siRNA Knockdown (sirna + knockdown)

Distribution by Scientific Domains

Selected Abstracts

Dan is required for normal morphogenesis and patterning in the developing chick inner ear

Takahiro Yamanishi
During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1. [source]

Non-muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)

Patrick G. Flynn
Abstract Non-muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF-alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365,1375, 2010. 2010 Wiley-Liss, Inc. [source]

Regulation of Wnt/,-catenin pathway by cPLA2, and PPAR,

Chang Han
Abstract Cytosolic phospholipase A2, (cPLA2,) is a rate-limiting key enzyme that releases arachidonic acid (AA) from membrane phospholipid for the production of biologically active lipid mediators including prostaglandins, leukotrienes and platelet-activating factor. cPLA2, is translocated to nuclear envelope in response to intracellular calcium increase and the enzyme is also present inside the cell nucleus; however, the biological function of cPLA2, in the nucleus remains unknown. Here we show a novel role of cPLA2, for activation of peroxisome proliferator-activated receptor-, (PPAR,) and ,-catenin in the nuclei. Overexpression of cPLA2, in human cholangiocarcinoma cells induced the binding of PPAR, to ,-catenin and increased their association with the TCF/LEF response element. These effects are inhibited by the cPLA2, siRNA and inhibitors as well as by siRNA knockdown of PPAR,. Overexpression of PPAR, or treatment with the selective PPAR, ligand, GW501516, also increased ,-catenin binding to TCF/LEF response element and increased its reporter activity. Addition of AA and GW501516 to nuclear extracts induced a comparable degree of ,-catenin binding to TCF/LEF response element. Furthermore, cPLA2, protein is present in the PPAR, and ,-catenin binding complex. Thus the close proximity between cPLA2, and PPAR, provides a unique advantage for their efficient functional coupling in the nucleus, where AA produced by cPLA2, becomes immediately available for PPAR, binding and subsequent ,-catenin activation. These results depict a novel interaction linking cPLA2,, PPAR, and Wnt/,-catenin signaling pathways and provide insight for further understanding the roles of these key molecules in human cells and diseases. J. Cell. Biochem. 105: 534,545, 2008. 2008 Wiley-Liss, Inc. [source]

Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses

Manchao Zhang
Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. 2007 Wiley-Liss, Inc. [source]

Functional analysis of CBP/p300 in embryonic orofacial mesenchymal cells

D.R. Warner
Abstract CREB binding protein (CBP) and the close structural homolog, p300, are nuclear coactivators of multiple signaling pathways that play important roles in embryonic development and cellular homeostasis. TGF, regulates the proliferation rate of many cell types and has been demonstrated to inhibit the growth rate of mouse embryonic maxillary mesenchymal (MEMM) cells. The role of CBP and p300 in TGF,-mediated control of proliferation of MEMM cells was thus investigated using an in vitro gene knockdown approach. TGF, reporter assays demonstrated that p300 mRNA knockdown via targeted siRNAs led to a reduction in the response to TGF,, whereas knockdown of CBP by the same approach had an insignificant effect. In MEMM cell proliferation assays, siRNA-mediated knockdown of CBP and/or p300 had little impact upon TGF,-mediated growth inhibition; however, the basal rate of proliferation was increased. Inhibition of p300 activity via overexpression of a dominant-negative mutant (p300,C/H3) led to significant inhibition of TGF,-mediated activation of p3TP-lux. As with the siRNA knockdown approach, p300,C/H3 also increased the basal rate of cell proliferation of MEMM cells. CBP/p300 siRNA knockdown had a significant but incomplete inhibition of TGF,-induction of matrix metalloproteinase-9 (gelatinase B) expression. These data demonstrate that p300 is involved in Smad-mediated transcription of p3TP-lux, however, its role (and that of CBP) in biological processes such as the control of cell proliferation and extracellular matrix metabolism is more complex and may be mediated via mechanisms beyond coactivator recruitment. J. Cell. Biochem. 99: 1374,1379, 2006. 2006 Wiley-Liss, Inc. [source]

miR-20b modulates VEGF expression by targeting HIF-1, and STAT3 in MCF-7 breast cancer cells,

Sandra Cascio
MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR-20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF-7 breast cancer cells under normoxic and hypoxia-mimicking conditions (CoCl2 exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24,h following CoCl2 treatment, and VEGF mRNA at 4,h of treatment. In addition, miR-20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre-miR-20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR-20b. Downregulation of VEGF mRNA by miR-20b under a 4,h of CoCl2 treatment was associated with reduced levels of nuclear HIF-1, subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1,, but not STAT3, was recruited to the VEGF promoter following the 4,h of CoCl2 treatment. This effect was inhibited by transfection of cells with pre-miR-20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl2 -mediated HIF-1, nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF-1 and STAT3 in a miR-20b-dependent manner. J. Cell. Physiol. 224:242,249, 2010 2010 Wiley-Liss, Inc. [source]

Alcohol Stimulates Activation of Snail, Epidermal Growth Factor Receptor Signaling, and Biomarkers of Epithelial,Mesenchymal Transition in Colon and Breast Cancer Cells

ALCOHOLISM, Issue 1 2010
Christopher B. Forsyth
Background:, Alcohol consumption is associated with the risk of progressive cancers including colon and breast cancer. The mechanisms for the alcohol-induced aggressive behavior of these epithelial cancer cells have not been fully identified. Epithelial,mesenchymal transition (EMT) is a developmental program recently shown to play a role in cancer progression and metastases. We hypothesized that alcohol might promote cancer progression by inducing EMT in cancer cells and tested this hypothesis by assessing alcohol-stimulated changes in phenotypic markers of EMT as well as the EMT transcription factor Snail and its related cell signaling. Methods:, Colon and breast cancer cell lines and a normal intestinal epithelial cell line were tested as well as colonic mucosal biopsy samples from alcoholic subjects. Cells were treated with alcohol and assessed for EMT-related changes using immunofluorescent microscopy, western blotting, reporter assays, RT-PCR, and knockdown of Snail with siRNA. Results:, We show alcohol upregulated the signature EMT phenotypic marker vimentin as well as matrix metalloprotease (MMP)-2, MMP-7, and MMP-9 and cell migration in colon and breast cancer cells,all characteristics of EMT. Alcohol also stimulated nuclear localization of Snail phosphorylated at Ser246, transcription from a Snail reporter plasmid, and Snail mRNA expression by RT-PCR. Snail siRNA knockdown prevented alcohol-stimulated vimentin expression. In vivo, Snail expression was significantly elevated in colonic mucosal biopsies from alcoholics. Also, we found alcohol stimulated activation of epidermal growth factor receptor (EGFR) signaling and an EGFR inhibitor blocked alcohol-induced cell migration and Snail mRNA expression. Conclusions:, Collectively, our data support a novel mechanism for alcohol promoting cancer progression through stimulating the EMT program in cancer cells via an EGFR-Snail mediated pathway. This study reveals new pathways for alcohol-mediated promotion of cancer that could be targeted for therapy or prevention of alcohol-related cancers. [source]

Regulation of bone morphogenetic protein signalling in human pulmonary vascular development,

M Southwood
Abstract The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH. Copyright 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source]

Reduced Activity of CD13/Aminopeptidase N (APN) in Aggressive Meningiomas Is Associated with Increased Levels of SPARC

Christian Mawrin
Abstract Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high-grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl-aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc-dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high-grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA-mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway-specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN-siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion. [source]

Constitutively activated FLT3 phosphorylates BAD partially through Pim-1

Kyu-Tae Kim
Summary Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukaemogenesis and their presence is associated with a poor prognosis in acute myeloid leukaemia (AML). Examining the anti- and pro-apoptotic proteins in constitutively activated FLT3 signalling in BaF3/ITD and MV4-11 cells, we found that the level of Bcl-2 antagonist of cell death (BAD) phosphorylation was greatly decreased in response to FLT3 inhibition. Both Ser-112 and Ser-136 of BAD are rapidly dephosphorylated after treatment with the FLT3 inhibitor CEP-701 in BaF3/ITD and MV4-11 cells. In confirmation of the cell line data, BAD was highly phosphorylated in both constitutively activated wild-type and mutant FLT3 primary AML samples, and rapidly dephosphorylated after treatment of the primary samples with CEP-701. Upstream proteins known to phosphorylate BAD include Akt, extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK), Pim-1 and Pim-2. We and other groups have shown that constitutively activated FLT3 induces multiple signalling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt, Erk/MAPK and Janus kinase/signal transducers and activators of transcription (Jak/STAT). Thus, BAD may be a nexus point upon which these multiple signalling pathways converge in FLT3-mediated cell survival. In support of this, siRNA knockdown of BAD expression in MV4-11 cells conferred resistance to CEP-701-mediated apoptosis. Our data suggests that Pim-1 is one of the principal kinases mediating the anti-apoptotic function of FLT3/ITD signalling via the phosphorylation of BAD. [source]

Wilms tumor gene protein 1 is associated with ovarian cancer metastasis and modulates cell invasion

CANCER, Issue 7 2008
Maria V. Barbolina PhD
Abstract BACKGROUND Although metastatic disease is the primary cause of death from epithelial ovarian carcinoma, to the authors' knowledge the cellular mechanisms that regulate intraperitoneal metastasis are largely unknown. Metastasizing ovarian carcinoma cells encounter a collagen-rich microenvironment because the submesothelial matrix is comprised mainly of interstitial collagens Types I and III. METHODS Immunohistochemistry using primary and metastatic ovarian carcinoma samples was employed to detect expression of Wilms tumor gene protein 1 (WT1). Three-dimensional (3D) collagen culture, real-time reverse transcriptase-polymerase chain reaction, and immunofluorescent staining were used to evaluate changes in WT1 RNA and protein expression in response to 3D collagen culture. Boyden chamber invasion assay, scratch-wound motility assay, and Western blot analysis were used to establish the function of WT1 in ovarian carcinoma cells. RESULTS To model intraperitoneal invasion in vitro, ovarian cancer cells were cultured in a 3D collagen microenvironment. 3D collagen culture resulted in robust induction of WT1 at the mRNA and protein levels. WT1 expression was prevalent in primary ovarian tumors and was retained in paired peritoneal metastases. Functional studies supported a role for WT1 in intraperitoneal invasion, because siRNA knockdown of WT1 expression reduced the ability of ovarian cancer cells to invade 3D collagen gels. CONCLUSIONS The data from the current study identify a novel regulatory mechanism for the control of WT1 expression and provide evidence for a functional role of WT1 protein in the control of cellular invasive activity. Cancer 2008. 2008 American Cancer Society. [source]