression of IBP was associated with the progression of breast cancer and that IBP protein levels were significantly elevated in matched distant metastases. High IBP levels also predict shorter overall survival of breast cancer patients. Furthermore, the forced expression of IBP decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing IBP in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. IBP silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2). Moreover, we identified a role for IBP in endogenous EMT induced by epidermal growth factor (EGF) and deletion of IBP attenuated EGF receptor (EGFR) signaling in breast cancer cells. Furthermore, IBP regulates the migration, invasion and matrix metalloprotease production in breast cancer cells as well as actin cytoskeleton rearrangement and the activation of GTP-Rac1, GTP-RhoA and GTP-Cdc42. Taken together, our findings demonstrate an oncogenic property for IBP in promoting the metastatic potential of breast cancer cells.

Association of Asef and Cdc42 expression to tubular injury in diseased human kidney.

BACKGROUND: Cdc42 is a small guanosine-5'-triphosphatase of the Rho family and plays essential roles in the establishment of cellular polarity and tight junctions in epithelial cells. Adenomatous polyposis coli-associated exchange factor (Asef) is a canonical guanine nucleotide exchange factor of Cdc42 and renders Cdc42 to be guanosine-5'-triphosphate bound and activated. The expression patterns and their significance in human renal diseases are unknown. METHODS: We examined the expression of Cdc42 and Asef in kidney biopsy specimens of 15 patients and in normal kidney tissue using immunofluorescence and correlated the expression patterns with the clinical characteristics. We also analyzed the coexpression pattern of Ki-67, a marker indicating cell division, and Asef in selected patients. RESULTS: Expression of Asef and Cdc42 together was associated with tubular injury with 100% specificity. Expression of Asef, regardless of Cdc42, also showed a significant diagnostic odds ratio for the presence of the injury. Expression of Asef was associated with lower estimated glomerular filtration rate at the time of biopsy and larger area of interstitial fibrosis. All Ki-67-expressing tubular cells expressed Asef. CONCLUSIONS: Induction of Asef and Cdc42 in the renal tubules is a cellular response to injury. Asef induction seems a necessary step for injured tubular cells to enter cell cycle.

Regulation of Cdc42 expression and signaling is critical for promoting corneal epithelial wound healing.

PURPOSE: Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42. METHODS: Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull-down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell-cycle proteins was identified by immunoprecipitation. RESULTS: In rabbit corneas, significant increase in Cdc42 expression that occurred 2 to 4 days after the injury coincided with wound closure, and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60% to 75% decrease in epithelial migration and growth. HGF, KGF, and EGF increased Cdc2 expression, activation, and its phosphorylation on ser71. Inhibition of growth factor-mediated PI-3K signaling resulted in the downregulation of Cdc42 expression and its phosphorylation. Increased association of cell-cycle proteins p27(kip) and cyclin-dependent kinase 4 (CDK4) with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors. CONCLUSIONS: Cdc42 is an important regulator of corneal epithelial wound repair. To promote healing, Cdc42 may interact with receptor tyrosine kinase-activated signaling cascades that participate in cell migration and cell-cycle progression.