Angiotensin AT(1) receptor inhibition-induced apoptosis by RhoA GTPase activation and pERK1/2 signaling pathways in neonatal obstructive nephropathy

Intrarenal renin-Angiotensin system (RAS) activity is increased during early development and is further enhanced by unilateral ureteral obstruction (UUO). We studied the involvement of mitogen-activated protein (MAP) kinase members and the RhoA GTPase signaling pathways on the regulation of renal cell response after AT1 Angiotensin II receptor inhibition in obstruction. Neonatal rats subjected to sham operation or complete UUO within the first 48 hours of life received saline vehicle, Losartan (AT1 inhibitor), or PD-123319 (AT2 inhibitor) during the first 14 days of life. Cortex tubular epithelial cell apoptotic response was shown by TUNEL and confirmed by electron microscopy associated with mitochondrial signaling pathway through the increased proapoptotic ratio Bax/BcL-2, and consequently increased caspase 3 expression and activity in obstructed kidney before and after Type 1 (AT1) receptor blockade. Non injury of contralateral kidney was shown. The convergence of two independent signal pathways, the RhoA GTPase and pERK and concurrent inhibition of JNK MAP kinase, were required for the apoptotic response in 14 day kidney obstructed tubular cells either with or without Losartan treatment. Absence of increased AT2 protein expression after AT1 receptor inhibition on day 14 of obstruction was shown. Selective AngiotensinAT2-receptor inhibition with PD-123319 had no protective effect on the renal response to complete 14 day UUO. We suggest a role of both RhoA GTPase activation and the opposing actions of the ERK and JNK-MAP kinase signaling pathways as events involved in tubular cell apoptosis regulation in neonatal UUO. The selective AT1-receptor inhibition had no effect on the renal cellular response in the kidney subjected to UUO for 14 days.     

The putative GTPase encoded by MTG3 functions in a novel pathway for regulating assembly of the small subunit of yeast mitochondrial ribosomes

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes. This gene codes for a member of the YqeH family of GTPases, which we have named MTG3 in keeping with the earlier convention. Mutations in MTG3 cause the accumulation of the 15 S rRNA precursor, previously shown to have an 80-nucleotide 5' extension. Sucrose gradient sedimentation of mitochondrial ribosomes from temperature-sensitive mtg3 mutants grown at the permissive and restrictive temperatures, combined with immunobloting with subunit-specific antibodies, indicate that Mtg3p is required for assembly of the 30 S but not 54 S ribosomal subunit. The respiratory deficient growth phenotype of an mtg3 null mutant is partially rescued by overexpression of the Mrpl4p constituent located at the peptide exit site of the 54 S subunit. The rescue is accompanied by an increase in processed 15 S rRNA. This suggests that Mtg3p and Mrpl4p jointly regulate assembly of the small subunit by modulating processing of the 15 S rRNA precursor.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.     

A novel Apaf-1-independent putative caspase-2 activation complex

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.     

Signals that dictate nuclear,nucleolar, and cytoplasmic shuttling of the gamma(1)34.5 protein of herpes simplex virus type 1

The gamma(1)34.5 protein of herpes simplex virus type 1 (HSV-1) is required for viral neurovirulence in vivo. In infected cells, this viral protein prevents the shutoff of protein synthesis mediated by double-stranded-RNA-dependent protein kinase PKR. This is accomplished by recruiting protein phosphatase 1 to dephosphorylate the alpha subunit of translation initiation factor eIF-2 (eIF-2 alpha). Moreover, the gamma(1)34.5 protein is implicated in viral egress and interacts with proliferating cell nuclear antigen. In this report, we show that the gamma(1)34.5 protein encoded by HSV-1(F) is distributed in the nucleus, nucleolus, and cytoplasm in transfected or superinfected cells. Deletion analysis revealed that the Arg-rich cluster from amino acids 1 to 16 in the gamma(1)34.5 protein functions as a nucleolar localization signal. The region from amino acids 208 to 236, containing a bipartite basic amino acid cluster, is able to mediate nuclear localization. R(215)A and R(216)A substitutions in the bipartite motif disrupt this activity. Intriguingly, leptomycin B, an inhibitor of nuclear export, blocks the cytoplasmic accumulation of the gamma(1)34.5 protein. L(134)A and L(136)A substitutions in the leucine-rich motif completely excluded the gamma(1)34.5 protein from the cytoplasm. These results suggest that the gamma(1)34.5 protein continuously shuttles between the nucleus, nucleolus, and cytoplasm, which may be a requirement for the different activities of the gamma(1)34.5 protein in virus-infected cells.     

Composition and structure of nucleolar skeleton (nucleolar matrix)

Purified nucleoli of HeLa cells were treated sequentially with nonionic detergent, nucleic acid enzyme, low salt and high salt. The residual nucleolar structure termed nucleolar skeleton (nucleolar matrix) was shown as a fine network under electron microscope with DGD embedding-unembedding technique. Such structures of BHK-21 cell and mouse liver cell are similar to that of HeLa cell. The protein composition of the nucleolar skeleton of HeLa cells was analyzed. The protein composition of such nucleolar residual shows obvious difference from the compositions of nuclear matrix and chromosome scaffold. The major protein composition of the nucleolar skeleton of HeLa cells contains 6–7 polypeptides. Their molecular weights are about 48, 43, 36 and 33 ku. Further studies show that actin and fibrillarin are two major protein components of nucleolar skeleton of HeLa cells.     

Growth compensatory role of sulindac sulfide-induced thrombospondin-1 linked with ERK1/2 and RhoA GTPase signaling pathways

Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.     

Multiple roles of Arf1 GTPase in the yeast exocytic and endocytic pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.     

Synthesis of novel 1-alkyl-8-substituted-3-(3-methoxypropyl) xanthines as putative A2B receptor antagonists

In order to identify a high-affinity, selective antagonist for the A_2B subtype adenosine receptor, more than 40 1,8-disubstituted-3-(3-methoxypropyl) xanthines were prepared and evaluated for their binding affinity at recombinant human adenosine receptors, mainly of the A_2A and A_2B subtypes. Some of the 1-ethyl-3-(3-methoxypropyl)-8-aryl substituted derivatives 15(a–m) showed moderate-to-high affinity at human A_2B receptors, with compound 15d showing A_2B selectivity over the other A receptors assayed (A₁, A_2A, A₃) of 34-fold or over.     

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed.     

The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics

The Cdc42-like GTPase Wnt responsive Cdc42 homolog 1 (Wrch1) has several atypical features; it has an N-terminal proline-rich extension that confers binding to SH3 domains, and it harbors an extremely high intrinsic nucleotide exchange activity, which overrides the normal GTPase activity. As a result, Wrch1 resides mainly in the active, GTP-loaded conformation under normal cellular conditions. We have previously shown that ectopic expression of Wrch1 in fibroblasts resulted in an altered cell morphology visible as a formation of filopodia, a loss of stress fibers, and a reduction in focal adhesions. Here, we show that Wrch1 binds to the nonreceptor tyrosine kinase Pyk2. The interaction required Wrch1 to be in a GTP conformation and also required an intact N-terminal proline-rich extension as well as an intact effector loop. Wrch1 requires Pyk2 in imposing the cytoskeletal effects, seen as the formation of filopodia, since treatment of cells with a Pyk2-specific small interfering RNA abrogated this response. Interestingly, we found that the presence and activity of Src were needed for the formation of a Wrch1-Pyk2 complex as well as for the Wrch1-induced formation of filopodia. We propose a model in which Pyk2 and Src function to coordinate the Wrch1-dependent effects on cytoskeletal dynamics.     

Characterization of the signaling pathways regulating alpha2beta1 integrin-mediated events by a pharmacological approach

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchymal-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on alpha2beta1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.     

Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2Cepsilon)

Although TAK1 signaling plays essential roles in eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory cytokine, how the IL-1-TAK1 signaling pathway is positively and negatively regulated remains poorly understood. In this study, we investigated the possible role of a novel protein phosphatase 2C (PP2C) family member, PP2Cepsilon, in the regulation of the IL-1-TAK1 signaling pathway. PP2Cepsilon was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cepsilon and PP2Calpha was found to be 26%. Ectopic expression of PP2Cepsilon inhibited the IL-1- and TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway. PP2Cepsilon dephosphorylated TAK1 in vitro. Co-immunoprecipitation experiments indicated that PP2Cepsilon associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6. Ectopic expression of a phosphatase-negative mutant of PP2Cepsilon, PP2Cepsilon(D/A), which acted as a dominant negative form, enhanced both the association between TAK1 and MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene. The association between PP2Cepsilon and TAK1 was transiently suppressed by IL-1 treatment of the cells. Taken together, these results suggest that, in the absence of IL-1-induced signal, PP2Cepsilon contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1.     

Thyroid hormones protect astrocytes from morphine-induced apoptosis by regulating nitric oxide and pERK 1/2 pathways

Unlike neurons and various other non-neuronal cells, astrocytes have been reported to be resistant to morphine induced cytotoxicity. The present work demonstrates that primary cultures of astrocytes are also sensitive to morphine toxicity depending upon the thyroidal status of the culture medium. Chronic morphine treatment of astrocytes, cultured under thyroid hormone (TH)-deficient conditions, induced apoptotic cell death which was characterized by nuclear condensation, DNA fragmentation and activation of caspase-3 like enzymes. Cell death was accompanied with increase in nNOS level, nitration of cellular proteins and down regulation of pAKT level. Phosphorylation of ERK1/2 showed a biphasic response, an initial induction followed by sustained decline during chronic morphine treatment and the initial induction of pERK1/2 level appeared to be critical for apoptosis in the cells. Interestingly, supplementation with normal levels of TH to cells attenuated morphine-induced apoptosis as well as the biphasic response of pERK1/2 in the astrocytes. However, in the presence of glutathione synthetase inhibitor L-buthionine-S,R-sulfoximine, TH failed to protect astrocytes. Overall, the study demonstrates a possible signaling mechanism of morphine induced toxicity to cells and suggests that alteration of glutathione homeostasis by TH protect astrocytes from morphine by regulating NO and pERK1/2 pathways in the cells.     

Fcf1p and Fcf2p are novel nucleolar Saccharomyces cerevisiae proteins involved in pre-rRNA processing

The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.     

Protein phosphatase with EF-hand domains 2 (PPEF2) is a potent negative regulator of apoptosis signal regulating kinase-1 (ASK1)

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H₂O₂, expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H₂O₂-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions.     

Nucleolar GTP-binding Protein-1 (NGP-1) Promotes G1 to S Phase Transition by Activating Cyclin-dependent Kinase Inhibitor p21Cip1/Waf1

Nucleolar GTP-binding protein (NGP-1) is overexpressed in various cancers and proliferating cells, but the functional significance remains unknown. In this study, we show that NGP-1 promotes G₁ to S phase transition of cells by enhancing CDK inhibitor p21^Cip-1/Waf1 expression through p53. In addition, our results suggest that activation of the cyclin D1-CDK4 complex by NGP-1 via maintaining the stoichiometry between cyclin D1-CDK4 complex and p21 resulted in hyperphosphorylation of retinoblastoma protein at serine 780 (p-RB^Ser-780) followed by the up-regulation of E2F1 target genes required to promote G₁ to S phase transition. Furthermore, our data suggest that ribosomal protein RPL23A interacts with NGP-1 and abolishes NGP-1-induced p53 activity by enhancing Mdm2-mediated p53 polyubiquitination. Finally, reduction of p-RB^Ser-780 levels and E2F1 target gene expression upon ectopic expression of RPL23a resulted in arrest at the G₁ phase of the cell cycle. Collectively, this investigation provides evidence that NGP-1 promotes cell cycle progression through the activation of the p53/p21^Cip-1/Waf1 pathway.