Dynamin 3 Research Articles

Renal Farnesoid X Receptor improves high fructose-induced salt-sensitive hypertension in mice by inhibiting DNM3 to promote nitro oxide production.

Farnesoid X Receptor (FXR) is highly expressed in renal tubules, activation of which attenuates renal injury by suppressing inflammation and fibrosis. However, whether renal FXR contributes to the regulation of blood pressure (BP) is poorly understood. This study aimed to investigate the anti-hypertensive effect of renal FXR on high-fructose-induced salt-sensitive hypertension and underlying mechanism. Hypertension was induced in male C57BL/6 mice by 20% fructose in drinking water with 4% sodium chloride in diet (HFS) for 8 weeks. The effects of FXR on NO production were estimated in vitro and in vivo . Compared with control, HFS intake elevated BP, enhanced renal injury and reduced renal NO levels as well as FXR expression in the kidney of mice. In the mouse renal collecting duct cells mIMCD-K2, FXR agonists promoted NO production by enhancing the expression of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS), whereas this effect was diminished by fxr knockdown. We further found that Dynamin 3 (DNM3), a binding protein with nNOS in the renal medulla, was inhibited by FXR and its deficiency elevated NO production in mIMCD-K2 cells. In HFS-fed mice, renal fxr overexpression significantly attenuated hypertension and renal fibrosis, regulated the expression of DNM3/nNOS/iNOS, and increased renal NO levels. Our results demonstrated that renal FXR prevents HFS-induced hypertension by inhibiting DNM3 to promote NO production. These findings provide insights into the role and potential mechanism of renal FXR for the treatment of hypertension.

Expression of DNM3 is associated with good outcome in colorectal cancer.

The aim of this study is to reveal the potential value of dynamin3 (DNM3) in colorectal cancer (CRC) evaluation of clinical diagnosis and prognosis. A total of 100 tissue samples were collected from 50 patients with stages I–IV, CRC tissues (n = 50) paired with non-cancerous adjacent colorectal tissues (n = 50). The expression levels of DNM3 were detected in 50 cases of CRC tissues and 50 cases of non-cancerous adjacent colorectal tissues by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical method (IHC) was conducted to semi-quantify the expression of DNM3 protein. Results showed that the relative expression of DNM3 mRNA in CRC tissues was 0.634-fold of that in non-cancerous adjacent colorectal tissues. The positive rate of DNM3 protein in CRC tissues (42.0%) was much lower than that in non-cancerous adjacent colorectal tissues (66.0%; P < 0.05). The expression level of DNM3 protein in CRC tissues was dependent on tumor size, degree of histological differentiation, and clinical stage (P < 0.05). The expression level of DNM3 mRNA in CRC tissues was significantly correlated with tumor size and pathology classification (P < 0.05). The research shows that detecting the expression of DNM3 helps in analyzing the tumor size, degree of histological differentiation, and clinical stage. Expression of DNM3 may be associated with good outcome in CRC.

Dynamin 3 Inhibits the Proliferation of Non-small-Cell Lung Cancer Cells by Suppressing c-MET-GBR2-STAT3 Complex Formation.

Dynamin 3 (DNM3) has gained increased attention ever since its potential as a tumor suppressor was reported. However, its action in lung cancer (LC) is undefined. In this study, the role of DNM3 in LC development was investigated. DNM3 expression was found to be downregulated in tumors of patients with LC, especially those with metastasis. The DNM3 downregulation enhanced the proliferative and metastatic ability of LC cells, whereas its upregulation had the opposite effects. In vivo xenograft experiments confirmed that lung tumors with lower DNM3 expression had higher growth and metastatic abilities. Mechanistic studies revealed that DNM3 interacts with growth factor receptor-bound protein 2 (GBR2), thereby interrupting tyrosine-protein kinase Met (c-MET)–GBR2–signal transducer and activator of transcription 3 (STAT3) complex formation, which suppressed STAT3 activation. Therefore, the absence of DNM3 frees GBR2 to activate STAT3, which regulates the expression of genes related to LC proliferation and metastasis (e.g., cyclin D1 and Snail family transcriptional repressor 1). Additionally, the c-MET inhibitor crizotinib effectively suppressed LC cell proliferation and migration in vitro and in vivo, even with DNM3 depleted. Therefore, our study has demonstrated the antitumor effect of DNM3 in LC and suggests that the inhibition of c-MET might be a promising strategy for treating those LC patients with low DNM3 expression.

Dynamin 3 overexpression suppresses the proliferation, migration and invasion of cervical cancer cells.

Dynamin 3 (DNM3) functions as a tumor suppressor in various malignancies. However, the underlying mechanism of DNM3 in cervical cancer remains to be elucidated. The present study aimed to indicate the function of DNM3 in cervical cancer. The expression of DNM3 in cervical tissues and cells was measured using bioinformatics analysis, immunohistochemistry and reverse transcription-quantitative PCR. The pcDNA3.1 plasmid was used to overexpress DNM3 in SiHa and C33A cells. The effects of DNM3 overexpression on cell proliferation, migration, invasion and apoptosis was detected by the CCK-8, clone formation, Transwell, flow cytometry and western blotting assays. In the present study, it was revealed that DNM3 was expressed at significantly low levels in cervical cancer tissues and cell lines compared with normal cervical tissues and cell lines. In addition, the low expression of DNM3 was significantly associated with high pathological grading of cervical cancer. The overall survival rate of patients with low DNM3 expression was significantly improved compared with patients with high DNM3 expression. In addition, the overexpression of DNM3 significantly inhibited the proliferation, migration and invasion of cervical carcinoma cells and induced cell apoptosis. The findings of the present study further revealed that the overexpression of DNM3 may inhibit cell migration and invasion by inactivating the epithelial mesenchymal transition process. In summary, the present study demonstrated that DNM3 was a tumor suppressor in cervical cancer progression and that it may serve as a potential prognostic biomarker for patients with cervical carcinoma.

Deletion at an 1q24 locus reveals a critical role of long noncoding RNA DNM3OS in skeletal development

BackgroundSkeletal development and maintenance are complex processes known to be coordinated by multiple genetic and epigenetic signaling pathways. However, the role of long non-coding RNAs (lncRNAs), a class of crucial epigenetic regulatory molecules, has been under explored in skeletal biology.ResultsHere we report a young patient with short stature, hypothalamic dysfunction and mild macrocephaly, who carries a maternally inherited 690 kb deletion at Chr.1q24.2 encompassing a noncoding RNA gene, DNM3OS, embedded on the opposite strand in an intron of the DYNAMIN 3 (DNM3) gene. We show that lncRNA DNM3OS sustains the proliferation of chondrocytes independent of two co-cistronic microRNAs miR-199a and miR-214. We further show that nerve growth factor (NGF), a known factor of chondrocyte growth, is a key target of DNM3OS-mediated control of chondrocyte proliferation.ConclusionsThis work demonstrates that DNM3OS is essential for preventing premature differentiation of chondrocytes required for bone growth through endochondral ossification.

Delineation of the 1q24.3 microdeletion syndrome provides further evidence for the potential role of non-coding RNAs in regulating the skeletal phenotype.

Microdeletions within 1q24 have been associated with growth deficiency, varying intellectual disability, and skeletal abnormalities. The candidate locus responsible for the various phenotypic features of this syndrome has previously been predicted to lie in the area of 1q24.3, but molecular evidence of the causative gene remains elusive. Here, we report two additional patients carrying the smallest reported 1q24 deletion to date. Patient 1 exhibited intrauterine growth retardation, shortening of the long bones, frontal bossing, microstomia, micrognathia, and a language acquisition delay. Her mother, Patient 2, displayed a broad forehead and nasal bridge, thick supraorbital ridges, and toe brachydactyly, along with learning disability and language acquisition delay. The microdeletion encompasses a 94 Kb region containing exon 14 and portions of the surrounding introns of the gene encoding dynamin 3 (DNM3), resulting in an in-frame loss of 38 amino acids. This microdeletion site also contains a long non-coding RNA (DNM3OS) and three microRNAs (miR-214, miR-199A2, and miR-3120). Following culture of patient-derived and control fibroblasts, molecular analyses were performed to determine expression levels of genes affected by the heterozygous deletion. Results show decreased expression of DNM3OS and miR-214-3p in patient fibroblasts cultured in an osteogenic induction medium. Overall, our data provide further evidence to support a functional role for non-coding RNAs in regulating the skeletal phenotype, and the potential of a functionally-impaired DNM3 protein causing the non-skeletal disease pathogenesis.

SiPRDX2-elevated DNM3 inhibits the proliferation and metastasis of colon cancer cells via AKT signaling pathway.

Purpose: We have previously reported that PRDX2 plays an oncogenic role in colon cancer. In this study, the mRNA expression profile of PRDX2 in HCT116 cells was investigated. Furthermore, we selected Dynamin 3 (DNM3), which is up-regulated by siPRDX2, to investigate its expression pattern and functions in colon cancer.Patients and methods: PRDX2 siRNA was transfected into HCT116 cells and the mRNA profile was tested by RNA-Sequencing. The expression of interest proteins was determined by Western blot. DNM3 expression in colon cancer tissues and para-carcinoma tissues was evaluated by Western blot and immunohistochemistry assays. Full-length cDNA of DNM3 was cloned into pcDNA3.1 and introduced into HCT116 and HT29 cells. Cell proliferation was tested by CCK-8 and colony formation assays. Cell invasion and migration were tested by transwell assays. Gelatin zymography was utilized for detection of MMP9 activity. Cell apoptosis was investigated with Annexin V/PI staining and flow cytometry and visualized with Hoechst/PI staining assay. All statistical analysis was performed with SPSS 17.0 software.Results: PRDX2 knockdown led to 210 up-regulated genes and 16 down-regulated genes in HCT116 cells. We also found that DNM3 expression was up-regulated following PRDX2 silencing in HCT116 and HT29 cells. In colon cancer patients, DNM3 was down-regulated and showed a significant association with pathologic grading. DNM3 overexpression inhibited cell proliferation and induced apoptosis in HCT116 and HT29 cells. Cell migration and invasion were also down-regulated in DNM3 overexpressing colon cancer cells, which might be due to the inhibition of MMP9 proteolytic activities. After thorough investigation of the potential mechanism involved, we hypothesized that DNM3 overexpression induced activation of the mitochondrial apoptosis pathway and inhibition of the AKT pathway.Conclusion: These data suggest that DNM3 is down-regulated in colon cancer, serving as a tumor suppressor. Our study provides new sights into the prognostic value and therapeutic application of DNM3 in colon cancer.

SNCA but not DNM3 and GAK modifies age at onset of LRRK2-related Parkinson's disease in Chinese population.

Recently, rs2421947 in DNM3 (dynamin 3) was reported as a genetic modifier of age at onset (AAO) of LRRK2 G2019S-related Parkinson's disease (PD) in a genome-wide association study in Arab-Berber population. Rs356219 in SNCA (α-synuclein) was also reported to regulate the AAO of LRRK2-related PD in European populations, and GAK (Cyclin G-associated kinase) rs1524282 was reported to be associated with an increased PD risk with an interaction with SNCA rs356219. G2019S variant is rare in Asian populations, whereas two other Asian-specific LRRK2 variants, G2385R and R1628P, are more frequent with a twofold increased risk of PD. In this study, we investigated whether rs2421947, rs356219 and rs1524282 modified AAO in LRRK2-related PD patients in Han Chinese population. We screened LRRK2 G2385R and R1628P variants in 732 PD patients and 1992 healthy controls, and genotyped DNM3 rs2421947, SNCA rs356219 and GAK rs1524282 among the LRRK2 carriers. The SNCA rs356219-G allele was found to increase the risk of PD in LRRK2 carriers (OR 1.50, 95%CI 1.08-2.01, P = 0.016), and the AAO of AG + GG genotypes was 4years earlier than AA genotype (P = 0.006). Nonetheless, no similar association was found in DNM3 rs2421947 and GAK rs1524282. Our results show that SNCA but not DNM3 or GAK is associated with AAO of LRRK2-PD patients in Chinese population.

Construction of a recombinant eukaryotic expression vector containing DNM3 gene and its expression in colon cancer cells.

IntroductionDynamin 3 (DNM3) is a large GTPase that possesses mechanochemical properties and has been shown to be involved in malignancies. However, most studies about DNM3 are observational, and knowledge of the precise molecular mechanism of DNM3 remains limited.Materials and methodsWe constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, which was then transfected into SW620 and LoVo cells. One cell line was divided into three groups. DNM3 mRNA and protein expression was analyzed by quantitative real-time PCR and Western blot assay. To investigate DNM3 biological activity in colon cancer SW620 and LoVo cell line, we performed cell proliferation, transwell migration, and invasion assay. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were detected by Western blot.ResultWe successfully constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, and stable DNM3 expression was observed in SW620 and LoVo cell lines. The vector overexpressing DNM3 inhibited the proliferation, weak invasion, and migration ability of colon cancer SW620 and LoVo cells relative to those in the control group (all P<0.001). DNM3 downregulated the protein expression of MMP-2 and MMP-9.ConclusionDNM3 may weaken the malignant behavior of colon cancer and may have promoted the invasion and migration of colon cancer by regulating the expression of MMP-2 and MMP-9.

Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits.

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr-/- mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.

Dynamin 3 suppresses growth and induces apoptosis of hepatocellular carcinoma cells by activating inducible nitric oxide synthase production.

Dynamin 3 (DNM3) is candidate tumor suppressor against hepatocellular carcinoma (HCC). Downregulation of DNM3 is more frequently identified in HCC tissues than in normal liver tissues. However, the mechanism underlying DNM3-mediated inhibition of HCC remains unclear. The present study demonstrated that DNM3 expression was decreased in human HCC tissues and cell lines. The downregulation of DNM3 promoted cell proliferation by increasing cell cycle-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 2 and CDK4. The upregulation of DNM3 induced HCC cell apoptosis and inhibited tumor growth. The present study also revealed that overexpression of DNM3 induced nitric oxide (NO) production and intracellular reactive oxygen species (ROS) accumulation. DNM3 overexpression also increased the protein expression level of inducible nitric oxide synthase (iNOS) in HCC cells and subcutaneous HCC tumor xenografts. The inhibition of iNOS by L-canavanine attenuated the DNM3-induced ROS accumulation and apoptotic cell death. In conclusion, the results indicate that DNM3 overexpression may induce apoptosis and inhibit tumor growth of HCC by activating iNOS production and the subsequent NO-ROS signaling pathways.

ETS1 Expression Is Upregulated in Chronic Myeloid Leukemia (CML) and the ETS1 Transcriptional Program Correlates with Disease Progression Towards Blast Crisis

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm characterized by the occurrence of the Ph1 chromosome in a primitive hematopoietic stem cell with a major amplification of myeloid compartment. Despite the major progress obtained by the use of tyrosine kinase inhibitors (TKI), resistances to these drugs occur with progression towards blast crisis (BC) during which there is an increase of BCR-ABL expression. To evaluate the potential targets of BCR-ABL in this context, a gene profiling analysis of the UT7-11 cell line overexpressing BCR-ABL fusion protein was performed using a whole transcriptome microarray. One of the highly upregulated genes was ETS1 with a fold change of +35.86. ETS1 proto-oncogene, the founder member of the ETS-domain family of transcription factors such as TEL and FLI1, is the human homolog of the avian erythroblastosis virus E26. They play a crucial role in stem cell biology, tumorigenesis and ETS1 has been shown to be involved in the regulation of granulocytic differentiation. Its role in CML pathophysiology has not been studied so far. We first showed by Western blots that ETS1 protein was highly increased in UT7-11 cells as compared to parental UT7 cells. Using a DOX-Inducible BCR-ABL system, we have shown that ETS1 expression was downregulated upon inhibition of BCR-ABL expression. ETS1 expression was a tyrosine kinase dependent event as its expression was reduced in the presence of TKI. To determine if ETS1 expression is upregulated in primary leukemic cells, we have analyzed leukemic cells of CML patients at diagnosis (n= 40) as compared to healthy controls (n= 30) showing an increase of ETS1 mRNA expression in primary CML cells in a highly significant manner (p < 0.0007). We then analyzed ETS1 targets using chip-sequencing in K562 cells, performed on HG19 human genome allowing to predict 3209 proximal promoters (-3000 upstream pb, + 50pb around Transcription Starting Sites) which represents a promoter enrichment of +33.68 (p-value=4.9E-324) as compared to the distribution of peaks in the whole genome. Chromosomes 19, 16, 17, 11, 12 and 1 were found over-represented with ETS1 bound events in Chip-sequencing. Integration of BCR-ABL transcriptome with ETS1 promoters identified by Chip-Sequencing led to the identification of 130 ETS1-targets activated by BCR-ABL, allowing reclassification of BCR-ABL transfected samples with transcriptome data by unsupervised classification. ETS1 transcriptional program regulated by BCR-ABL analysis performed on CD34+ from CML patients during progression of the disease (GSE47927) allowed discrimination of the different states of the progression (from the chronic phase to accelerated phase and BC p-value=1.63E-33). This analysis was also found significant by Gene Set Enrichment Analysis ETS1_BC specific geneset (NES=+1.52, p-value<0.0001). Among this ETS1 program specific of BC, we identified 2 targets that are discriminant for CD34+ cells of CML patients without cytogenetic response under Imatinib therapy. These two genes were Dynamin 3 (DNM3) and LIMS1/ PINCH1. Dynamin 3 is a member of the motor proteins implicated in cell motility and cytokinesis and it is implicated in megakaryocyte development whereas LIMS1 / PINCH1 is essentially involved in cell migration and adhesion. In order to validate ETS1 and its target expressions in a novel cohort of CML patients, we have analyzed the expression of DNM3 and PINCH1 in whole blood samples in CML patients ( n= 60 ) as compared to healthy controls (n= 32). We have found that, similar to ETS1, DNM3 and LIMS1 / PINCH1 mRNA were highly upregulated in primary CML and this increase was highly significant. In this new cohort of patients, DNM3 transcript levels were inversely correlated to WBC count (r=-0.32, p-value=0.011) and especially in male gender (r=-0.43, p-value=0.012). The expression of LIMS1 was inversely correlated with the age at diagnosis and the major molecular response at 12 months. Thus, our results show for the first time the major upregulation of ETS1 transcriptional program in CML. ETS1 transcriptional program has been found to correlate with the gene expression pattern involved in the progression of the disease in the transcriptomic analysis of CML CD34+ cells. The novel druggable targets that we identified in the BCR-ABL-activated transcriptional program is currently under investigation to determine their use in patients with resistance to targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

DNM3 Attenuates Hepatocellular Carcinoma Growth by Activating P53.

BackgroundPrimary hepatocellular carcinoma is one of the most common malignant tumors in China and its mortality rate shows no sign at present of ceasing to rise. In our previous study, we found that the mRNA level of Dynamin3 (DNM3), a member of the Dynamin family, is significantly lower in hepatocellular carcinoma tissues than in non-tumor tissues. The aim of this study was to investigate the expression pattern and potential function of DNM3 in hepatocellular carcinoma.Material/MethodsFirst, we determined the expression ofDNM3 in human hepatocellular carcinoma tissues and cell lines. We then studied the biological function of DNM3 on hepatocellular carcinoma cells by proliferation assay and colony formation assay. Flow cytometry was used to study the effect of DNM3 on cell cycle and apoptosis.ResultsExpression of DNM3 was significantly downregulated in hepatocellular carcinoma tissues and was associated with vein invasion and tumor metastasis. In addition, upregulation of DNM3 reduced hepatocellular carcinoma cell proliferation and colony formation, induced hepatocellular carcinoma cell G0/G1 phase arrest, and stimulated hepatocellular carcinoma cell apoptosis. We also found that DNM3 may exert its anti-proliferative effect through upregulating p53.ConclusionsOur findings suggest that DNM3 attenuates the proliferation and induces apoptosis of gastric cancer cells. Modulation of DNM3 may prove to be an efficient method of hepatocellular carcinoma treatment.

Erythropoietin-mediated expression of placenta growth factor is regulated via activation of hypoxia-inducible factor-1α and post-transcriptionally by miR-214 in sickle cell disease.

Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated invivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation.

BackgroundDown-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation.ResultsTo identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells.ConclusionsWe identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0118-4) contains supplementary material, which is available to authorized users.

Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation

Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells. We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

Dynamin 3: a new candidate tumor suppressor gene in hepatocellular carcinoma detected by triple combination array analysis

BackgroundTo identify genes associated with hepatocellular carcinoma (HCC) pathogenesis, we developed a triple combination array strategy comprising methylation, gene expression, and single nucleotide polymorphism (SNP) array analysis.MethodsSurgical specimens obtained from a 68-year-old female HCC patient were analyzed by triple combination array, and identified Dynamin 3 (DNM3) as a candidate tumor suppressor gene in HCC. Subsequently, samples from 48 HCC patients were evaluated for DNM3 methylation and expression status using methylation specific polymerase chain reaction (PCR; MSP) and semi-quantitative reverse transcriptase (RT)-PCR, respectively. The relationship between clinicopathological factors and DNM3 methylation status was also investigated.ResultsDNM3 was shown to be hypermethylated (methylation value 0.879, range 0–1.0) in cancer tissue compared with adjacent normal tissue (0.213) by methylation array in the 68-year-old female patient. Expression arrays revealed decreased expression of DNM3 in cancerous tissue. SNP arrays revealed that the copy number of chromosome 1q24.3, in which DNM3 resides, was normal. MSP revealed hypermethylation of the DNM3 promoter region in 33 of 48 tumor samples. A trend toward decreased DNM3 expression was observed in patients with DNM3 promoter methylation (P = 0.189). Furthermore, patients with reduced expression of DNM3 in tumor tissues exhibited worse prognosis with decreased disease specific survival compared to patients without decreased expression (P = 0.014).ConclusionThe present study indicates that a triple combination array strategy is an effective method to detect novel genes related to HCC. We propose that DNM3 is a tumor suppressor gene in HCC.

Distinct Functional Effects for Dynamin 3 During Megakaryocytopoiesis

Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.

Dynamin 3 participates in the growth and development of megakaryocytes

High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of <or=0.001 with a fold change cutoff of three or more in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2+/-2.1 (mean+/-standard deviation). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2, and DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time quantitative polymerase chain reaction confirmed that DNM3 increased by 20.7-+/-3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in proplatelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs, with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from culture-derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Overexpression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony-forming cells and colony-forming unit-megakaryocytes, and a concomitant increase in the expression of nuclear factor erythroid 2 (NF-E2) and beta-tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.

Dynamin 3 Is Differentially Expressed during Human Megakaryocyte Development.

We found that expression of dynamin 3 (DNM3) is increased significantly when human CD34+/CD38lo cells are induced to develop along the megakaryocyte (MK) lineage. DNM3 is a member of a family of mechanochemical enzymes which participate in membrane dynamics. Of the 3 family members (DNM1, DNM2 and DNM3), cDNA microarray analysis showed that only DNM3 significantly increased by 9.5±1.8-fold (mean±SEM; p≤0.001). mRNAs for DNM1 and DNM2 were detectable, but their expression levels remained essentially unchanged during MK differentiation. Consistent with our microarray data, real time quantitative RT-PCR confirmed that DNM3 expression levels increased by as much as 20.0±7.3 fold during megakaryocytopoiesis. Western Blot analysis showed that protein expression patterns for each of the 3 dynamins differ in MKs/platelets. DNM3 protein expression levels are greater in MKs than in platelets. DNM1 was not detectable in MKs, but was present in platelets. Although DNM2 protein was detectable in both MKs and platelets, the relative mobility for DNM2 was slightly lower in platelets than MKs. Indirect immunofluorescent microscopy showed that DNM3 was predominantly in the cytoplasm with localization to sites that appear to be regions of pro-platelet formation. The cDNA encoding DNM3 was amplified from culture derived human MKs, cloned and sequenced. Sequence analysis revealed that the cloned product matched a DNM3 transcriptional splice variant in the Ensemble Human GeneView database (i.e. ENST00000367731). Together these results are the first evidence indicating that members of the dynamin family of mechanochemical proteins are present in human MKs and suggest that they may play an important role in MK membrane dynamics.