Co-transfected COS-7 Cells Research Articles

Augmented leptin-induced trefoil factor 3 expression and epidermal growth factor receptor transactivation differentially influences neoplasia progression in the stomach and colorectum of dietary fat-induced obese mice

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

CFTR-associated ligand is a negative regulator of Mrp2 expression.

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.

IQGAP1 modulates the proliferation and invasion of thyroid cancer cells in response to estrogen.

Thyroid cancer is an endocrine malignancy with a high incidence rate, which is affected by female hormones, particularly estrogens, in its growth and progression. IQ-domain GTPase-activating protein 1 (IQGAP1) is overexpressed in a range of types of cancer and is reported to interact with estrogen receptor α (ERα) in breast cancer cells. However, the association between IQGAP1 and ERα in thyroid cancer cells remains to be elucidated. In this study, the role of IQGAP1 in thyroid cancer cells was examined. The expression of IQGAP1 (190 kDa) was analyzed using western blot analysis, which indicated that IQGAP1 was overexpressed in thyroid cancer tissues and FTC133 cells. However, IQGAP1 knockdown in the FTC133 cells led to a significant downregulation in ERα transcriptional activity, cell proliferation, cell adhesion and cell invasion under 17β-estradiol (E2) conditions. Furthermore, ERα knockdown inhibited the enhanced protein expression levels of phosphorylated ERK1/2 and cyclin D1, which were induced by the overexpression of IQGAP1. Co-immunoprecipitation was also performed in thyroid cancer cells and the results suggested that IQGAP1 directly interacted with ERα in the FTC133 cells and the co-transfected COS-7 cells. Taken together, these findings revealed that IQGAP1 may directly interact with ERα and serve as a signal integrator, mediating ERα transcriptional activity, cell proliferation and cell invasion during the progression of thyroid cancer.

Methylsulfonylmethane Suppresses Breast Cancer Growth by Down-Regulating STAT3 and STAT5b Pathways

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1α, VEGF, BrK, and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90α, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis. Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.

Abstract 9842: Bone Morphogenetic Protein-2 Decreases MicroRNA 30b-c to Promote Vascular Smooth Muscle Cell Calcification

Bone morphogenetic protein-2 (BMP2) initiates vascular smooth muscle cell (SMC) calcification by upregulating expression of the osteoblast transcription factor Runx2. MicroRNAs (miRs) are negative regulators of gene expression; however, the role of miRs in regulating Runx2 expression remains unknown. Accordingly, we treated human coronary artery SMC with BMP-2 (100 ng/ml), and confirmed a 13-fold (p<0.01) increase in Runx2 mRNA levels at 4 h and a 1.7-fold (p<0.02) increase in protein at 24 h. A miR microarray revealed that BMP2 decreased miR-30 family expression 5.1-fold (p<0.01), and real-time PCR verified this finding: BMP2 decreased expression of miR-30b by 4.4-fold (p<0.01) and miR-30c by 3.4-fold (p<0.01). Downregulation of miR-30b-c by transfection of SMC with anti-miR-30b-c increased Runx2 protein by 1.9-fold (p<0.01) or 3.0-fold (p<0.01), respectively. By contrast, forced expression of miR-30b-c by transfection with pre-miR-30b-c attenuated BMP2-induced expression of Runx2 (1 ± 0 vs. 0.6 ± 0.3 vs. 0.5 ± 0.2 arb units, p <0.02). Next, we cotransfected COS7 cells with a luciferase reporter plasmid containing the Runx2 3' untranslated region (UTR) and pre-miR-30b-c. In this assay system, forced expression of miR-30b-c decreased luminosity (1.0 ± 0 vs. 0.6 ± 0.1 vs. 0.6 ± 0.1 fl. units, p<0.04) indicating that miR-30b-c bind to the Runx2 3'-UTR to modulate expression. Downregulation of miR-30b-c also increased expression of the Runx2-dependent calcification protein osteocalcin (1.0 ± 0 vs. 1.7 ± 0.5 vs. 2.2 ± 0.4 arb. units, p<0.03). Von Kossa staining of SMC transfected with anti-miR-30b-c for 14 d revealed that SMC calcification was increased by 1.7-fold (p<0.03) or 1.8-fold (p<0.03) while forced expression of miR-30b-c prevented calcification (1.2 ± 0 vs. 0.9 ± 0.1 vs. 0.9 ± 0.1 arb units, p<0.01). In BMP2-treated SMC, downregulation of miR-30b or miR-30c increased calcification 1.7± 0.3-fold (p<0.03) or 2.3 ± 0.7-fold (p<0.03) while forced expression of miR-30b-c prevented calcification (0.9 ± 0.1 vs. 1.0 ± 0.1 vs. 1.0 ± 0.1 arb units, p<0.01). These data demonstrate that miR-30b-c regulate Runx2 expression to influence SMC mineralization, and suggest that strategies to regulate expression of these miRNA may limit vascular calcification.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Calcium/Calmodulin-Dependent Kinase II Mediates the Phosphorylation and Activation of NADPH Oxidase 5

Excessive synthesis of reactive oxygen species contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Noxes). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We have reported that the activity and calcium-sensitivity of Nox5 can also be modulated by direct phosphorylation. However, the kinases that phosphorylate Nox5 have not been identified, and thus, the goal of this study was to determine whether calcium-activated kinases such as calcium/calmodulin-dependent kinase II (CAMKII) are involved. We found that Nox5 activity in bovine aortic endothelial cells was suppressed by two doses of the CAMKII inhibitor 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93). In cotransfected COS-7 cells, wild-type and constitutively active CAMKII, but not a dominant-negative, robustly increased basal Nox5 activity. The ability of CAMKII to increase Nox5 activity was also observed with fixed calcium concentrations in an isolated enzyme activity assay. CAMKII did not elevate intracellular calcium or activate other Nox enzymes. In vitro phosphorylation assays revealed that CAMKII can directly phosphorylate Nox5 on Thr494 and Ser498 as detected by phosphorylation state-specific antibodies. Mass spectrometry (MS) analysis revealed the phosphorylation of additional, novel sites at Ser475, Ser502, and Ser675. Of these phosphorylation sites, mutation of only Ser475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate Ser475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Together, these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of Ser475.

Abstract 3077: The SR protein kinase Clk1 phosphorylates SPF45 and regulates its localization, degradation and alternative splicing activity

Abstract Background: Alternative splicing (AS) of pre-mRNA is a major source of protein diversity in eukaryotic proteomes. Alterations in splicing factor expression have been shown in numerous cancers and reversible phosphorylation of splicing factors by SR protein kinases can regulate their localization and activity. The AS factor SPF45 (Splicing Factor 45) has low expression in normal tissues, but is overexpressed in several cancers. SPF45 overexpression was reported to induce multidrug resistance. There are no reports on how SPF45 activity is regulated by phosphorylation. Methods: To determine if Clk1 (Cdc2-like kinase 1) regulates SPF45 alternative splicing activity, we generated a delta-fas minigene and cotransfected COS-1 cells with delta-fas, wild-type or mutant SPF45, and active or kinase dead Clk1. Clk1 and SPF45 effects on delta-fas splicing were determined by RT-PCR. Clk1 inhibition was achieved by the chemical inhibitor TG003 or by Clk1 siRNA. Half life of endogenous, transiently-transfected and stably transfected SPF45 was determined by cycloheximide treatment and western blotting. Proteosome inhibition was achieved with MG132. Recombinant Clk1 was used to phosphorylate recombinant SPF45 in vitro. Phosphorylation sites were identified by (LC)-electrospray ionization (ESI) -tandem mass spectrometry (MS/MS) and confirmed by in vitro kinase assays. Confocal immunofluorescence was performed to determine the subnuclear localization of wild type and mutant SPF45 upon manipulation of Clk1 expression or activity. Results: Clk1 expression significantly increased SPF45-mediated delta-fas alternative splicing, while Clk1 inhibition with kinase-dead Clk1, Clk1 siRNA, or TG003 significantly inhibited SPF45 AS activity. Clk1 expression enhanced expression of SPF45 protein, but not mRNA, while Clk1 inhibition decreased SPF45 protein expression. Clk1 inhibition increased SPF45 degradation through a proteosome-independent pathway. Clk1 phosphorylated SPF45 in vitro on Ser202 and Ser204 and mutation of these residues partially inhibited SPF45 AS activity, although Clk1 still partially induced AS activity of a SPF45-S202/204 mutant, suggesting that the effects of Clk1 on SPF45 were both Ser202/204 dependent and independent. Confocal immunofluorescence studies demonstrated that expression of active Clk1 caused a redistribution of SPF45 from nuclear speckles to a diffuse pattern within the nucleus in a Ser202/Ser204 dependent manner, while inhibition of Clk1 blocked these effects. Conclusion: Clk1 is the first kinase demonstrated to phosphorylate SPF45, increasing its expression and alternative splicing activity while changing SPF45 subnuclear localization. These data provide new insights into the regulation of SPF45 and raise the possiblity that inhibition of Clk1 could be used as a therapeutic target for SPF45-mediated drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2011-3077

Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn

BackgroundTau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.ResultsIn this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.ConclusionsFyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.

Regulation of M3 Muscarinic Receptor Expression and Function by Transmembrane Protein 147

The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.

Stimulation of Platelet-derived Growth Factor Receptor β (PDGFRβ) Activates ADAM17 and Promotes Metalloproteinase-dependent Cross-talk between the PDGFRβ and Epidermal Growth Factor Receptor (EGFR) Signaling Pathways

Binding of the platelet-derived growth factor (PDGF)-B to its receptor PDGFRbeta promotes proliferation, migration, and recruitment of pericytes and smooth muscle cells to endothelial cells, serving to stabilize developing blood vessels. The main goals of this study were to determine whether the extracellular domain of the PDGFRbeta can be proteolytically released from cell membranes and, if so, to identify the responsible sheddase and determine whether activation of the PDGFRbeta stimulates its shedding and potentially that of other membrane proteins. We found that the PDGFRbeta is shed from cells by a metalloproteinase and used loss-of-function experiments to identify ADAM10 as the sheddase responsible for constitutive and ionomycin-stimulated processing of the PDGFRbeta. Moreover, we showed that ligand-dependent activation of the PDGFRbeta does not trigger its own shedding by ADAM10, but instead it stimulates ADAM17 and shedding of substrates of ADAM17, including tumor necrosis factor alpha and transforming growth factor alpha. Finally, we demonstrated that treatment of mouse embryonic fibroblasts with PDGF-B triggers a metalloproteinase-dependent cross-talk between the PDGFRbeta and the epidermal growth factor receptor (EGFR)/ERK1/2 signaling axis that is also critical for PDGF-B-stimulated cell migration, most likely via ADAM17-dependent release and activation of ligands of the EGFR. This study identifies the principal sheddase for the PDGFRbeta and provides new insights into the mechanism of PDGFRbeta-dependent signal transduction and cross-talk with the EGFR.

An Arginine Stretch Limits ADAM10 Exit from the Endoplasmic Reticulum

A disintegrin and metalloproteinase 10 (ADAM10) is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a number of proteins implicated in the pathogenesis of diseases ranging from cancer to Alzheimer Disease. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its shedding activity. Here we report the identification of an endoplasmic reticulum (ER) retention motif within the ADAM10 intracellular C-terminal tail. Sequential deletion/mutagenesis analyses showed that an arginine-rich ((723)RRR) sequence was responsible for the retention of ADAM10 in the ER and its inefficient surface trafficking. Mutating the second arginine to alanine was sufficient to allow ER exit and surface expression in both heterologous cells and hippocampal neurons. As synapse-associated protein 97 (SAP97) binds ADAM10 at its cytoplasmic tail and facilitates forward ADAM10 trafficking in neurons, we tested whether SAP97 could modulate ER export. However, neither expression nor Ser-39 phosphorylation of SAP97 in heterologous cells or hippocampal neurons were sufficient to allow the ER exit of ADAM10, suggesting that other signaling pathways or alternative binding partners are responsible for ADAM10 ER exit. Together, these results identify a novel mechanism regulating the intracellular trafficking and membrane delivery of ADAM10.

Interaction of retinal guanylate cyclase with the α subunit of transducin: potential role in transducin localization

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

Investigation of Phosphorylation Site Responsible for CaLP (P. fucata) Nucleo-cytoplasmic Shuttling Triggered by Overexpression of p21Cip1

Calmodulin (CaM) is a highly conserved and ubiquitous Ca(2+)-binding protein regulating intracellular Ca(2+) concentration by acting as a sensor of this divalent cation in eukaryotic cells. Being such a very important signal sensor, CaM is susceptible to undergo many posttranslational modifications. One of these important modifications is its phosphorylation. Our previous investigations showed that CaM and calmodulin-like protein (CaLP) cloned from Pinctada fucata have many different characteristics in spite of their high similarity to each other. We have narrowed down that the C-terminal domains of CaM and CaLP are responsible for their discrepant subcellular localizations and shuttling of CaLP when it is co-transfected with p21(Cip1), which is commonly considered as an important cell cycle regulating protein. In this study, we first predicted the potential phosphorylation site responsible for the shuttling and confirmed by fluorescence confocal microscopy. Together with fluorescence activated cell sorter analysis, we further investigated the releasing ability of wild type and point mutated CaLP from arrested cell cycle caused by p21(Cip1) overexpression. By performing pull-down analysis and phosphorylation status of CaLP in cytoplasm fraction of transfected COS-7 cells with CaLP alone and phosphorylation status of CaLP in nuclear fraction of co-transfected COS-7 cells with CaLP and p21(Cip), we propose that the CaLP staying in the cytoplasm is in the state of phosphorylation, but when p21(Cip1) is overexpressed in mammalian cells, some signal triggers CaLP dephosphorylation and translocation into the nucleus.

537 Reg IV Induces β-Catenin Nuclear Translocation to Induce Cell Proliferation in Human Colon Adenocarcinomas

PCR (RT-PCR) was conduced using total RNA from jejunal mucosal tissues. In Vitro studies were conducted in COS-7 cells transfected with cDNA constructs for HA-Klf5 and FLAGβ-catenin. Luciferase activities were conducted using the TOPFLASH reporter plasmid which contains a β-catenin responsive element. RESULTS: 100% of Apc mice developed intestinal adenomas by 18 weeks of age, whereas only 53% of Apc/Klf5 mice developed adenomas. In addition, Apc mice developed an average of 34 ± 5 adenomas per mouse, compared to an average of 2 ± 1 in the Apc/Klf5mice (n=15, P < 0.0001). Immunohistochemical staining of Klf5 revealed that Klf5 levels were lower in the normal-appearing intestinal tissues of Apc/Klf5 mice compared to wild-type or Apc mice. Staining of β-catenin showed reduced nuclear/cytoplasmic localization of β-catenin in normal-appearing tissues of the Apc/Klf5 compared to Apc mice. In addition, mRNA levels of two transcriptional targets of β-catenin, cyclin D1 and EphrinB2, were reduced in intestines from Apc/Klf5 compared to Apc mice. In Vitro studies in co-transfected COS-7 cells showed a direct interaction of Klf5 and β-catenin by immunoprecipitation. In addition, nuclear localization of β-catenin was enhanced when Klf5 and β-catenin were co-expressed compared to expression of β-catenin alone. Finally, while Klf5 did not increase TOPFLASH luciferase activity, co-expression of Klf5 and β-catenin synergistically enhanced the ability of β-catenin to induce TOPFLASH luciferase activity. CONCLUSIONS: Our study demonstrates that Klf5 is a major contributor to intestinal adenoma formation in the Apc mouse model. The involvement of Klf5 is through its effects on β-catenin localization and activity.

MyD88-5 and Neuronal Toxicity

The first four members of the myeloid differentiation marker 88 (MyD88) family of cytosolic adaptor proteins are well known for their roles in mediating Toll-like receptor (TLR) signaling in the immune system. Kim et al . set out to characterize the more enigmatic fifth member of this family, MyD88-5. Northern and Western blotting demonstrated a higher abundance of MyD88-5 mRNA and protein in the brains of mice and humans than in any other organ. In human blood, real-time reverse transcription polymerase chain reaction (RT-PCR) assays showed that MyD88-5 mRNA was preferentially found in lymphocytes and not in myeloid cells. The authors developed a transgenic mouse that expressed a green fluorescent protein (GFP)-tagged MyD88-5 protein. Confocal microscopic analysis of brain sections showed that GFP fluorescence was strongest in the hippocampus and cerebellum and found only in neurons, within which MyD88-5-GFP was cytoplasmic and had a punctate distribution. When expressed in COS-1 cells, MyD88-5-GFP strongly colocalized with mitochondria. In cotransfected COS-1 cells, the authors demonstrated the colocalization of MyD88-5-GFP with c-Jun N-terminal kinase 3 (JNK3) at mitochondria, an association that was also shown in coimmunoprecipitation studies in COS-1 cells and in hippocampal lysates. JNK3 promotes apoptosis in hippocampal cells after cerebral ischemia (oxygen and nutrient deprivation), and the authors found that cell death was reduced in ischemic hippocampal slices from MyD88-5-deficient mice compared with those from wild-type mice. This study reveals the role of MyD88-5 in mediating neuronal cell death after injury and raises the interesting question of what role MyD88-5 might play in lymphocytes. Y. Kim, P. Zhou, L. Qian, J.-Z. Chuang, J. Lee, C. Li, C. Iadecola, C. Nathan, A. Ding, MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival. J. Exp. Med. 204 , 2063-2074 (2007). [Abstract] [Full Text]

The p21-activated Kinase 3 Implicated in Mental Retardation Regulates Spine Morphogenesis through a Cdc42-dependent Pathway

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.

The role of native signal peptides in trafficking RAMP2 to the plasma membrane

Receptor activity-modifying protein 2 (RAMP2) forms a heterodimer with the calcitonin receptor-like receptor (CL) to produce an adrenomedullin receptor. Recently, splice variants in the signal peptide region of RAMP2 have been identified in pufferfish and rats (FASEB J 20 437.7). The splice variant rat RAMP2b has a 26 amino acid deletion in a highly hydrophobic region of the signal peptide. Our goal was to determine whether the rat RAMP2b trafficked to the plasma membrane. We transiently co-transfected COS-7 cells with an N-terminal HA-tagged CL and either the wild-type RAMP2, containing the intact signal peptide, or the RAMP2b. Forty-eight hours after transfection, non-permeabilized live cells were immunostained with HA and RAMP2 primary antibodies to determine if epitopes of the proteins were present on the outer surface of the plasma membrane. A RAMP2 mutant, lacking the signal peptide, was also constructed and tested. Our studies showed that both the rat RAMP2 and RAMP2b trafficked to the cell membrane in the presence or absence of CL, but that the RAMP2 mutant without the signal peptide did not reach the plasma membrane, even though it was detected inside the cells. (Support by Creighton University Medical Center Health Futures Foundation, MAS)

Dominant-Negative Effect of NPM-RAR on NPM Mediated Regulation of p53.

We have been studying the variant Acute Promyelocytic Leukemia (APL) translocations with a view towards identifying common molecular pathways underlying APL. The t(5;17) variant of APL fuses the N-terminal 117 amino acids of nucleophosmin (NPM) with the C-terminal 402 amino acids of the retinoic acid receptor alpha (RAR). NPM plays a role in several important pathways within cells beyond its originally described function in the transport of ribonucleoproteins. It serves a chaperone function in transport of proteins such as p53, Tat, Rb, YY1, IRF-1, and Arf. NPM binds to and becomes a target of CDK2/cyclin E to play a role in regulation of centrosomal duplication. The importance of NPM in myeloid leukemias has been highlighted by the recent finding that mutation of the NPM C-terminus is the most common genetic event in non-APL acute myeloid leukemia. We have previously shown that the RAR portion of NPM-RAR acts as a dominant-negative towards wild-type RAR. We now investigate the hypothesis that NPM-RAR serves as a dominant negative for NPM and partially contributes to the leukemic phenotype through disruption of NPM-dependent cellular functions. We have focused our studies on the effects of NPM-RAR on p53. It has previously been shown that NPM directly binds to p53 to increase p53 stability and p53-dependent transcription. The p53-interaction domain maps to a region of NPM that is not contained in NPM-RAR. We hypothesized that NPM-RAR might bind directly with NPM, disrupt NPM association with p53, and thereby indirectly affect p53 activity. We first used pull-down assays of Maltose Binding Protein (MBP)-NPM or MBP-NPM-RAR fusions incubated with 35S-NPM to determine that the N-terminal domain of NPM-RAR is sufficient to interact with NPM. To investigate whether NPM-RAR alters NPM interaction with p53, we transfected COS cells with expression vectors encoding NPM, p53 and either NPM-RAR or control plasmid (pBluescript). Complexes were precipitated with an anti-NPM antibody (that recognizes epitopes not contained in NPM-RAR) and immunoblotted with an anti-p53 antibody. We found a decrease in the p53 signal captured by NPM in the lysate expressing NPM-RAR, compared to the control lysate. To further investigate the effects of NPM-RAR on p53, we made use of a transcriptional assay utilizing a reporter gene under control of a p53 response element. We co-transfected COS cells with a mix containing NPM-RAR, p53, and NPM expression plasmids, along with a p53-luciferase reporter (containing a 20 base pair p53 response element derived from the p21 promoter), and a ß-gal transfection control plasmid, or a similar mix in which NPM-RAR was replaced by an equivalent amount of carrier DNA (pBluescript). We found that the co-expression of NPM-RAR led to a decrease in the luciferase activity. These results support our hypothesis that NPM-RAR binding to NPM interferes with NPM's ability to regulate p53.

Nephrogenic Syndrome of Inappropriate Antidiuresis: A Novel Disorder in Water Balance in Pediatric Patients

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We report findings in 2 unrelated male infants whose clinical presentation and laboratory findings were consistent with SIADH, but who exhibited unmeasurable arginine vasopressin (AVP) levels on repeated occasions. We hypothesized that these infants had a novel gain of function defect in the AVP-signaling pathway. DNA sequencing of each patient’s vasopressin V 2 receptor (V 2R) gene identified mutations (R137C or R137L) in each. R137H mutations have been reported previously to cause nephrogenic diabetes insipidus. To further characterize the effects of these mutations, we re-created each mutation by site-directed mutagenesis in a vasopressin V 2R expression vector and cotransfected COS-7 cells with wild-type and mutant vasopressin V 2R vectors and a cyclic adenosine monophosphate–responsive luciferase reporter plasmid. The luciferase activity was induced 7.5-fold (R137L mutant; P = 0.0037) and 4-fold (R137C mutant; P = 0.013) more than the wild-type vasopressin V 2R, which is the empty vector or the inactivating R137H mutant. This novel gain of function mutation in the vasopressin V 2R can cause constitutive activation of the receptor and resultant hyponatremia. These findings represent a previously unrecognized genetic disease, which was designated as nephrogenic syndrome of inappropriate antidiuresis. A number of questions have emerged, including the following: (1) What is the frequency? (2) Are there nonrenal manifestations? (3) Are heterozygotes affected? (4) What is the optimal therapy? and (5) How do these mutations cause constitutive activation of the receptor?