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BBA - Molecular Cell Research (v.1813, #8)

Title Page/Section Editors (pp. i).

C/EBPα and C/EBPβ binding proteins modulate hepatocyte apoptosis through iNOS signaling pathway by Kewei Wang; John J. Brems; Richard L. Gamelli; Ai-Xuan Holterman (pp. 1395-1403).

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) involve many pathophysiologic conditions. The expression of iNOS is regulated at multiple stages. Presently, the regulatory details of iNOS signaling are still unclear. This study aimed to investigate the regulatory role of C/EBPα and C/EBPβ in iNOS signaling pathway. By employing the techniques such as EMSA, ChIP assay, site-directed mutagenesis, and siRNA silencing, the relationship between iNOS and C/EBPα/C/EBPβ in rat hepatocytes was clarified. iNOS promoter was the direct transcriptional targets of the C/EBPα, C/EBPβ, and NF-κB binding proteins. There was the interactive influence between NF-κB and C/EBPα/C/EBPβ. The expression of iNOS was modulated by C/EBPα/C/EBPβ transcription factors. Moreover, the iNOS expression mediated glycochenodeoxycholate (GCDC)-induced apoptosis in hepatocytes. C/EBPα/C/EBPβ binding proteins could affect the GCDC-induced apoptosis through iNOS cascade. These findings indicate that C/EBPα and C/EBPβ regulate the iNOS expression, which may further modify cell responses such as apoptosis and cell survival.► iNOS promoter was the direct transcriptional targets of the C/EBPα, C/EBPβ, and NF-κB. ► There was the interaction between NF-κB and C/EBPα/C/EBPβ. ► The expression of iNOS was modulated by C/EBPα/C/EBPβ transcription factors. ► iNOS involve GCDC induced apoptosis in hepatocytes. ► C/EBPα/C/EBPβ could regulate the GCDC-induced apoptosis through iNOS signaling cascade.

Keywords: Apoptosis; Hepatocyte; iNOS; C/EBPα; C/EBPβ; Signal transduction

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells by João Agostinho Machado-Neto; Patricia Favaro; Mariana Lazarini; Fernando Ferreira Costa; Sara T. Olalla Saad; Fabiola Traina (pp. 1404-1411).

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.► The effect of IRS1 silencing, by shRNA-lentiviral, in K562 cells was evaluated. ► IRS1 silencing decreased cell proliferation and colony formation. ► Inhibition of IRS1 arrested cells in the G0/G1 phase of cell cycle in K562 cells. ► IRS1 silencing resulted down modulation of Akt, P70S6K and ERK1/2 activity. ► IRS1 silencing downregulates Akt/mTOR and MAPK signaling in BCR-ABL cells.

Keywords: IRS1; Akt/mTOR; MAPK; K562 cell; BCR-ABL; Proliferation

Apoptosis signal-regulating kinase 1 is an intracellular inducer of p38 MAPK-mediated myogenic signalling in cardiac myoblasts by Tae Gyu Choi; Jisun Lee; Joohun Ha; Sung Soo Kim (pp. 1412-1421).

Myogenic differentiation is an essential process for the myogenesis in response to various extracellular stimuli. p38 MAPK is a core signalling molecule in myogenic differentiation. The activation of p38 MAPK is required for myogenic differentiation; however, the mechanism for this activation remains undefined. ASK1 is a member of the MAP3K family that activates both JNK and p38 MAPK pathways in response to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. Here, we reported that TNFα was significantly released from H9c2 cardiac myoblast in differentiation medium. Furthermore, the oxidant H₂O₂ acted as a messenger in the TNFα signalling pathway to disrupt the complex of ASK1–Trx, which was followed by the activation of ASK1 in cardiac myogenic differentiation. Subsequently, the activated ASK1 stimulated MKK3/6–p38MAPK signalling cascade to induce specific myogenic differentiation. In addition, exogenous TNFα added to the medium at physiological levels enhanced the ASK1–p38 MAPK signalling pathway through the increased generation of H₂O₂. Interestingly, inhibition of p38 MAPK abrogated the production of H₂O₂, suggesting that there might be a positive feedback loop in the myogenic–redox signalling pathway. These results indicate that ASK1 is a new intracellular regulator of activation of the p38 MAPK in cardiac myogenic differentiation.► Activity of ASK1–p38 MAPK cascade is upregulated in cardiac muscle differentiation. ► TNFα is spontaneously released from differentiating myoblast. ► Exogenous TNFα enhances cardiac myogenic differentiation in physiological level. ► TNFα-induced H₂O₂ disrupts ASK1/Trx complex to process myogenic differentiation. ► ASK1 promotes p38 MAPK-mediated myogenic differentiation in H₂O₂-dependent.

Keywords: TNFα; H; ₂; O; ₂; p38 MAPK; ASK1; Trx; Myogenic differentiation

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells by Ruiguang Ge; Xuesong Sun; Dongxian Wang; Qinglu Zhou; Hongzhe Sun (pp. 1422-1427).

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.► Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro. ► Hpn fibrils inhibit the proliferation of gastric epithelial AGS cells. ► Hpn fibrils induce cell arrest in the G2/M phase. ► Hpn fibrils disrupt mitochondrial bioenergetics.

Keywords: Abbreviations; A; β; β; -amyloid; AD; Alzheimer's disease; ΔΨ; _m; mitochondrial transmembrane potential; FBS; fetal bovine serum; H. pylori; Helicobacter pylori; HRC; histidine-rich Ca; ²⁺; binding protein; IAPP; islet amyloid polypeptide; JC-1; 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazolylcarbocyanine iodide; LUV; large unilamellar vesicle; MALT; mucosa-associated lymphoid tissue; PBS; phosphate-buffered saline; PI; propidium iodide; polyQ; poly(glutamine); PrP; prion protein; ThT; thioflavin T; TOM; translocase of the outer membrane Helicobacter pylori; Amyloid; Hpn; Gastric epithelial cell; Mitochondria

A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors by Alexander Annenkov; Anne Rigby; Sandra Amor; Dun Zhou; Nasim Yousaf; Bernhard Hemmer; Yuti Chernajovsky (pp. 1428-1437).

In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional.► The aim is to produce neural progenitor cells capable of insulin-like-growth-factor (IGF)-independent growth. ► This study characterises a chimeric receptor (ChR) with the IGF receptor type 1 signalling domain. ► The ectodomain of the ChR is a single chain antibody to myelin oligodendrocyte glycoprotein (MOG), a brain-specific ligand. ► ChR activation is shown by increased phosphotyrosine content and dimerization in response to MOG. ► CG4 oligodendrocyte progenitors transduced with the ChR and expressing endogenous MOG grow independently of insulin.

Keywords: Receptor tyrosine kinase; Insulin-like growth factor receptor type I; Regenerative medicine; Gene therapy; Oligodendrocyte progenitor; Chimeric receptor

Arginine vasopressin controls p27^Kip1 protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells by Fabio L. Forti; Hugo A. Armelin (pp. 1438-1445).

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27^Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27^Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27^Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27^Kip1 protein as well as phosphorylation of the p27^Kip1 protein at both Ser10 and Thr187.► Inhibition of mouse Y1 malignant cells proliferation by AVP depends on PKC activation and high levels of K-Ras-GTP. ► AVP activates ERK pathway independent on PKC activity but inhibits PI3K/Akt via activation in a PKC-dependent manner. ► AVP stabilises, increases and/or maintains expression of the p27^Kip1 protein antagonizing the mitogenic activity of FGF2. ► Phosphorylation of p27^Kip1 protein at Ser10 and Thr187 residues is modulated by AVP±FGF2. ► PMA mimics most AVP effects through PKC, and dependently on K-Ras malignant phenotype of Y1 cells.

Keywords: Arginine vasopressin; p27; ^Kip1; Mouse Y1 adrenocortical cells; K-Ras; Protein kinase C

Extracellular ATP induces spikes in cytosolic free Ca²⁺ but not in NADPH oxidase activity in neutrophils by Jens Christian Brasen; Lars Folke Olsen; Maurice B. Hallett (pp. 1446-1452).

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca²⁺ concentration, we simultaneously measured Ca²⁺ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca²⁺ spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca²⁺ occurred in the absence of Ca²⁺ influx, but the amplitude was damped by inhibition of Ca²⁺ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca²⁺ but, despite subsequent spikes in Ca²⁺ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca²⁺ in a very reproducible way and we propose that the Ca²⁺ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca²⁺.► In this study we have measured Ca²⁺ and NADPH oxidase activity in neutrophils. ► ATP induces spikes in cytosolic Ca²⁺ concentration. ► NADPH oxidase does not show rapid spikes. ► Ca²⁺ is an on-switch for NADPH oxidase.

Keywords: Abbreviations; [Ca; ²⁺; ]; _cyt; cytosolic Ca; ²⁺; concentration; DAG; diacyl glycerol; fMLP; N; -Formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; HE; hydroethidine; HRP; horseradish peroxidase; PKC; protein kinase C; PB; peripheral blood; PMA; phorbol-12-myristate-13-acetateATP; Calcium; Ca; ²⁺; Spikes; Neutrophils; NADPH oxidase

Bimodal regulation of FoxO3 by AKT and 14-3-3 by Melissa Dobson; Gopalakrishnan Ramakrishnan; Stephanie Ma; Ludmila Kaplun; Vitaly Balan; Rafael Fridman; Guri Tzivion (pp. 1453-1464).

FoxO3 is a member of FoxO family transcription factors that mediate cellular functions downstream of AKT. FoxO3 phosphorylation by AKT generates binding sites for 14-3-3, which in-turn regulates FoxO3 transcriptional activity and localization. We examine here the functional significance of AKT–FoxO3 interaction and further detail the mechanistic aspects of FoxO3 regulation by AKT and 14-3-3. Our data show that AKT overexpression increases the steady-state levels of FoxO3 protein in a manner dependent on AKT activity and its ability to bind FoxO3. Characterization of the AKT–FoxO3 interaction shows that the three AKT phosphorylation-site-recognition motifs (RxRxxS/T) present on FoxO3, which are required for FoxO3 phosphorylation, are dispensable for AKT binding, suggesting that AKT has a docking point on FoxO3 distinct from the phosphorylation-recognition motifs. Development of a FoxO3 mutant deficient in 14-3-3 binding (P34A), which can be phosphorylated by AKT, established that 14-3-3 binding and not AKT phosphorylation per se controls FoxO3 transcriptional activity. Intriguingly, 14-3-3 binding was found to stabilize FoxO3 by inhibiting its dephosphorylation and degradation rates. Collectively, our data support a model where both AKT and 14-3-3 positively regulate FoxO3 in addition to their established negative roles and that 14-3-3 availability could dictate the fate of phosphorylated FoxO3 toward degradation or recycling.► AKT binding to FoxO3 is independent of the RxRxxS/T AKT phosphorylation recognition motif. ► AKT activation is required for FoxO3 binding. ► AKT expression increases FoxO3 steady-state protein levels. ► 14‐3‐3 binding stabilizes phosphorylated FoxO3. ► FoxO3 P34A mutation impairs 14‐3‐3 binding, resulting in a constitutively active form.

Keywords: AKT; 14-3-3; FoxO; Forkhead domain; Transcription factors; Protein phosphorylation

Baicalein, a component of Scutellaria baicalensis, induces apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells by Hiroki Takahashi; Monica C. Chen; Hung Pham; Eliane Angst; Jonathan C. King; Jenny Park; Ethan Y. Brovman; Hideyuki Ishiguro; Diane M. Harris; Howard A. Reber; Oscar J. Hines; Anna S. Gukovskaya; Vay Liang W. Go; Guido Eibl (pp. 1465-1474).

Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in several cancers, including pancreatic cancer (PaCa). However, the precise molecular mechanisms have not been fully defined. SB extract and SB-derived polyphenols (wogonin, baicalin, and baicalein) were used to determine their anti-proliferative mechanisms. Baicalein significantly inhibited the proliferation of PaCa cell lines in a dose-dependent manner, whereas wogonin and baicalin exhibited a much less robust effect. Treatment with baicalein induced apoptosis with release of cytochrome c from mitochondria, and activation of caspase-3 and -7 and PARP. The general caspase inhibitor zVAD-fmk reversed baicalein-induced apoptosis, indicating a caspase-dependent mechanism. Baicalein decreased expression of Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, presumably through a transcriptional mechanism. Genetic knockdown of Mcl-1 resulted in marked induction of apoptosis. The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression, suggesting a critical role of Mcl-1 in this process. Our results provide evidence that baicalein induces apoptosis in pancreatic cancer cells through down-regulation of the anti-apoptotic Mcl-1 protein.► Scutellaria baicalensis (SB) and SB-derived polyphenols possess anti-proliferative activities in pancreatic cancer. ► Baicalein decreased mRNA and protein expression of the anti-apoptotic Bcl-2 family protein Mcl-1 and induced apoptosis. ► Mcl-1 knock-down induced apoptosis through caspase cascade, but Bcl-2 or Bcl-xL knock-down had no or only a slight effect. ► The effect of baicalein on apoptosis was significantly attenuated by Mcl-1 over-expression.

Keywords: Mcl-1; Baicalein; Apoptosis; Pancreatic cancer; Bcl-2 family protein

Regulation of heparanase by albumin and advanced glycation end products in proximal tubular cells by Valentina Masola; Giovanni Gambaro; Elena Tibaldi; Maurizio Onisto; Cataldo Abaterusso; Antonio Lupo (pp. 1475-1482).

Diabetic nephropathy is one of the main causes of end-stage renal disease, in which the development of tubular damage depends on factors such as high glucose levels, albuminuria and advanced glycation end-product. In this study, we analyzed the involvement of heparanase, a heparan sulfate glycosidase, in the homeostasis of proximal tubular epithelial cells in the diabetic milieu. In vitro studies were performed on a wild-type and stably heparanase-silenced adult tubular line (HK2) and HEK293. Gene and protein expression analyses were performed in the presence and absence of diabetic mediators. Albumin and advanced glycation end-product, but not high glucose levels, increased heparanase expression in adult tubular cells via the AKT/PI3K signaling pathway. This over-expression of heparanase is then responsible for heparan sulfate reduction via its endoglycosidase activity and its capacity to regulate the heparan sulfate-proteoglycans core protein. In fact, heparanase regulates the gene expression of syndecan-1, the most abundant heparan sulfate-proteoglycans in tubular cells. We showed that heparanase is a target gene of the diabetic nephropathy mediators albumin and advanced glycation end-product, so it may be relevant to the progression of diabetic nephropathy. It could take part in several processes, e.g. extracellular-matrix remodeling and cell–cell crosstalk, via its heparan sulfate endoglycosidase activity and capacity to regulate the expression of the heparan sulfate-proteoglycan syndecan-1.► Albumin and AGE, but not high glucose levels, increase heparanase expression in tubular cells. ► Heparanase up-regulation is mediated by PI3K/AKT pathway. ► Heparanase reduces heparan sulfate via its endoglycosidase activity. ► Heparanase reduces heparan sulfate throw the gene expression regulation of syndecan-1.

Keywords: Heparanase; Albuminuria; AGE; PI3K/AKT; Syndecan-1; Diabetic nephropathy

Acidic NAADP-releasable Ca²⁺ compartments in the megakaryoblastic cell line MEG01 by Natalia Dionisio; Albarran Letizia Albarrán; Lopez José J. López; Alejandro Berna-Erro; Ginés M. Salido; Régis Bobe; Juan A. Rosado (pp. 1483-1494).

A novel family of intracellular Ca²⁺-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca²⁺ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca²⁺ release from the acidic compartments.The present study is aimed to investigate NAADP-mediated Ca²⁺ release from intracellular stores in the megakaryoblastic cell line MEG01.Changes in cytosolic and intraluminal free Ca²⁺ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR.Treatment of MEG01 cells with the H⁺/K⁺ ionophore nigericin or the V-type H⁺-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca²⁺ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca²⁺ from acidic lysosomal-like Ca²⁺ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca²⁺ efflux from the acidic lysosomal-like Ca²⁺ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca²⁺-induced Ca²⁺ release (CICR) from the complementary Ca²⁺ compartment.Our results show for the first time NAADP-evoked Ca²⁺ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.► Two separate Ca²⁺ compartments in the megakaryoblastic cell line MEG01: the ER and an acidic lysosomal-like Ca²⁺ compartment. ► There is NAADP evoked Ca²⁺ release from acidic compartments and CICR in MEG01 cells. ► NAADP-induced Ca²⁺ release occurs through the activation of TPC1 and TPC2 in the megakaryoblastic cell line.

Keywords: MEG01; NAADP; Two-pore channels; Acidic Ca; ²⁺; stores; TG; Bafilomycin A1

Intracellular α_2C-Adrenoceptors: Storage depot, stunted development or signaling domain? by Maqsood A. Chotani; Nicholas A. Flavahan (pp. 1495-1503).

G-protein coupled receptors (GPCRs) are generally considered to function as cell surface signaling structures that respond to extracellular mediators, many of which do not readily access the cell's interior. Indeed, most GPCRs are preferentially targeted to the plasma membrane. However, some receptors, including α_2C-Adrenoceptors, challenge conventional concepts of GPCR activity by being preferentially retained and localized within intracellular organelles. This review will address the issues associated with this unusual GPCR localization and discuss whether it represents a novel sub-cellular niche for GPCR signaling, whether these receptors are being stored for rapid deployment to the cell surface, or whether they represent immature or incomplete receptor systems.► We review the dilemma of GPCRs with a predominant intracellular localization. ► The major focus is on α2C-Adrenoceptors. ► GPCRs can be preferentially retained during biosynthesis in the ER and Golgi. ► Retention can reflect incomplete processing or targeted protein interactions. ► Appropriate stimuli can mobilize GPCRs to the surface or activate them in situ.

Keywords: Signal transduction; Endoplasmic Reticulum; Golgi; Cold

Release of podocalyxin into the extracellular space by Fernandez Darío Fernández; Susana Larrucea; Adam Nowakowski; Miguel Pericacho; Roberto Parrilla; Matilde S. Ayuso (pp. 1504-1510).

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.► Podocalyxin was detected in the Golgi, plasma membrane and as cytoplasmic vesicles. ► Podocalyxin-containing vesicles seems to move along the filopodia to finally be excreted. ► Podocalyxin was detected in the insoluble and soluble fractions of extracellular medium. ► Insoluble extracellular Podocalyxin was released of as a cargo of microvesicles. ► Soluble extracellular podocalyxin was enhanced by stimulation of metalloproteinases.

Keywords: Human podocalyxin; Fate of podocalyxin-GFP; Extracellular podocalyxin; CHO cells; Tera-1 cells

Long and short distance movements of β₂-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2–β₂-adrenoceptor fusion by Ali İ. Kaya; Ugur Özlem Uğur; Altuntas Olga Altuntaş; Kemal Sayar; H. Ongun Onaran (pp. 1511-1524).

Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (>5μm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human β2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~4μm), ~90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~0.1μm²/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4–40μm), although a slowly diffusing fraction of receptors (with D<0.01μm²/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state.► Mobility of β2-adrenoceptors follows a hindered diffusion in the cell membrane. ► This process is apparently independent of the receptor activity. ► Large scale movements of the receptor is explainable by its mesoscopic diffusion. ► Mathematical and experimental techniques are provided to investigate such processes. ► Results are discussed in terms of regulation of receptor functions.

Keywords: Abbreviations; β; ₂; AR; β; ₂; -adrenoceptor; FRAP; fluorescence recovery after photobleaching; GFP; green fluorescent protein; GPCR; G protein-coupled receptor; FCS; fluorescence correlation spectroscopy; SPT; single particle (or fluorescent molecule) trackingβ-adrenoceptor; Lateral diffusion; Dendra2; Receptor mobility; G protein-coupled receptor; Compartmentalization

Proteolytic action of kallikrein-related peptidase 7 produces unique active matrix metalloproteinase-9 lacking the C-terminal hemopexin domains by Vishnu C. Ramani; Gur P. Kaushal; Randy S. Haun (pp. 1525-1531).

The gelatinases, matrix metalloproteinase (MMP)-9 and -2, are produced as latent, inactive enzymes that can be proteolytically activated by a number of proteases. In many normal and pathological conditions, where the expression of MMPs is deregulated, changes in the expression of other proteases have also been reported. Human kallikrein-related peptidase 7 (KLK7), a chymotryptic-like serine protease, is overexpressed in many different types of neoplastic conditions, which have also been shown to express high levels of both MMP-9 and -2. Since the activation of MMPs by KLK7 has never been examined, we sought to determine whether KLK7 can activate these MMPs. To test this hypothesis KLK7 was incubated with the recombinant MMPs and the products of the reaction were analyzed for their activity. Incubation of proMMP-9 with KLK7 resulted in the production of a novel truncated, active MMP-9 lacking the C-terminal hemopexin domains. In contrast, KLK7 degraded, but did not activate, proMMP-2. The novel activation of proMMP-9 by KLK7 was further confirmed using conditioned medium prepared from an MMP-9-expressing cell line, MDA-MMP-9. Our results clearly establish that KLK7 activates proMMP-9 to produce a novel truncated, active MMP-9 product not generated by other proteases. These findings suggest that KLK7 may play an important role in the activation of MMP-9 in tumors that express high levels of both these proteases and the resulting truncated MMP may possess altered substrate specificities compared with full-length MMP-9 activated by other proteases.► KLK7 and MMP-9 are expressed in pancreatic, breast, cervical, and ovarian cancers. ► KLK7 cleaves proMMP-9 to produce a unique truncated, active gelatinase. ► Active MMP-9 lacking the hemopexin domains may have altered substrate specificities.

Keywords: Gelatinase; Pancreatic cancer; Proteolytic cascade; Zymogen activation

αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects muscle myoblasts from TNF-α induced cytotoxicity by Amit S. Adhikari; Bhairab N. Singh; K. Sridhar Rao; Ch Mohan Rao (pp. 1532-1542).

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB.We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKβ and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.► αB-crystallin and TNF-α are upregulated during myoblast differentiation. ► αB-crystallin associates with and enhances IKKβ activity, facilitating I-kBα degradation.► Ser-59 phosphorylation of αB-crystallin is necessary to activate NF-κB pathway. ► Upregulation of αB-crystallin leads to elevation of antiapoptotic protein Bcl 2. ► αB-crystallin protects C2C12 cells from TNF-α induced cytotoxicity.

Keywords: HSP; αB-crystallin; TNF-α; NF-κB; Apoptosis; Differentiation

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells by Ryuichiro Kimura; Chie Ishikawa; Takayoshi Rokkaku; Ralf Janknecht; Naoki Mori (pp. 1543-1553).

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.Display Omitted► Osteosarcoma patients develop pulmonary metastases, but its mechanisms remain unclear. ► MMP-1 and CXCR4 were notably expressed in a highly metastatic osteosarcoma cell line. ► MAPK pathways activated AP-1 (c-Jun/Fra-1), and thereby regulated MMP-1 expression. ► The MAPK/AP-1/MMP-1 axis plays a central role in osteosarcoma invasion.

Keywords: Abbreviations; AP-1; activator protein-1; CXCR4; C–X–C chemokine receptor type 4; ECM; extracellular matrix; ELISA; enzyme-linked immunosorbent assay; EMSA; electrophoretic mobility shift assay; ERK; extracellular signal-regulated kinase; ETS; E-twenty six; FCS; fetal calf serum; Fra-1; Fos-related antigen-1; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; IL; interleukin; IL-2Rα; IL-2 receptor α chain; JNK; c-Jun NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; MNNG; N-methyl-N′-nitro-N-nitrosoguanidine; NF-κB; nuclear factor-κB; PBS; phosphate-buffered saline; PEA3; polyomavirus enhancer-A binding protein-3; Rb; retinoblastoma protein; RT-PCR; reverse transcription-PCR; SD; standard deviation; SDF-1α; stromal cell-derived factor-1α; siRNA; small interfering RNA; WST-8; water-soluble tetrazolium-8; WT; wild typeMMP-1; Osteosarcoma; Invasion; Pulmonary metastasis; c-Jun; Fra-1

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway by Natasha L. Grimsey; Catherine E. Goodfellow; Mike Dragunow; Michelle Glass (pp. 1554-1560).

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.► First direct evidence of human CB2 recycling back to the cell surface after agonist-induced internalization. ► Rab5 implicated in human CB2 internalization. ► Rab11, but not Rab4, implicated in human CB2 recycling.

Keywords: Abbreviations; CB1; cannabinoid receptor 1; CB2; cannabinoid receptor 2; DMEM; Dulbecco's modified Eagle medium; FBS; fetal bovine serum; GPCR; G-protein coupled receptor; HA; hemagglutinin; HEK; human embryonic kidney; PBS; phosphate buffered saline; WT; wild-typeCannabinoid receptor CB2; Cell Surface receptor; Endocytosis; Protein trafficking; Rab GTP-binding protein

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BBA - Molecular Cell Research (v.1813, #8)