Immobilon-P Membrane Research Articles

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Interdigitated Capacitive Immunosensors With PVDF Immobilization Layers

We have explored for the first time PVDF Immobilon-P membranes, attached to interdigitated capacitor microelectrodes, as 3-D matrixes for the immobilization of antibodies in an interdigitated capacitive immunosensor format. The thick membranes allow distribution of antibodies across the entire region probed by the measuring electric field applied to the microelectrodes. This new approach is an alternative to the use of nanometer electrodes necessary to detect smaller layers of antibodies. As a proof-of-concept, a horseradish peroxidase (HRP) antibody/antigen affinity pair allowed for specific detection of the antigen in blocked membranes containing the antibody. Immobilization of antibody and penetration of antigen inside the PVDF membrane was assessed by fluorescence measurements and by an HRP enzymatic assay, respectively. The new interdigitated capacitive immunosensor was able to discriminate between buffer alone and HRP antigen in buffer, as proven by capacitance measurements.

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

A Highly Efficient Multifunctional Tandem Affinity Purification Approach Applicable to Diverse Organisms

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Expression of hypoxia-related glucose transporters GLUT1 and GLUT3 in benign, malignant and non-neoplastic thyroid lesions

The enhancement of glucose metabolism in neoplastic cells is mediated by the overexpression of key glycolytic enzymes and glucose transporters (GLUTs). In particular, an increased expression of hypoxia-related GLUT1 and GLUT3 has been found in a variety of malignancies. The aim of this study was to examine the expression levels of GLUT1 and GLUT3 in benign and malignant thyroid tumors, as well as in non-neoplastic lesions. Analysis of the mRNA expression levels of solute carrier family2, member1 (SLC2A1) and solute carrier family2, member3 (SLC2A3) (genes coding GLUT1 and GLUT3, respectively) was performed by the real-time PCR method with fluorescent probes. GLUT1 and GLUT3 protein expression levels were determined in thyroid specimens by immunodetection after separation of proteins on 10% polyacrylamide slab gels and electrotransfer onto Immobilon-P membranes. The majority of papillary carcinoma samples showed a higher expression of GLUT1 and GLUT3 in comparison with follicular carcinoma cases and non-neoplastic thyroid lesions. A tendency towards an increased expression of GLUT1 and GLUT3 was observed in papillary carcinoma cases with more advanced disease stages. Moreover, a significant correlation was noted between the hypoxia-related GLUT1 and GLUT3 expression determined by both methods. In conclusion, our findings suggest that GLUT1 and GLUT3 play an important role in the pathology of thyroid glands.

Insulin-like Growth Factor Receptor-1 and Nuclear Factor κB Are Crucial Survival Signals That Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.

Degradation of p21Cip1 through Anaphase-promoting Complex/Cyclosome and Its Activator Cdc20 (APC/CCdc20) Ubiquitin Ligase Complex-mediated Ubiquitylation Is Inhibited by Cyclin-dependent Kinase 2 in Cardiomyocytes

Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.

Synthesis, Maturation, and Trafficking of Human Na+-Dicarboxylate Cotransporter NaDC1 Requires the Chaperone Activity of Cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids

Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone

Mitoquinone (MitoQ) is a synthetically modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We found that MitoQ is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ treatment led to irreversible inhibition of clonogenic growth of breast cancer cells through a combination of autophagy and apoptotic cell death mechanisms. Relatively limited cytotoxicity was seen with the parent ubiquinone coenzyme Q(10.) Inhibition of cancer cell growth by MitoQ was associated with G(1)/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. The possible role of oxidative stress in MitoQ activity was investigated by measuring the products of hydroethidine oxidation. Increases in ethidium and dihydroethidium levels, markers of one-electron oxidation of hydroethidine, were observed at cytotoxic concentrations of MitoQ. Keap1, an oxidative stress sensor protein that regulates the antioxidant transcription factor Nrf2, underwent oxidation, degradation, and dissociation from Nrf2 in MitoQ-treated cells. Nrf2 protein levels, nuclear localization, and transcriptional activity also increased following MitoQ treatment. Knockdown of Nrf2 caused a 2-fold increase in autophagy and an increase in G(1) cell cycle arrest in response to MitoQ but had no apparent effect on apoptosis. The Nrf2-regulated enzyme NQO1 is partly responsible for controlling the level of autophagy. Keap1 and Nrf2 act as redox sensors for oxidative perturbations that lead to autophagy. MitoQ and similar compounds should be further evaluated for novel anticancer activity.

LKB1 Suppresses p21-activated Kinase-1 (PAK1) by Phosphorylation of Thr109 in the p21-binding Domain

The serine/threonine protein kinase LKB1 is a tumor suppressor gene mutated in Peutz-Jeghers syndrome patients. The mutations are found also in several types of sporadic cancer. Although LKB1 is implicated in suppression of cell growth and metastasis, the detailed mechanisms have not yet been elucidated. In this study, we investigated the effect of LKB1 on cell motility, whose acquisition occurs in early metastasis. The knockdown of LKB1 enhanced cell migration and PAK1 activity in human colon cancer HCT116 cells, whereas forced expression of LKB1 in Lkb1-null mouse embryonic fibroblasts suppressed PAK1 activity and PAK1-mediated cell migration simultaneously. Notably, LKB1 directly phosphorylated PAK1 at Thr(109) in the p21-binding domain in vitro. The phosphomimetic T109E mutant showed significantly lower protein kinase activity than wild-type PAK1, suggesting that the phosphorylation at Thr(109) by LKB1 was responsible for suppression of PAK1. Consistently, the nonphosphorylatable T109A mutant was resistant to suppression by LKB1. Furthermore, we found that PAK1 was activated in the hepatocellular carcinomas and the precancerous liver lesions of Lkb1(+/-) mice. Taken together, these results suggest that PAK1 is a direct downstream target of LKB1 and plays an essential role in LKB1-induced suppression of cell migration.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Parathyroid Hormone Receptor Directly Interacts with Dishevelled to Regulate β-Catenin Signaling and Osteoclastogenesis

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.

Embryonic Expression of Cyclooxygenase-2 Causes Malformations in Axial Skeleton

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.