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BBA - Molecular Cell Research (v.1773, #4)

Editorial Board (pp. ii).

mRNA specific subcellular localization represents a crucial step for fine-tuning of gene expression in mammalian cells by Marisol Corral-Debrinski (pp. 473-475).

mRNA subcellular distribution and translational control are key player mechanisms for post-transcriptional gene expression regulation. In the last decade it has become increasingly clear that these processes are associated with various human diseases. Understanding the interconnected multistep process of mRNA localization and its involvement in organelle biogenesis and in the overall spatial structure of eukaryotic cells will be an important step towards the long-term goal of curing individual molecular defects. In a recent issue, Russo et al. [The 3′-untranslated region directs ribosomal protein-encoding mRNAs to specific cytoplasmic regions, Biochim. Biophys. Acta, Mol. Cell Res. 1763 (8) (2006) 833–843] reported interesting findings on the mechanisms that direct mRNAs encoding different ribosomal proteins to specific cytoplasmic regions in human cells.

Keywords: Subcellular mRNA localization; 3′UTR; Mitochondria; Ribosome biogenesis; Gene expression regulation

Cell surface dynamics of CFTR: The ins and outs by Tsukasa Okiyoneda; Gergely L. Lukacs (pp. 476-479).

Ganeshan et al. (Biochem Biophys. Acta 1173 (2007) 192–200) recent study documents the effect of N-WASP inhibition and actin cytoskeleton disruption on the constitutive internalization and recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) channel. The results implicate the cytoskeleton network as a potential modulator of immobilized CFTR pool size at the plasma membrane and the recycling efficiency of endocytosed channel back to the cell surface.

Keywords: Endocytosis; Recycling; Actin; WASP; Sorting

Cardiomyocyte necrosis: Alternative mechanisms, effective interventions by Nektarios Tavernarakis (pp. 480-482).

Necrotic death of cardiac myocytes is a major contributor to heart failure associated with several cardiac pathologies such as ischemia and reperfusion injury. Preventing cardiomyocyte necrosis is an important challenge towards the development of effective strategies, aiming to battle cardiovascular disorders. While, necrotic cell death was traditionally considered a passive and chaotic process, emerging evidence indicates that specific molecular mechanisms underlie cellular destruction during necrosis. Elucidation of these mechanisms will facilitate therapeutic intervention against heart failure.

Keywords: Ischemia; Lipid; Mitochondria; Necrotic cell death; Oxidative stress; Peroxidation; Reperfusion

CCK causes PKD1 activation in pancreatic acini by signaling through PKC-δ and PKC-independent pathways by Marc J. Berna; K. Martin Hoffmann; Jose A. Tapia; Michelle Thill; Andrea Pace; Samuel A. Mantey; Robert T. Jensen (pp. 483-501).

Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependant increase in serine phosphorylation by activation of high- and low-affinity CCK_A receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-ζ pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-δ, but not PKC-ε, or treatment with PKC-δ translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCK_A receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways.

Keywords: PKD1 activation; Pancreas; Cholecystokinin; Gastrointestinal hormone; Growth factor; PKC

FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation by Linda Gibbs; Laurence Legeai-Mallet (pp. 502-512).

Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development.

Keywords: Abbreviations; ACH; achondroplasia; BFA; Brefeldin A; ER; endoplasmic reticulum; FGFR; Fibroblast Growth Factor Receptor; HCH; hypochondroplasia; TDI and TDII; thanatophoric dysplasia types I and II; TK; tyrosine kinase; WT; wild typeChondrodysplasia; Receptor; Kinase; Signaling; Trafficking

Visualization of CD146 dimerization and its regulation in living cells by Pengcheng Bu; Jie Zhuang; Jing Feng; Dongling Yang; Xun Shen; Xiyun Yan (pp. 513-520).

Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescence-tagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.

Keywords: CD146; Dimerization; FRET; NF-kappa B

Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei by Nathalie Galland; Fabian Demeure; Véronique Hannaert; Emilie Verplaetse; Didier Vertommen; Patrick Van Der Smissen; Pierre J. Courtoy; Paul A.M. Michels (pp. 521-535).

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.

Keywords: Abbreviations; AAA+ proteins; ATPases associated with various cellular activities; ALD; aldolase; GAPDH; glycosomal glyceraldehyde-3-phosphate dehydrogenase; PEX; peroxin; PTS; peroxisome-targeting signal; PYK; pyruvate kinase; Tet; tetracycline; TIM; triosephosphate isomerase Trypanosoma; Glycosome biogenesis; Peroxin; RNA interference

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells by Elly S.W. Ngan; K.Y. Lee; Francesco Y.L. Sit; H.C. Poon; Jacqueline K.Y. Chan; M.H. Sham; Vincent C.H. Lui; Paul K.H. Tam (pp. 536-545).

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

Keywords: Prokineticin-1; Neural crest cell; Proliferation; Differentiation

Nuclear trafficking of the POZ-ZF protein Znf131 by Nickett S. Donaldson; Yasmin Daniel; Kevin F. Kelly; Monica Graham; Juliet M. Daniel (pp. 546-555).

Znf131 is a member of the BTB/POZ family of transcription factors with roles in development and carcinogenesis. Like many members of this protein family, Znf131 displays robust nuclear localization in cultured cells, but the mechanism(s) of Znf131 nuclear trafficking is unknown. Here, we report the mechanism of Znf131 nuclear localization. Visual inspection of the Znf131 amino acid sequence revealed three basic regions (BR-1, -2 and -3) with the potential to serve as nuclear localization signals (NLS). Of the three basic regions, only BR-1 functioned independently to efficiently target heterologous β-gal-GFP fusion proteins to HeLa cell nuclei. However, a Znf131 truncation mutant containing BR-2 and BR-3 efficiently targeted heterologous β-gal-GFP fusion proteins to HeLa cell nuclei. Mutational analysis of full-length GFP-tagged Znf131 revealed that loss of any one BR alone did not prevent Znf131 nuclear localization. This apparent redundancy in NLS activity was due to the fact that intact BR-1 or BR-2 alone could target full-length Znf131 to nuclei. Consequently, simultaneous mutation of BR-1 and BR-2 abolished full-length Znf131 nuclear localization. Therefore, BR-1 and BR-2 are functional NLSs for Znf131 and as such are designated NLS-1 and NLS-2. Finally, wild type Znf131, and not a Znf131 NLS-defective mutant (NLS-1m/NLS-2m) interacted preferentially with the nuclear import receptor Importin-α3 in vitro.

Keywords: Znf131; Transcription factor; POZ-ZF; Nuclear import; NLS

Possible involvement of RecQL4 in the repair of double-strand DNA breaks in Xenopus egg extracts by Yuji Kumata; Shusuke Tada; Yumie Yamanada; Takashi Tsuyama; Takayuki Kobayashi; Yu-Peng Dong; Kyoko Ikegami; Hiromu Murofushi; Masayuki Seki; Takemi Enomoto (pp. 556-564).

Mutations in RecQL4 are a causative factor in Rothmund–Thomson syndrome, a human autosomal recessive disorder characterized by premature aging. To study the role of RecQL4, we employed a cell-free experimental system consisting of Xenopus egg extracts. RecQL4 loading onto chromatin was observed regardless of the presence or absence of EcoRI. However, in the absence of EcoRI, RecQL4 loading was suppressed by geminin, an inhibitor of pre-replicative complex formation, while in the presence of EcoRI, it was not affected. These results suggest that under the former condition, RecQL4-loading depended on DNA replication, while under the latter, the interaction occurred in response to double-stranded DNA breaks (DSBs) induced by EcoRI. DSB-induced RecQL4 loading depended on the function of the ataxia-telangiectasia mutated protein, DNA-dependent protein kinase (DNA-PK), and replication protein A, while there were only minor changes in DNA replication-associated RecQL4 loading upon suppression of these proteins. Furthermore, analyses using a chromatin-immunoprecipitation assay and quantification of γH2AX after induction of DSBs suggested that RecQL4 is loaded adjacent to Ku heterodimer-binding sites on damaged chromatin, and functions in the repair of DSBs.

Keywords: RecQL4; Double-strand DNA break repair; Ku heterodimer; DNA-dependent protein kinase (DNA-PK); Non-homologous end-joining (NHEJ)

TRPV6 mediates capsaicin-induced apoptosis in gastric cancer cells—Mechanisms behind a possible new “hot” cancer treatment by Justine Chow; Manith Norng; Jing Zhang; Jianyuan Chai (pp. 565-576).

Capsaicin is an organic compound in chili peppers which are consumed by over one quarter of the world's population daily. Studies have shown that capsaicin can induce apoptosis in some cancer cells by unknown mechanisms. In this study, both gastric cancer and normal epithelial cells were treated with capsaicin and examined for apoptosis by Annexin V binding. Our results showed that capsaicin induces apoptosis in both cells, although cancer cells are more susceptible. This susceptibility is dependent on the availability of TRPV6, a calcium-selective channel protein, as overexpression of TRPV6 in normal cells increased capsaicin-induced apoptosis and knockdown of TRPV6 in cancer cells suppressed this action. Our results further demonstrated that capsaicin increases mitochondrial permeability through activation of Bax and p53 in a JNK-dependent manner. Conclusions: (1) TRPV6, rather than TRPV1 (the well-known capsaicin receptor), mediates capsaicin-induced apoptosis in gastric cells; (2) abundance of TRPV6 in gastric cells determines their live or death under capsaicin treatment; and (3) capsaicin induces apoptosis by stabilization of p53 through JNK activation. Together, our data suggest that capsaicin may be a promising dietary candidate for cancer chemoprevention.

Keywords: TRPV6; Capsaicin; Apoptosis; Gastric cancer

Overexpression of IAP-2 attenuates apoptosis and protects against myocardial ischemia/reperfusion injury in transgenic mice by Chu Chang Chua; Jinping Gao; Ye-Shih Ho; Ye Xiong; Xingshun Xu; Zhongyi Chen; Ronald C. Hamdy; Balvin H.L. Chua (pp. 577-583).

Inhibitors of apoptosis proteins (IAPs) are key intrinsic regulators of caspases-3 and -7. During ischemia, IAP-2 is upregulated dramatically, while the other IAPs show little or no change. To test whether IAP-2 prevents cardiac apoptosis and injury following ischemia/reperfusion, we generated a line of transgenic mice that carried a mouse IAP-2 transgene. High levels of mouse IAP-2 transcripts and 70 kDa IAP-2 were expressed in the hearts of transgenic mice, whereas IAP-1 and XIAP levels remained the same. Immunohistochemical studies revealed more intense staining of IAP-2 in the myocytes of transgenic mouse hearts. To assess the role of IAP-2 in I/R injury, the transgenic mice were subjected to ligation of the left descending anterior coronary artery ligation followed by reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (30±2% vs. 44±2%, respectively, P<0.05). This protection was accompanied by a decrease of the serum level of troponin I in the transgenic mice. IAP-2 transgenic hearts had significantly fewer TUNEL-positive cardiac cells, which indicated an attenuation of apoptosis. Our results demonstrate that overexpression of IAP-2 renders the heart more resistant to apoptosis and I/R injury.

Keywords: Caspases; Inhibitor of apoptosis proteins; Ischemia/reperfusion injury; Heart

Multisite phosphorylation of adipocyte and hepatocyte phosphodiesterase 3B by Rebecka Lindh; Faiyaz Ahmad; Svante Resjö; Peter James; Jeong S. Yang; Henry M. Fales; Vincent Manganiello; Eva Degerman (pp. 584-592).

Phosphodiesterase 3B (PDE3B) is an important component of insulin and cAMP-dependent signalling pathways. In order to study phosphorylation of PDE3B, we have used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes and H4IIE hepatoma cells. Phosphorylation of PDE3B after treatment of cells with insulin, cAMP-increasing agents, or the phosphatase inhibitor, calyculin A was analyzed by two-dimensional tryptic phosphopeptide mapping and mass spectrometry. We found that PDE3B is multisite phosphorylated in adipocytes and H4IIE hepatoma cells in response to all these stimuli. Several sites were identified; serine (S)273, S296, S421, S424/5, S474 and S536 were phosphorylated in adipocyte as well as H4IIE hepatoma cells whereas S277 and S507 were phosphorylated in hepatoma cells only. Several of the sites were phosphorylated by insulin as well as cAMP-increasing hormones indicating integration of the two signalling pathways upstream of PDE3B, maybe at the level of protein kinase B.

Keywords: PDE3B; Phosphorylation; Adipocyte; Hepatocyte; Insulin; cAMP

Calcineurin mediates acetylcholinesterase expression during calcium ionophore A23187-induced HeLa cell apoptosis by Hui Zhu; Wei Gao; Hua Jiang; Jun Wu; Yu-fang Shi; Xue-Jun Zhang (pp. 593-602).

We previously reported that acetylcholinesterase plays a critical role in apoptosis and its expression is regulated by Ca²⁺ mobilization. In the present study, we show that activated calpain, a cytosolic calcium-activated cysteine protease, and calcineurin, a calcium-dependent protein phosphatase, regulate acetylcholinesterase expression during A23187-induced apoptosis. The calpain inhibitor, calpeptin, and the calcineurin inhibitors, FK506 and cyclosporine A, inhibited acetylcholinesterase expression at both mRNA and protein levels and suppressed the activity of the human acetylcholinesterase promoter. In contrast, overexpression of constitutively active calcineurin significantly activated the acetylcholinesterase promoter. Furthermore, we identify a role for the transcription factor NFAT (nuclear factor of activated T cells), a calcineurin target, in regulating the acetylcholinesterase promoter during ionophore-induced apoptosis. Overexpression of human NFATc3 and NFATc4 greatly increased the acetylcholinesterase promoter activity in HeLa cells treated with A23187. Overexpression of constitutive nuclear NFATc4 activated the acetylcholinesterase promoter independent of A23187, whereas overexpression of dominant-negative NFAT blocked A23187-induced acetylcholinesterase promoter activation. These results indicate that calcineurin mediates acetylcholinesterase expression during apoptosis.

Keywords: Abbreviations; AChE; Acetylcholinesterase, acetylcholine acetyl hydrolase; CnA; calcineurin A; CnB; calcineurin B; CsA; cyclosporine A; DMSO; dimethyl sulphoxide; NFAT; nuclear factor of activated T cellsAcetylcholinesterase; Apoptosis; Calpain; Calcineurin; NFAT

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BBA - Molecular Cell Research (v.1773, #4)