NET-1 Expression Research Articles

18F-meta-fluorobenzylguanidine (18F-mFBG) to monitor changes in norepinephrine transporter expression in response to therapeutic intervention in neuroblastoma models

Targeted radiotherapy with 131I-mIBG, a substrate of the human norepinephrine transporter (NET-1), shows promising responses in heavily pre-treated neuroblastoma (NB) patients. Combinatorial approaches that enhance 131I-mIBG tumour uptake are of substantial clinical interest but biomarkers of response are needed. Here, we investigate the potential of 18F-mFBG, a positron emission tomography (PET) analogue of the 123I-mIBG radiotracer, to quantify NET-1 expression levels in mouse models of NB following treatment with AZD2014, a dual mTOR inhibitor. The response to AZD2014 treatment was evaluated in MYCN amplified NB cell lines (Kelly and SK-N-BE(2)C) by Western blot (WB) and immunohistochemistry. PET quantification of 18F-mFBG uptake post-treatment in vivo was performed, and data correlated with NET-1 protein levels measured ex vivo. Following 72 h AZD2014 treatment, in vitro WB analysis indicated decreased mTOR signalling and enhanced NET-1 expression in both cell lines, and 18F-mFBG revealed a concentration-dependent increase in NET-1 function. AZD2014 treatment failed however to inhibit mTOR signalling in vivo and did not significantly modulate intratumoural NET-1 activity. Image analysis of 18F-mFBG PET data showed correlation to tumour NET-1 protein expression, while further studies are needed to elucidate whether NET-1 upregulation induced by blocking mTOR might be a useful adjunct to 131I-mIBG therapy.

LncRNA CTC-497E21.4 promotes the progression of gastric cancer via modulating miR-22/NET1 axis through RhoA signaling pathway

Long non-coding RNAs (lncRNAs) have emerged as important roles in gastric cancer (GC). However, the role of the dysregulated lncRNAs in GC remained large unknown. We investigated the clinical significance, biological function and mechanism of CTC-497E21.4 in GC. Firstly, RTFQ-PCR was used to detect the expression of CTC-497E21.4 in GC. Furthermore, knockdown of CTC-497E21.4 was conducted to assess the effect of CTC-497E21.4 in vitro and vivo. Subcellular localization of CTC-497E21.4 was determined by nuclear plasmolysis PCR and FISH. We also predicted CTC-497E21.4 binding miRNAs and downstream target genes and evaluated its regulation of miR-22 by acting as a ceRNA. CTC-497E21.4 was upregulated in GC tissues and GC cell lines (P < 0.05), and the expression was associated with depth of invasion, lymph node metastasis, and neurological invasion. Besides, knockdown of CTC-497E21.4 inhibited cell proliferation, invasion and promoted cell cycle arrest in vitro and inhibited tumorigenesis in vivo. Mechanistic investigations indicated that CTC-497E21.4 acted as a ceRNA for miR-22 and regulated NET1 expression. CTC-497E21.4/miR-22-3p/NET1 participated in the RhoA signaling pathway in the GC progression. CTC-497E21.4 competed with miR-22 to regulate the expression of NET1 and regulated the malignant progression of GC through RhoA signaling pathway.

Inhibition of skin squamous cell carcinoma proliferation and promote apoptosis by dual silencing of NET-1 and survivin.

The simultaneous silencing of multiple upregulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. In the present study, skin squamous cell carcinoma (SSCC) tissue microarray was constructed and the expression of NET-1 and survivin was identified. The high expression of NET-1 and survivin gene in SSCC was confirmed as an important event for the formation and development of the cancer. A total of 100 primary SSCC patients were included in the present study. Expression of NET-1 and survivin in cancer cells was evaluated immunohistochemically in tissue microarrays. The interaction between NET-1 and survivin in SSCC by co-immunoprecipitation was subsequently verified by producing the siRNA sequence targeting the single gene (siRNA-NET-1 and siRNA-survivin) as well as NET-1 and survivin gene (one-chain-double-target siRNA). The levels of NET-1 and survivin mRNA and protein expression in A431 cells were detected by RT-qPCR and western blotting, and the expression of related genes including vascular endothelial growth factor (VEGF), cortactin, Bcl-2, caspase-3 and -8 was identified using RT-qPCR. The protein localization and expression of NET-1 and survivin in A431 cells were documented by immunohistochemistry and immuno-fluorescence staining. The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry (FCM). The tissue microarray showed that NET-1 and survivin were highly expressed in SSCC, while the correlation analysis showed NET-1 expression was positively associated with survivin. In addition, we reported that using the one-chain-double-target siRNA conjugate composed of NET-1 and survivin siRNA sequences in the same backbone inhibited proliferation and promoted apoptosis of SSCC. The one-chain-double-target siRNA showed further downregulation on NET-1 and survivin mRNA and protein levels compared with NET-1 siRNA or survivin siRNA. It also exhibited greater suppression on proliferation and triggering of apoptosis in A431 cells than NET-1 siRNA or survivin siRNA. This result may be explained by the significant downregulation of VEGF, cortactin and Bcl-2, and upregulation of caspase-3 and -8. NET-1 and survivin were overexpressed in SSCC and an interaction between NET-1 and survivin was identified. The one-chain-double-target siRNA appears to be superior in inhibiting cell proliferation and promoting apoptosis compared with the single target siRNA. NET-1 and survivin may have correlative signaling pathways with VEGF, cortactin, Bcl-2, caspase-3 and -8. Simultaneous silencing of NET-1 and survivin using one-chain-double-target siRNA thus provides an advantageous alternative in the development of therapeutics for SSCC.

The RhoGEF Net1 is required for normal mammary gland development.

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland.

Expression and function of NET-1 in human skin squamous cell carcinoma

To evaluate the clinicopathological significance of NET-1 in human skin squamous cell carcinoma (SSCC). The expression of NET-1 and Ki67 protein was detected using immunostaining from 60 SSCC cases, 50 SIN samples and ten normal skin tissues. The vectors expressing NET-1, siRNA NET-1 and shRNA NET-1 were constructed, as well as negative controls (target-off). In transfected A431 cells, the expression of NET-1 was detected by qRT-PCR, Western blot and immunofluorescence staining; the proliferation and migration of cells was evaluated by MTT, flow cytometry, wound healing and transwell chamber assays. The stable cell lines transfected with shRNANET-1 was inoculated in nude mice for in vivo study. (1) The levels of NET-1 were significantly higher in SSCC (96.67 %) and SIN III (93.75 %) than that in SIN I and II (41.18 %), (P < 0.05). NET-1 expression was significantly enhanced in spindle-cell SSCC (75 %) versus other histological types (P < 0.05). (2) The expression of NET-1 in A431 cells transfected with siRNANET-1 or shRNANET-1 was significantly decreased; the proliferation and migration of these cells were obviously inhibited as compared to controls (P < 0.05). (3) The growth of subcutaneous tumors was significantly inhibited associated with reduction in the expression of NET-1 vs. the negative control or untreated group (P < 0.05). The overexpression of NET-1 in tumor cells may be closely related to the malignant phenotype of SSCC. NET-1 RNAi used in this study can specifically and effectively downregulate NET-1 gene expression; thus SSCC proliferation, invasion and tumor growth were attenuated. NET-1 might be one of the potential targets for SSCC therapy.

Rho GTPase-independent regulation of mitotic progression by the RhoGEF Net1.

Neuroepithelial transforming gene 1 (Net1) is a RhoA-subfamily-specific guanine nucleotide exchange factor that is overexpressed in multiple human cancers and is required for proliferation. Molecular mechanisms underlying its role in cell proliferation are unknown. Here we show that overexpression or knockdown of Net1 causes mitotic defects. Net1 is required for chromosome congression during metaphase and generation of stable kinetochore microtubule attachments. Accordingly, inhibition of Net1 expression results in spindle assembly checkpoint activation. The ability of Net1 to control mitosis is independent of RhoA or RhoB activation, as knockdown of either GTPase does not phenocopy effects of Net1 knockdown on nuclear morphology, and effects of Net1 knockdown are effectively rescued by expression of catalytically inactive Net1. We also observe that Net1 expression is required for centrosomal activation of p21-activated kinase and its downstream kinase Aurora A, which are critical regulators of centrosome maturation and spindle assembly. These results identify Net1 as a novel regulator of mitosis and indicate that altered expression of Net1, as occurs in human cancers, may adversely affect genomic stability.

Inhibition of hepatocellular carcinoma growth and angiogenesis by dual silencing of NET-1 and VEGF

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we used dual gene targeted siRNA (DGT siRNA) conjugate composed of NET-1 and VEGF siRNA sequences in the same backbone could inhibit growth and angiogenesis HCC. DGT siRNA showed a further down regulation on VEGF mRNA and protein levels compared with NET-1 siRNA or VEGF siRNA, but not on NET-1 expression. It also exhibited greater suppression on proliferation and trigger of apoptosis in HepG2 cells than NET-1 siRNA or VEGF siRNA; this could be explained by the significant down regulation of cyclin D1 and Bcl-2. A lower level of ANG2 mRNA and protein was detected in HUVEC cultured with supernatant of HepG2 cells treated with DGT siRNA than that of VEGF siRNA or NET-1 siRNA, resulting in much more inhibited angiogenesis of HUVEC. Tumor growth was inhibited and microvessel density dropped in the xenograft tumor models compared to the untreated controls. NET-1 and VEGF silencing play a key role in inhibiting hepatocellular cell proliferation, promoting apoptosis, and reducing angiogenesis. Simultaneous silencing of NET-1 and VEGF using DGT siRNA construct may provide an advantageous alternative in development of therapeutics for Hepatocellular carcinoma.

PTU-173 Neuroepithelial cell transforming gene 1 in adenocarcinoma of the oesophago-gastric junction: expression, biology and prognostic significance in a large well characterised cohort

IntroductionNeuroepithelial Transforming Gene 1 (NET1) is a protein involved in tumour invasion and metastasis and is associated with poor prognosis in a number of human cancers. We aimed to determine...

Neuroepithelial Transforming Gene 1 (Net1) Binds to Caspase Activation and Recruitment Domain (CARD)- and Membrane-associated Guanylate Kinase-like Domain-containing (CARMA) Proteins and Regulates Nuclear Factor κB Activation

The molecular complexes containing CARMA proteins have been recently identified as a key components in the signal transduction pathways that regulate activation of nuclear factor κB (NF-κB) transcription factor. Here, we used immunoprecipitation coupled with mass spectrometry to identify cellular binding partners of CARMA proteins. Our data indicate that the Rho guanine nucleotide exchange factor Net1 binds to CARMA1 and CARMA3 in resting and activated cells. Net1 expression induces NF-κB activation and cooperates with BCL10 and CARMA proteins in inducing NF-κB activity. Conversely, shRNA-mediated abrogation of Net1 results in impaired NF-κB activation following stimuli that require correct CARMA-BCL10-MALT1 complex formation and functioning. Microarray expression data are consistent with a positive role for Net1 on NF-κB activation. Thus, this study identifies Net1 as a CARMA-interacting molecule and brings important information on the molecular mechanisms that control NF-κB transcriptional activity.

Direct effect of morphine on breast cancer cell function in vitro: role of the NET1 gene

Experimental data suggest that postoperative analgesia in general and opioids in particular may affect the risk of metastases after primary cancer surgery. Perioperative single-gene activation may also spark metastatic disease. The NET1 gene promotes migration in adenocarcinoma cells. We investigated opioid receptor expression in both breast cancer cell lines and the direct effect of morphine and NET-1 on breast cancer cell migration in vitro. Proliferation and migration of oestrogen receptor-negative MDA-MB-231 and oestrogen receptor-positive MCF7 breast cancer cells were studied after incubation with morphine 10-100 ng ml(-1) and control. NET1 gene expression was determined by polymerase chain reaction. The effect of NET1 on cell migration was determined using gene silencing with siRNA and stimulation with lysophosphatidic acid (LPA). The effect of morphine on NET1 expression and migration of cells with silenced NET1 was investigated. The NET1 gene was expressed in both cell lines and stimulated by LPA (2.9-fold in MCF7 and 78-fold in MDA-MB-231). NET1 expression was decreased by 96% after gene silencing in both cell lines with corresponding changes in migration. Despite the lack of opioid receptor expression, morphine increased the expression of NET1 (by 94% in MCF7 and by 263% in MDA-MB-231 cells). Morphine also increased migration by 17-27% and 7-53% in MCF7 and MDA-MB-231, respectively. Silencing the NET1 gene reversed the effect of morphine on migration. The NET1 gene, but not opioid receptors, is expressed in breast adenocarcinoma cells and may facilitate their migration. Morphine increased both expression of NET1 and cell migration but not when NET1 was silenced, implying that NET1 contributes to mediating the direct effect of morphine on breast cancer cell migration.

Oscillatory Dynamics of Cell Cycle Proteins in Single Yeast Cells Analyzed by Imaging Cytometry

Progression through the cell division cycle is orchestrated by a complex network of interacting genes and proteins. Some of these proteins are known to fluctuate periodically during the cell cycle, but a systematic study of the fluctuations of a broad sample of cell-cycle proteins has not been made until now. Using time-lapse fluorescence microscopy, we profiled 16 strains of budding yeast, each containing GFP fused to a single gene involved in cell cycle regulation. The dynamics of protein abundance and localization were characterized by extracting the amplitude, period, and other indicators from a series of images. Oscillations of protein abundance could clearly be identified for Cdc15, Clb2, Cln1, Cln2, Mcm1, Net1, Sic1, and Whi5. The period of oscillation of the fluorescently tagged proteins is generally in good agreement with the inter-bud time. The very strong oscillations of Net1 and Mcm1 expression are remarkable since little is known about the temporal expression of these genes. By collecting data from large samples of single cells, we quantified some aspects of cell-to-cell variability due presumably to intrinsic and extrinsic noise affecting the cell cycle.

Functions of NET-1 gene in skin squamous cell carcinoma cell line (A431): a siRNA study

To investigate the effect of NET-1 siRNA on NET-1 expression and on the proliferation and infiltration of skin squamous cell carcinoma cell line A431. Four recombinant vectors of pU6H1-GFP-siRNAs NET-1 were transfected into A431 cells. The levels of NET-1 mRNA expression were measured by semi-quantitative RT-PCR for selecting the most effective one in the four kind vectors of pU6H1-GFP-siRNA-NET-1. The controls consisted of pU6H1-GFP-siRNA-target off with random double-stranded RNA, pcDNA3.1 sense and other controls including the empty vectors (pU6H1-GFP and pcDNA3.1), NET-specific siRNA alone, lipofection reagent alone, target off siRNA alone, or untreated cells. After transfection, levels of NET-1 mRNA and protein expression were detected using semi-quantitive RT-PCR and Western blotting, respectively. The intracellular location of NET-1 protein was documented by immunofluorescence staining followed by laser scanning confocal microscopy. Proliferation rates of A431 cells were determined by MTT assay and flow cytometry. Abilities of migration and infiltration of A431 cells were determined by wound healing effect and transwell migration assay, respectively. siRNAs coding NET-1 sequences (19-23nt) were confirmed between H1 and U6 promoters of pU6H1-GFP vector by sequencing. The transfection efficiency of pU6H1-GFP in A431 cells was about 80% as the percentage of GFP expression cells in total cells. Transfection with either pU6H1-GFP-siRNA NET-1 or pcDNA3.1 antisense NET-1 gave rise to an obvious reduction in the expression of NET-1 mRNA and protein in A431 cells. The abilities of proliferation, migration and infiltration of A431 cells were significantly reduced 48 hours after transfection with either pU6H1-GFP-siRNA NET-1 or pcDNA3.1 antisense NET-1, compared with the vector controls including pU6H1-GFP or pcDNA3.1 (P < 0.01, respectively). pU6H1-GFP-siRNA NET-1 showed a stronger inhibition effect on proliferation, migration and infiltration of A431 cells over pcDNA3.1 antisense NET-1 (P < 0.05). Transfection with pcDNA3.1 sense NET-1 resulted in an obvious increase in the expression level of NET-1 mRNA and protein, but without inhibition effect to proliferation, migration and infiltration of A431 cells.No obviously inhibition effects were observed when transfected with pU6H1-GFP-siRNA-target or the controls. RNAi targeting NET-1 gene effectively down-regulates the expression of NET-1 in A431 cells, leading to an inhibition of proliferation, migration and infiltration. RNAi technique appears to be superior to antisense oligonucleotide technique in the suppression of gene expression.

Interaction of the RhoA Exchange Factor Net1 with Discs Large Homolog 1 Protects It from Proteasome-mediated Degradation and Potentiates Net1 Activity

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor beta, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

Coexpression of α6β4 Integrin and Guanine Nucleotide Exchange Factor Net1 Identifies Node-Positive Breast Cancer Patients at High Risk for Distant Metastasis

Preclinical data indicate that alpha6beta4 integrin signaling through Ras homolog gene family, member A, plays an important role in tumor cell motility. The objective of this study was to determine whether the combined expression of alpha6beta4 integrin and neuroepithelioma transforming gene 1 (Net1), a guanine nucleotide exchange factor specific for Ras homolog gene family member A, is associated with adverse clinical outcome in breast cancer patients. Immunohistochemical expression of each protein was evaluated in a tumor tissue microarray prepared from the primary tumors of 94 node-positive patients with invasive breast carcinoma treated with total mastectomy and doxorubicin-based chemotherapy without radiation with a median follow-up of 12.5 years. Associations between staining results and multiple clinicopathologic variables were investigated. Although there was no significant association between alpha6beta4 integrin or Net1 expression and clinical outcome when each marker was considered individually, coexpression of alpha6beta4 and Net1 was associated with decreased distant metastasis-free survival (P = 0.030). In the subset of patients with hormone receptor-positive tumors, coexpression of alpha6beta4 and Net1 was associated with a decrease in distant metastasis-free and overall survival (P < 0.001 and P = 0.006, respectively). Although an association between human epidermal growth factor receptor 2 expression and coexpression of alpha6beta4 and Net1 (P = 0.008) was observed, coexpression of alpha6beta4 and Net1 (hazard ratio, 1.63; P = 0.02) and lymphovascular invasion (hazard ratio, 2.35; P = 0.02) were the only factors independently associated with the development of distant metastasis in multivariate analysis. These findings suggest that coexpression of alpha6beta4 integrin and Net1 could be a useful biomarker for aggressive disease in node-positive breast cancer patients.

Association of NET-1 Gene Expression With Human Hepatocellular Carcinoma

NET-1 is a member of the NET-x family. To explore the potential role of NET-1 in hepatocellular carcinoma (HCC), the expression of NET-1 and the relationship with HCC were examined for the first time. We found that NET-1 was frequently expressed in HCC and the peritumor tissue. The relative amounts of NET-1 mRNA in HCC and peritumor tissue were 0.645 +/- 0.37 and 0.466 +/- 0.30, respectively, indicating a higher expression level in HCC than in the peritumor (P < .05). NET-1 protein is usually located on the cell membrane and in the cytoplasm of HCC cells. NET-1 immunoreactivity was found in 126 out of 130 samples of HCC tissue (96.92%). An association of NET-1 expression with cytological variants, histopathological grading, and clinical stages of HCC was also found (P < .05). Detection of NET-1 gene expression in liver biopsy may provide useful information about the biological behavior of HCC.

The guanine exchange factor Net1 is induced upon TGF-beta stimulation during transdifferentiation of hepatic stellate cells

Activation of hepatic stellate cells (HSC), the key fibrogenic cell type inside the liver, is mediated by TGF-beta. One hallmark of TGF-beta mediated transdifferentiation is the reorganization of the actin cytoskeleton. Actin reorganization requires activity of RhoA GTPase, which is activated by a Smad-dependent mechanism after stimulation with TGF-beta. Moreover RhoA activity may be related to the amount of Net1 protein, a specific guanine exchange factor for RhoA. We are interested in antifibrotic strategies, ideally by suppressing only the fibrotic action of TGF-beta, without compromising normal defence, tumor suppression, and repair functions. In our model we use adenoviral gene delivery of Smad7, an intracellular antagonist of parts of TGF-beta signal transduction, to prevent or at least reduce liver fibrosis. To elucidate the action of Smad7 on RhoA activity, we determined Net1 expression levels on mRNA and protein level in transdifferentiating HSC. Net1 expression was increased shortly (1 to 4 hours) after TGF-beta simulation and at later time points (7 days) of transdifferentiation. HSC expressing high levels of Smad7 showed not only a marked reduction of Net1 expression but also a pronounced decrease of actin fiber formation as evaluated by immunocytochemical staining for alpha smooth muscle actin and stress fibers (phalloidin). This suggests that the Smad7 dependent reduction of Net1 leads to less active RhoA and thereby keeping HSCs in a more quiescent state upon TGF-beta stimulation compared to controls. To test for possible adverse effects of Smad7 gene therapy, we are currently investigating changes in expression of genes which are associated with migratory activity of cancer cells, e.g. E-Cadherin and PKC-alpha.

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing.

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.

The Activity of Guanine Exchange Factor NET1 Is Essential for Transforming Growth Factor-β-mediated Stress Fiber Formation

To examine signaling pathways underlying transforming growth factor-beta (TGF-beta)-mediated changes in cell morphology, we used a microarray system to identify downstream target genes that may play a role in this process. Through this approach, we found that the NET1 gene was induced upon TGF-beta treatment in several cell types. NET1 is a guanine nucleotide exchange factor for RhoA whose activity has been implicated in stress fiber formation. In the Swiss 3T3 cell line, TGF-beta induces NET1 expression, and this correlated with an increase in stress fiber formation. Overexpression of the wild type NET1 gene increases stress fiber formation, and overexpression of a dominant negative NET1 mutant (L392E) prevented TGF-beta dependent increase in stress fiber formation. Furthermore, treatment of the cells with a RhoA kinase inhibitor Y-27632 blocks TGF-beta-induced stress fiber formation. By using a stable cell line expressing dominant negative Smad3, we found that the Smad signaling pathway is essential for the induction of NET1, which in turn leads to the increase of Rho activity. Taken together, those data suggest that induction of NET1 is important for the increase of Rho activity upon TGF-beta treatment, which may represent the critical trigger for a variety of downstream events in different cells. Our results support the presence of a novel signaling pathway by which TGF-beta may regulate the formation of stress fibers and reorganization of cytoskeletal structures.

Expression and regulation of a netrin homologue in the zebrafish embryo

Proteins of the Netrin family have been implicated in axon guidance in both C. elegans and vertebrates. Here, we report the cloning and expression analysis of a zebrafish netrin homologue ( net1). net1 is expressed in the floor plate and the anterior ventral neural tube. Its expression is ectopically induced by misexpression of sonic hedgehog ( shh) and a dominant negative mutant of the regulatory subunit of protein kinase A (dnReg). Ectopic activation of net1, however, is restricted to distinct regions in the brain. Upon overexpression of shh or dnReg in cyclops mutants, which have strongly impaired net1 expression in the ventral neural tube, rescue of net1 expression was observed in the brain but not in the spinal cord. Ectopic expression of dnReg and Shh protein can be detected at high levels throughout injected embryos from pre-gastrula stages onwards suggesting that the competence of the neural plate to respond to Shh signalling activity differs regionally. Similar to net1, axial, the zebrafish homologue of mammalian HNF3β, which is also expressed along the ventral neural tube, is ectopically induced in the brain of embryos injected with dnReg mRNA. Neurons differentiate normally within domains of ectopic net1 and axial expression. Thus, dorsal neuronal differentiation appears to be unaffected despite co-expression of a gene program specific for the ventral neural tube. This also suggests that these ectopically expressing regions have not differentiated into floor plate.