Autophagy,Bafilomycin and Cell Death: The “A-B-Cs” of Plecomacrolide-Induced Neuroprotection

Bafilomycin A1 (BafA1), which is a member of the plecomacrolide sub-class of macrolide antibiotics, has differential, concentration-dependent effects on neuronal cell viability. When used at high concentrations, BafA1 inhibits vacuolar ATPase (V-ATPase), promotes the accumulation of autophagic vacuoles and triggers Bax-dependent apoptosis. These effects are similar to those induced by the lysosomotropic agent chloroquine. Conversely, at concentrations below its reported ability to completely inhibit V-ATPase, BafA1 dramatically attenuates chloroquine-induced apoptosis. The protective effects of BafA1 appear to be independent of the chloroquine-induced accumulation of autophagosomes. Rather, BafA1 appears to inhibit events downstream of chloroquine-induced autophagosome accumulation, such as the loss mitochondrial or lysosomal integrity. Our finding that BafA1 inhibits the death of neurons induced by autophagic stress suggests a potentially novel mechanism of action apart from its ability to inhibit V-ATPase. Here we provide further evidence of neuroprotection against chloroquine-induced death by plecomacrolide antibiotics that are structurally similar to BafA1, including bafilomycin B1 and concanamycin A, and discuss potential mechanism(s) of neuroprotection against autophagic stress.

Addendum to:

Bafilomycin A1 Inhibits Chloroquine-Induced Death of Cerebellar Granule Neurons

John J. Shacka, Barbara J. Klocke, Masahiro Shibata, Yasuo Uchiyama, Geeta Datta, Robert E. Schmidt and Kevin A. Roth

Mol Pharmacol 2006; 69:1125-36     

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

Regulation of cAMP-dependent Protein Kinases: THE HUMAN PROTEIN KINASE X (PrKX) REVEALS THE ROLE OF THE CATALYTIC SUBUNIT αH-αI LOOP*

cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIβ but not by substrate inhibitors RIIα or RIIβ. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2–4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.     

Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend”

Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α₁-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was “fished out” (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.     

Functional Analysis of the Transmembrane Domains of Presenilin 1: PARTICIPATION OF TRANSMEMBRANE DOMAINS 2 AND 6 IN THE FORMATION OF INITIAL SUBSTRATE-BINDING SITE OF γ-SECRETASE*

γ-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2, which mediates intramembrane proteolysis of a range of type I transmembrane proteins. We previously analyzed the functional roles of the N-terminal transmembrane domains (TMDs) 1–6 of PS1 in the assembly and proteolytic activity of the γ-secretase using a series of TMD-swap PS1 mutants. Here we applied the TMD-swap method to all the TMDs of PS1 for the structure-function analysis of the proteolytic mechanism of γ-secretase. We found that TMD2- or -6-swapped mutant PS1 failed to bind the helical peptide-based, substrate-mimic γ-secretase inhibitor. Cross-linking experiments revealed that both TMD2 and TMD6 of PS1 locate in proximity to the TMD9, the latter being implicated in the initial substrate binding. Taken together, our data suggest that TMD2 and the luminal side of TMD6 are involved in the formation of the initial substrate-binding site of the γ-secretase complex.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

G��q-mediated Activation of GRK2 by Mechanical Stretch in Cardiac Myocytes: THE ROLE OF PROTEIN KINASE C*

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of β-adrenergic receptor (β-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and β-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by “fetal” gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating β-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the Gα_q-coupled angiotensin II AT₁ receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-Gα_q coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKCα also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKCα in transgenic mice led to impaired β-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired β-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and β-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.     

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3): POTENTIAL ROLE IN BORTEZOMIB-MEDIATED ANTICANCER THERAPY*

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome.     

Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/β-CATENIN SIGNALING PATHWAY*

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β-catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway.     

p90 Ribosomal S6 Kinase 1 (RSK1) and the Catalytic Subunit of Protein Kinase A (PKA) Compete for Binding the Pseudosubstrate Region of PKAR1��: ROLE IN THE REGULATION OF PKA AND RSK1 ACTIVITIES*

Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIα, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIα. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIα. This explains the lack of an interaction between active RSK1 and PKARIα. Full-length RSK1 bound to PKARIα with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93–99) of PKARIα. Overexpressed RSK1 dissociated PKAc from PKARIα, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIα interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIα for their interactions, RSK1/PKARIα association requires all four Arg residues (Arg-93–96) in the pseudosubstrate site of PKARIα. A peptide (Wt-PS) corresponding to residues 91–99 of PKARIα competed for binding of RSK1 with PKARIα both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIα, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIα, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIα by associating with RSK1 regulates its activation and its biological functions.     

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages: IN VITRO AND IN SILICO STUDIES

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.     

CLCA Splicing Isoform Associated with Adhesion through β1-Integrin and Its Scaffolding Protein: SPECIFIC EXPRESSION IN UNDIFFERENTIATED EPITHELIAL CELLS*

We previously found that a rat CLCA homologue (rCLCA-f) modulates Ca²⁺-dependent Cl⁻ transport in the ductal cells of the rat submandibular gland. CLCA proteins have been shown to be multifunctional, with roles in, for example, cell adhesion. Here, we describe the mRNA and protein expressions of a splicing isoform of rat rCLCA (rCLCA-t). This isoform is a 514-amino acid protein containing a C-terminal 59-amino acid that is distinct from the rCLCA-f sequence. Immunohistochemistry revealed rCLCA-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation. In a heterologous expression system, rCLCA-t was found to be a membrane protein present predominantly in the perinuclear region, and not to be either present on the cell surface or secreted. rCLCA-t failed to enhance ionomycin-induced Cl⁻ conductance (unlike rCLCA-f). When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was evidently modulated by intracellular Ca²⁺, protein kinase C, and β₁-integrin. rCLCA-t was found to associate with RACK1 (receptor for activated C kinase) and to reduce expression of mature β₁-integrin. Treatment of rat skin with rCLCA-t siRNA increased the expression of β₁-integrin in the stratum basale of the epidermis. These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells.     

Differential Inhibitor of G�¦� Signaling to AKT and ERK Derived from Phosducin-like Protein: EFFECT ON SPHINGOSINE 1-PHOSPHATE-INDUCED ENDOTHELIAL CELL MIGRATION AND IN VITRO ANGIOGENESIS*

Differential inhibitors of Gβγ-effector regions are required to dissect the biological contribution of specific Gβγ-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gβγ-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gβγ, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3β, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane and the signaling to AKT in response to Gβγ overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kγ and Gβγ. Other PhLP-derived constructs interacted with Gβγ but were not effective inhibitors of Gβγ signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gβγ-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gβγ-PI3Kγ interaction.Heterotrimeric G protein signaling depends on the actions of GTP-loaded Gα and free Gβγ, the two functional components of the heterotrimer, leading to the generation of second messengers and cell specific functional events (1, 2). Differential inhibitors of Gβγ are required to dissect the biological impact of different Gβγ-dependent effectors. Gβγ actions can be blocked by competition with peptides derived from its effectors. For example, the effect of Gβγ on adenylyl cyclase II, G protein-activated inward rectifier K⁺ channel, G protein-coupled receptor kinase 2, and phospholipase Cβ3, is attenuated by a peptide from adenylyl cyclase II (3). In addition, RACK1 (receptor for activated C kinase 1) selectively inhibits the effect the chemokine receptor CXCR2 on the activation of phospholipase Cβ2 and adenylyl cyclase II in HEK293 cells, without affecting other functions of Gβγ (4). Recently, Smrcka and colleagues characterized the effect of small molecule inhibitors of Gβγ, suggesting their potential application in therapeutic strategies targeting particular Gβγ-dependent pathways (5). Emerging possibilities to target this heterodimer in pathological situations such as inflammation and angiogenesis are based on the role of Gβγ in cell survival and chemotaxis. To the best of our knowledge, no molecular tool is yet available to differentially inhibit Gβγ signaling to AKT.³Gβγ is a key transducer of sphingosine 1-phosphate (S1P)-elicited angiogenic signals promoting endothelial cell migration, proliferation, and survival (6–12). Multiple Gβγ-dependent effectors are potentially involved in the molecular events required for endothelial cell migration. These include lipid kinases such as PI3Kγ and PI3Kβ (13), and a novel family of Rac guanine nucleotide exchange factors, represented by P-REX1, which is activated by Gβγ and phosphatidylinositol 3,4,5-trisphosphate (14–16). Gβγ signaling is frequently attributed to pertussis toxin-sensitive G_i coupled receptors, and it has been consistently revealed by the antagonistic effect of the carboxyl-terminal region of G protein-coupled receptor kinase 2, which sequesters Gβγ thereby inhibiting all its intracellular actions (17). In addition, mutational analysis of Gβ revealed that different residues, all of them mapping to the interface of contact between Gβγ and Gα, are important for the activation of distinct Gβγ effector molecules (18).Phosducin was originally identified as a phosphoprotein restricted to the retina and pineal gland forming a complex with Gβγ (19, 20). It was considered a protein kinase A-sensitive regulator of G protein-mediated signaling (21, 22). Further studies identified a family of phosducin-like proteins (PhLPs) (23, 24). Phosducin and Gα share affinity for the same region of Gβγ, as revealed by the structural analysis of Gβγ in complex with Gα or phosducin and by in vitro binding experiments (25). This area of interaction includes some of the residues considered necessary for the activation of Gβγ-dependent effectors (18, 26). It was initially postulated that phosducin and related proteins, by interfering with the availability of free Gβγ, exert an inhibitory role on Gβγ signaling. However, recent genetic evidence raised an apparently conflicting situation; the knockout of PhLP in fungi resulted in a phenotype equivalent to the absence of Gβγ, contrary to its expected role as an inhibitor (27). Novel experimental evidence indicated that PhLP has a positive effect on Gβγ signaling due to its participation in the assembly of the heterodimer, helping to stabilize free Gβ subunits leaving the ribosome after synthesis (28–31).Despite the positive role of full-length PhLP in the assembly of Gβγ heterodimers, it is still possible that different fragments of this protein, which could retain their interaction with distinct regions of Gβγ, might function as inhibitors of Gβγ signaling. Accordingly, we characterized here the effect of different PhLP-derived constructs on the signaling pathways elicited by S1P or HGF in endothelial cells. In addition, we explored the mechanism by which PhLP-M1-G149 interferes with Gβγ preventing the activation of AKT.     

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β: THE CRITICAL ROLE OF FINGERS DOMAIN MOVEMENTS AND A NOVEL NON-COVALENT STEP DURING NUCLEOTIDE SELECTION*

During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.     

Enhanced Anti-tumor Activity by the Combination of the Natural Compounds (?)-Epigallocatechin-3-gallate and Luteolin: POTENTIAL ROLE OF p53*

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3–5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser¹⁵ phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.     

Ciliary Neurotrophic Factor Protects Mice Against Streptozotocin-induced Type 1 Diabetes through SOCS3: THE ROLE OF STAT1/STAT3 RATIO IN β-CELL DEATH*

Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.     

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK,ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser^176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser^32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser^217/221), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), and p38 (Thr¹⁸⁰/Tyr¹⁸²) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser⁵³⁶-phosphorylated (p65⁵³⁶) and Lys³¹⁰-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr³⁵⁹/Ser³⁶³ in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65⁵³⁶ kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.     

GEF-H1 Mediates Tumor Necrosis Factor-��-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY*

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-α, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-α on the Rho pathway in tubular cells (LLC-PK₁ and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-α-induced alterations of paracellular permeability. We show that TNF-α induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-α-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-α-induced activation of these proteins. Importantly TNF-α enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-α-induced paracellular permeability increase was absent in LLC-PK₁ cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-α-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.Tumor necrosis factor-α (TNF-α)² is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury (1). Subsequently it is cleaved by the metalloprotease TNF-α convertase enzyme to release a 17-kDa soluble peptide (for a review, see Ref. 2). TNF-α has two receptors, the constitutively expressed, ubiquitous TNF receptor 1 and the inducible TNF receptor 2.An increasing body of evidence supports a key role for TNF-α in both acute renal injury and chronic kidney diseases (for reviews, see Refs. 3 and 4). Although TNF-α is almost undetectable in normal kidneys, elevated intrarenal, serum, or urine concentrations have been reported in various pathological states including ischemia-reperfusion, endotoxinemia, and early diabetic nephropathy (5–8). Moreover kidney injury in various pathological states was prevented or mitigated by inhibition of TNF-α production, by addition of neutralizing antibodies, or in TNF receptor knock-out mice (for a review, see Ref. 3). The central role of TNF-α in mediating kidney injury is therefore well established. Importantly TNF-α can be produced in the kidney not only by infiltrating macrophages and lymphocytes but by resident cells including the tubular epithelium. For example, in reperfusion injury TNF-α expression precedes macrophage infiltration and localizes mostly to the tubules (3, 7). Tubular TNF-α production is also enhanced by endotoxin and hypoxia (9–12). Although effects of locally released TNF-α on the tubular epithelium could contribute to its deleterious actions, the underlying mechanisms have been incompletely explored.Although a large number of studies have focused on the inflammatory and apoptotic signaling initiated by TNF-α in various cells, its cytoskeletal effects remain much less explored. In recent years Rho and its effector, Rho kinase (ROK), key regulators of both the actin cytoskeleton and myosin phosphorylation (13), have emerged as important mediators of TNF-α effects in endothelial cells (14–18). Similar effects in the tubular epithelium, however, have not been established. Even more importantly, the upstream signaling that connects the TNF receptor to activation of the Rho pathway remains completely unknown. Like other small GTPases, Rho cycles between an inactive (GDP-bound) and active (GTP-bound) form (13). The exchange of GDP to GTP during activation is stimulated by GDP-GTP exchange factors (GEFs). The diverse family of Rho GEFs contains >70 members in humans (19), making it challenging to identify the specific factors involved in mediating Rho activation through receptor-mediated stimuli. In the case of TNF-α, neither the particular Rho GEF involved nor the mechanism of its regulation has been identified in any of the cell systems studied.A rise in epithelial paracellular permeability through the intercellular junctions is a prominent event during inflammation (“leaky epithelium”) (for reviews, see Refs. 20 and 21). In addition, the junctions maintain the polarized phenotype of epithelial cells that is necessary for directional transport processes and constitute an important signaling platform that transmits environmental cues to the cells. Therefore, the consequences of junction disruption during inflammation might go beyond the compromised barrier functions. Interestingly TNF-α has been reported to affect the permeability of the tubular epithelium. Mullin et al. (22) have reported that in a tubular cell line TNF-α induced a temporary elevation in transepithelial resistance followed by a drop in transepithelial resistance and increased paracellular permeability. The transepithelial resistance decrease was blocked by genistein, a general tyrosine kinase inhibitor; however, the exact mechanism underlying the observed permeability changes remained incompletely explored.The actin cytoskeleton and especially phosphorylation of myosin light chain (MLC) was shown to be essential for the permeability increase caused by pathogens, cytokines, and growth factors in various epithelial and endothelial systems (for reviews, see Refs. 21, 23, and 24). Interestingly although myosin phosphorylation mediates the TNF-α-elicited permeability changes in intestinal cells (25, 26), phospho-MLC was reported not to be involved in the TNF-α-induced permeability rise in endothelial cells (17). The possible role of the Rho pathway and myosin phosphorylation in the TNF-α-induced permeability changes in the tubular epithelium therefore remains to be established.The aim of this study was to explore the signaling pathways through which TNF-α causes cytoskeleton remodeling and elevates paracellular permeability in kidney tubular cells. Our findings show that TNF-α induces rapid activation of RhoA that leads to Rho/Rho kinase-dependent actin remodeling and myosin phosphorylation. Using an affinity precipitation assay followed by mass spectrometry, we identified GEF-H1 as a TNF-α-activated GEF. We showed that GEF-H1 mediates the TNF-α-induced stimulation of Rho and its effectors. In addition, activation of the GEF-H1/Rho pathway by TNF-α was downstream of ERK signaling and required GEF-H1 phosphorylation on Thr-678. Finally using a dominant negative MLC mutant, we showed that myosin phosphorylation is essential for the TNF-α-induced elevation in paracellular permeability.