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BBA - Molecular Cell Research (v.1803, #8)

Editorial Board (pp. i).

Be different—The diversity of peroxisomes in the animal kingdom by M. Islinger; M.J.R. Cardoso; M. Schrader (pp. 881-897).

Peroxisomes represent so-called “multipurpose organelles” as they contribute to various anabolic as well as catabolic pathways. Thus, with respect to the physiological specialization of an individual organ or animal species, peroxisomes exhibit a functional diversity, which is documented by significant variations in their proteome. These differences are usually regarded as an adaptational response to the nutritional and environmental life conditions of a specific organism. Thus, human peroxisomes can be regarded as an in part physiologically unique organellar entity fulfilling metabolic functions that differ from our animal model systems. In line with this, a profound understanding on how peroxisomes acquired functional heterogeneity in terms of an evolutionary and mechanistic background is required. This review summarizes our current knowledge on the heterogeneity of peroxisomal physiology, providing insights into the genetic and cell biological mechanisms, which lead to the differential localization or expression of peroxisomal proteins and further gives an overview on peroxisomal biochemical pathways, which are specialized in different animal species and organs. Moreover, it addresses the impact of proteome studies on our understanding of differential peroxisome function describing the utility of mass spectrometry and computer-assisted algorithms to identify peroxisomal target sequences for the detection of new organ- or species-specific peroxisomal proteins.

Keywords: Abbreviations; ABCD; peroxisomal ABC transporter; ACOX; acyl-CoA oxidase; AECI/II; alveolar type I/II cells; BAAT; bile acyl-CoA:amino acid; N; -acetyltransferase; CNS; central nervous system; d/l; -PBE; peroxisomal; d/l; -bifunctional enzyme; DHAPAT; dihydroxyacetonephosphate acyltransferase; DHCA; dihydroxycholestanoic acid; MFP; peroxisomal multifunctional protein; PMP; peroxisomal membrane protein; PPAR; peroxisome proliferator-activating receptor; PTS; peroxisomal targeting signal; ROS; reactive oxygen species; SCP; sterol carrier protein; THCA; trihydroxycholestanoic acid; VLCFA; very long chain fatty acid; X-ALD; X-linked adrenoleukodystrophyPeroxisomes; Molecular evolution; Differential protein expression; Metabolism; Tissue variation; Species differences

FADD–calmodulin interaction: A novel player in cell cycle regulation by Giuliana Papoff; Nadia Trivieri; Roberta Crielesi; Francesca Ruberti; Sonia Marsilio; Giovina Ruberti (pp. 898-911).

Analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8–9 and 10–11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIα), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIα substrates and that in synchronized HeLa cells, FADD, CaM and CKIα co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD−/− T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIα may have an important role in the regulation of cell fate.

Keywords: Abbreviations; GST; glutathione S-transferase; mFADD; murine FADD; dFADD; Drosophila; FADD; CaM; calmodulin; DED; death effector domain; DD; death domain; DISC; death inducing signaling complex; CKIα; casein kinase I alpha; TNFRI; tumor necrosis factor receptor IFADD; Calmodulin; Casein kinase Iα; Phosphorylation; Mitosis; Apoptosis

Study of the in vivo phosphorylation of E2F1 on Ser403 by Sebastian Real; Lilia Espada; Carme Espinet; Santidrian Antonio F. Santidrián; Albert Tauler (pp. 912-918).

Multiple E2F1 phosphorylation sites have been described as targets of different kinases, yet their in vivo implication is uncertain. We previously reported that GSK3β is able to phosphorylate E2F1 in vitro at Ser403 and Ser433. Recently, it has been shown that both residues are also direct targets of p38 MAP kinase. In order to determine whether Ser403 phosphorylation occurs in vivo and to elucidate its role in E2F1 transcription activity, we developed a phospho-E2F1(Ser403) antibody for use in in vivo detection studies. Our results demonstrate that endogenous E2F1 is phosphorylated in vivo on Ser403, however neither GSK3β nor p38 MAP kinase are responsible for this event. E2F1 phosphorylation on Ser403 is induced after treatment with doxorubicin in a dose response manner. The transcriptional response of E2F1 to doxorubicin is lower in an E2F1 Ser/Ala403 mutated construct relative to the wild type, suggesting a role for Ser403 phosphorylation in DNA damage conditions. Comparative study between the expression of the bcl2 gene family induced by the wild type and E2F1 Ser/Ala403 mutant revealed a statistically different pattern between both conditions. These results suggest that phosphorylation of Ser403 could influence the selection and regulation of E2F1 target genes.

Keywords: E2F1; Doxorubicin

Advanced glycation end products inhibit Na⁺ K⁺ ATPase in proximal tubule epithelial cells: Role of cytosolic phospholipase A₂α and phosphatidylinositol 4-phosphate 5-kinase γ by Marisa A. Gallicchio; Leon A. Bach (pp. 919-930).

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na⁺ K⁺ ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40μM) induced³H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A₂(cPLA₂) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [⁸⁶Rb⁺] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA₂α inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the α-adaptin but not β2- or μ2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kγ siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kγ to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA₂α activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.

Keywords: Abbreviations; BSA; bovine serum albumin; LLC-PK1; porcine kidney proximal tubule epithelial cells; HK2; human kidney proximal tubule epithelial cells; SFM; serum free medium; GM; growth medium; NKA; sodium potassium ATPase; PIP2; phosphatidylinositol 4,5 bisphosphate; ROS; reactive oxygen species; cPLA; ₂; α; cytosolic phospholipase A; ₂; alpha; AGEs; advanced glycation end products; PIP5Kγ; phosphatidylinositol 4-phosphate 5-kinase γ; AP-2; adaptor protein complex 2Diabetes; Glycation; Nephropathy; Cytosolic phospholipase A; ₂; α; Na; ⁺; K; ⁺; ATPase; Phosphatidylinositol 4-phosphate 5-kinase γ, endocytosis

Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation by Heini Ruhanen; Sarah Borrie; Gyorgy Szabadkai; Henna Tyynismaa; Aleck W.E. Jones; Dongchon Kang; Jan-Willem Taanman; Takehiro Yasukawa (pp. 931-939).

Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.

Keywords: Single-stranded DNA binding protein; mtSSB; Mitochondrial DNA; 7S DNA; DNA replication; Mitochondrial nucleoid

Ligation of β₄ integrins activates PKB/Akt and ERK1/2 by distinct pathways—relevance of the keratin filament by Stefan Kippenberger; Matthias Hofmann; Zoller Nadja Zöller; Thaci Diamant Thaçi; Muller Jutta Müller; Roland Kaufmann; August Bernd (pp. 940-950).

In normal epithelial cells hemidesmosomes mediate stable adhesion to the underlying basement membrane. In carcinoma cells a functional and spatial dissociation of the hemidesmosomal complex is observed stimulating the hypothesis that the β₄ integrin may trigger essential signalling cascades determining cell fate. In the present study we dissected the signalling pathways giving rise to PKB/Akt and ERK1/2 activation in response to β₄ ligation by 3E1. It was found that the activation of PKB/Akt is sensitive towards alterations of the keratin filament as demonstrated by using KEB-7 cells that carry a keratin mutation typical for epidermolysis bullosa simplex. Similar results were achieved by chemically induced keratin aggregations. Of note, the signalling to ERK1/2 was not affected. ERK1/2 activation utilizes an EGF-R transactivation mechanism as shown by dominant-negative expression experiments and also by treatment with a specific inhibitor (AG1478). Downstream from the EGF-R the activation of ERK1/2 takes the prototypical signalling cascade via Shc, Ras and Raf-1 as demonstrated by dominant-negative expression experiments. Taken together our data define a new model of β₄-dependent PKB/Akt and ERK1/2 activation demonstrating the keratin filament as a structure necessary in signal transmission.

Keywords: Hemidesmosome; Integrin; Signalling; Keratin; Epidermolysis bullosa; Carcinogenesis

AKT loss in human epithelial cells treated with severe hypoxia by Adrian Harold Box; Sun-Myoung Kim; Douglas James Demetrick (pp. 951-959).

Cancer cells which can survive and or proliferate in hypoxia may be resistant to anti-cancer treatment. In our previous work, we showed that we could group cell lines treated with severe hypoxia into either hypoxia-induced cell cycle arrest-sensitive or resistant phenotypes, and hypoxia-induced cell death (HCD)-sensitive or resistant phenotypes. We showed that the resistant phenotypes were associated with high levels of active-AKT in late hypoxia and sensitive cells were associated with decreased or undetectable levels of AKT in late hypoxia. We have now extended our findings to numerous other cell lines. We show that HCD and loss of AKT is cell density dependent, and both AKT1 and AKT2 isoforms are lost in late hypoxia. Loss of AKT is most likely due to regulated degradation, as transcription of AKT isoforms is unchanged in hypoxia, and AKT is not significantly translocated to the nucleus to account for its disappearance from cytoplasmic lysates. Interestingly, inhibitors of proteosome, calpain or caspase-mediated proteolysis did not significantly block AKT loss. Inhibition of autophagy using diverse lysosome-targeted autophagy inhibitors also did not block AKT loss, however autophagy inhibitors which block general PI3K activity, such as 3-methyladenine or LY294002, were effective inhibitors of AKT loss in late hypoxia. Interestingly, those inhibitors also blocked HCD in an HCD-sensitive cancer cell line. Inhibitors of proteolytic pathways which did not block AKT loss also did not block HCD in HeLa. Our investigations support a model by which AKT is a major switch involved in regulating hypoxia-induced cell death.

Keywords: AKT; Hypoxia; Proteolysis; Autophagy; Inhibitor

Calcineurin signaling and PGC-1α expression are suppressed during muscle atrophy due to diabetes by Tiffany K. Roberts-Wilson; Ramesh N. Reddy; James L. Bailey; Bin Zheng; Ronald Ordas; Jennifer L. Gooch; S. Russ Price (pp. 960-967).

PGC-1α is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1α expression and oxidative capacity are decreased in skeletal muscle of patients and animals undergoing atrophy, suggesting that PGC-1α participates in the regulation of muscle mass. PGC-1α gene expression is controlled by calcium- and cAMP-sensitive pathways. However, the mechanism regulating PGC-1α in skeletal muscle during atrophy remains unclear. Therefore, we examined the mechanism responsible for decreased PGC-1α expression using a rodent streptozotocin (STZ) model of chronic diabetes and atrophy. After 21days, the levels of PGC-1α protein and mRNA were decreased. We examined the activation state of CREB, a potent activator of PGC-1α transcription, and found that phospho-CREB was paradoxically high in muscle of STZ-rats, suggesting that the cAMP pathway was not involved in PGC-1α regulation. In contrast, expression of calcineurin (Cn), a calcium-dependent phosphatase, was suppressed in the same muscles. PGC-1α expression is regulated by two Cn substrates, MEF2 and NFATc. Therefore, we examined MEF2 and NFATc activity in muscles from STZ-rats. Target genes MRF4 and MCIP1.4 mRNAs were both significantly reduced, consistent with reduced Cn signaling. Moreover, levels of MRF4, MCIP1.4, and PGC-1α were also decreased in muscles of CnAα^−/− and CnAβ^−/− mice without diabetes indicating that decreased Cn signaling, rather than changes in other calcium- or cAMP-sensitive pathways, were responsible for decreased PGC-1α expression. These findings demonstrate that Cn activity is a major determinant of PGC-1α expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass.

Keywords: Skeletal muscle; Atrophy; Calcineurin; PGC-1α; Diabetes; Streptozotocin

The nuclear import of the constitutive androstane receptor by importin/Ran-GTP systems by Yuichiro Kanno; Yukari Miyazaki; Yoshio Inouye (pp. 968-974).

The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. The CAR is normally located in the cytoplasmic compartment of untreated liver cells and translocates to the nucleus after exposure to phenobarbital (PB) or PB-like chemicals. Previously, we identified two nuclear localization signals (NLS) in the rat constitutive androstane/active receptor (CAR), NLS1, which is located in the hinge region, and NLS2, which overlaps with the ligand-binding domain. However, the nuclear import mechanism of CAR is unclear. In this study, we show that nuclear import of CAR is regulated by importin/Ran-GTP systems. The regulation of CAR nuclear import by a Ran-GTP concentration gradient was confirmed using the dominant negative, GTPase-deficient form of Ran (RanQ69L), suggesting the involvement of transport receptors of the importinβ family. IPO13 was shown to be involved in the PB-mediated nuclear translocation of CAR, which was found to be susceptible to inhibition by a dominant negative mutant of IPO13 in primary hepatocytes.

Keywords: Abbreviations; CAR; constitutive androstane/active receptor; XRS; xenochemical response signal; PB; phenobarbital; NPC; nuclear pore complexes; IPO13; importin 13; NLS; nuclear localization signal; NES; nuclear export signal; GR; glucocorticoid receptor; AR; androgen receptor; LBD; ligand-binding domainConstitutive androstane receptor; Nuclear receptor; Importin 13

TM4SF5 accelerates G1/S phase progression via cytosolic p27^Kip1 expression and RhoA activity by Hyeonjung Kim; Minkyung Kang; Sin-Ae Lee; Tae Kyoung Kwak; Oisun Jung; Hyo Jeong Lee; Sung-Hoon Kim; Jung Weon Lee (pp. 975-982).

Transmembrane 4 L six family member 5 (TM4SF5) causes epithelial–mesenchymal transition (EMT) for aberrant cell proliferation. However, the effects of TM4SF5 expression on cell cycle are unknown so far. In this study, using hepatocytes that either ectopically or endogenously express TM4SF5 and human hepatocarcinoma tissues, the role of TM4SF5 in G1/S phase progression was examined. We found that TM4SF5 expression accelerated G1/S phase progression with facilitated cyclin D1 and E expression and Rb phosphorylation. Furthermore, TM4SF5 enhanced trafficking of CDK4 and cyclin D1 into the nucleus and induced complex formation between them. However, TM4SF5-facilitated G1/S phase progression was blocked by silencing of p27^Kip1 using siRNA or by infection of active RhoA. Pharmacological inhibition of ROCK accelerated the G1/S phase progression of control TM4SF5-unexpressing cells. Altogether, these observations suggest that TM4SF5 accelerates G1/S phase progression with facilitated CDK4/cyclin D1 entry into the nucleus, which might be supported by TM4SF5-mediated actin reorganization through cytosolic p27^Kip1 expression and Rho GTPase activity.

Keywords: Cytosolic p27; ^Kip1; Cell cycle; Cancer; RhoA; Tetraspan

Differential involvement of caveolin-1 in brown adipocyte signaling: Impaired β₃-adrenergic, but unaffected LPA, PDGF and EGF receptor signaling by Charlotte L. Mattsson; Emma R. Andersson; Jan Nedergaard (pp. 983-989).

Caveolae and caveolin have been implicated as being involved in the signal transduction of many receptors, including the EGF, PDGF, LPA and β₃-adrenergic receptors. To investigate the role of caveolin-1 (Cav1) in these signaling pathways in brown adipose tissue, primary brown adipocyte cultures from Cav1-ablated mice and wild-type mice were investigated. In pre-adipocytes, Cav1-ablation affected neither the G-protein coupled LPA receptor signaling to Erk1/2, nor the receptor tyrosine kinases PDGF- or EGF-receptor signaling to Erk1/2. Mature primary Cav1(−/−) brown adipocytes accumulated lipids and expressed aP2 to the same extent as did wild-type cells. However, the cAMP levels induced by the β₃-adrenergic receptor agonist CL316,243 were lower in the Cav1(−/−) cultures, with an unchanged EC₅₀ for CL316,243. Also the response to the direct adenylyl cyclase agonist forskolin was reduced. Thus, in brown adipocytes, Cav1 is apparently required for an intact response to adenylyl cyclase-linked agonists/activators, whereas other signaling pathways examined function without Cav1.

Keywords: Caveolin-1; Brown adipocyte; β; ₃; -adrenergic receptor; LPA; EGF; PDGF

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BBA - Molecular Cell Research (v.1803, #8)