BBA - Molecular Cell Research (v.1803, #12)

The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells by Ioannis Sanidas; Vassiliki Kotoula; Eleni Ritou; Jasmijn Daans; Christof Lenz; Mario Mairhofer; Makrina Daniilidou; Andrea Kolbus; Volker Kruft; Peter Ponsaerts; Eleni Nikolakaki (1319-1331).
SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.►The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells. ►Overexpression of SRPK1a induces erythroid differentiation of K562 cells. ►Down-regulation of SRPK1 in K562 cells results similarly in erythroid differentiation. ►SRPK1a-associated proteins have been previously identified in ribosomal complexes.
Keywords: SRPK1a; SRPK1; K562 cell; Differentiation; Erythroid progenitor cell;

Vrp1p–Las17p interaction is critical for actin patch polarization but is not essential for growth or fluid phase endocytosis in S. cerevisiae by Ming Hwa Wong; Lei Meng; Rajamuthiah Rajmohan; Shangjuan Yu; Thirumaran Thanabalu (1332-1346).
Vrp1p (yeast WIP) forms a protein complex with Las17p (yeast WASP), however the physiological significance of the interaction has not been fully characterized. Vrp1p residues, 788MPKPR792 are essential for Vrp1p–Las17p interaction. While C-Vrp1p364–817 complements all the defects of the vrp1Δ strain, C-Vrp1p364–817 5A (788AAAAA792) does not complement any of the defects, due to its inability to localize to cortical patches. Targeting C-Vrp1p364–817 5A to membranes using CAAX motif (C-Vrp1p364–817 5A-CAAX) rescued the growth and endocytosis defect but not the actin patch polarization defect of vrp1Δ. Vrp1p can localize to cortical patches, either by binding to Las17p through LBD (Las17 Binding Domain, Vrp1p760–817) or independent of Las17p through residues in N-Vrp1p1–364. Unlike Vrp1p, Vrp1p5A localizes poorly to cortical patches and complements all the defects of vrp1Δ strain except actin patch polarization at elevated temperature. N-Vrp1p1–364 complements all the defects of vrp1Δ strain except the actin patch polarization defect while N-Vrp1p1–364–LBD fusion protein complements all the defects. Thus our results show that while both Vrp1p and Las17p are essential for many cellular processes, the two proteins do not necessarily have to bind to each other to carry out these cellular functions. However, Las17p–Vrp1p interaction is essential for actin patch polarization at elevated temperature.► Vrp1p residues 787–797 are essential for Vrp1p–Las17p interaction. ► The motif 788MPKPR792 is essential for the direct interaction between Vrp1p and Las17p. ► Fusion of CAAX motif to C-Vrp1p364–817 5A mutant can restore its activity in growth and endocytosis. ► N-Vrp1p1–364 fused to LBD complements the actin patch polarization defect of vrp1Δ strain.
Keywords: Arp2/3; Myosin; WASP; WIP; Cortical actin patch; Membrane traffic;

Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors by Sylwia Łukasiewicz; Agnieszka Polit; Sylwia Kędracka-Krok; Krzysztof Wędzony; Marzena Maćkowiak; Marta Dziedzicka-Wasylewska (1347-1358).
In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT2A receptor and the dopamine D2 receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT2AR and D2R, pointing toward the formation of hetero-5-HT2AR/D2R dimers and homo-5-HT2AR/5-HT2AR dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D2R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT2AR) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT2AR and D2R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.►Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors. ►Homo-dimerization of serotonin 5-HT2A receptors. ►Co-localization of serotonin 5-HT2A and dopamine D2 receptors in the rat brain. ►The influence of serotonin 5-HT2A and dopamine D2 receptors agonists an antagonists on dimerization process. ►Involvement of ic3 of dopamine D2 receptor and C-tail of serotonin 5-HT2A receptors in hetero-dimerization.
Keywords: Serotonin 5-HT2A receptor; Dopamine D2 receptor; Arg-rich motif; GPCR oligomerization; FRET; Fluorescent proteins;

Activation of the sonic hedgehog signaling controls human pulmonary arterial smooth muscle cell proliferation in response to hypoxia by Guansong Wang; Zhiyuan Zhang; Zhi Xu; Hongjin Yin; Li Bai; Zhuang Ma; Mark A. DeCoster; Guisheng Qian; Guangyu Wu (1359-1367).
The hedgehog signal pathway plays a crucial role in the angiogenesis and vascular remodeling. However, the function of this pathway in the pulmonary vascular smooth cell proliferation in response to hypoxia remains unknown. In this study, we have demonstrated that the main components of the hedgehog pathway, including sonic hedgehog (SHH), patched1 (PTCH1), smoothened (SMO), GLI and hypoxia-inducible factor 1 (HIF1) are expressed in the human pulmonary arterial smooth muscle cells (HPASMCs). Interestingly, hypoxia significantly enhanced the expression of SHH and HIF1, facilitated the translocation of GLI1 into the nuclei, and promoted the proliferation of HPASMCs. Furthermore, direct activation of the SHH pathway through incubation with the purified recombinant human SHH or with purmorphamine and SAG, two Smo agonists, also enhanced the proliferation of HPASMCs. Importantly, the treatment with anti-SHH and anti-HIF1 antibodies or cyclopamine, a specific SMO inhibitor, markedly inhibited the nuclear translocation of GLI1 and cell proliferation in the HPASMCs induced by hypoxia and activation of the SHH pathway. Moreover, the treatment with cyclopamine increased apoptosis in the hypoxic HPASMCs. These data strongly demonstrate for the first time that the SHH signaling plays a crucial role in the regulation of HPASMC growth in response to hypoxia.► Hypoxia induces upregulation and secretion of SHH in the human pulmonary arterial smooth muscle cells (HPASMCs). ► Hypoxia increases HIF1 expression and hypoxia-induced SHH expression depends on HIF1 in the HPASMCs. ► Hypoxia enhances nuclear translocation of GLI1 in the HPASMCs. ► Hypoxia induces HPASMC proliferation. ► The SHH signaling pathway regulates apoptosis in the HPASMCs subjected to hypoxia.
Keywords: Anoxia; Hedgehog signal pathway; Sonic hedgehog; GLI; Cell proliferation; Human pulmonary arterial smooth muscle cells;

Hepatocytes in primary culture undergo apoptosis upon exposure to glycochenodeoxycholate (GCDC). The signaling mechanisms of GCDC-induced apoptosis remain unclear. To investigate the role of antiapoptotic genes, we compared apoptotic response in primary hepatocytes following GCDC treatment. The hepatocytes from adult Sprague–Dawley rats were cultured in collagen-coated dishes and treated with GCDC in varying concentrations, or the same concentration at different time intervals. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, DNA fragmentation assay, and caspase assays. Expression of apoptosis-related genes and proteins was evaluated by RT-PCR, quantitative real-time PCR (qRT-PCR), and Western blotting, respectively. The DNA-binding property of a nuclear protein was assessed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. An interesting result was that GCDC caused hepatocyte apoptosis to display a biphasic phenomenon at a dosage of 50 μM, whereas it was not found at higher dosages such as 200 μM. GCDC stimulated the expression of antiapoptotic Survivin, which also presented a biphasic response. The activation of nuclear factor-kappaB (NF-κB) corresponded with the up-regulation of Survivin. The inhibitor of NF-κB, BAY 11-7082, suppressed the expression of Survivin and simultaneously eliminated the biphasic response. The expression of Survivin was transcriptionally mediated by the activation of NF-κB, as shown by EMSA and ChIP assay. Conclusions: These results demonstrated that a low dosage of GCDC induced the hepatocyte apoptosis to exhibit the biphasic response, which was regulated by the expression of Survivin through NF-κB signaling pathway.►GCDC induces the hepatocyte apoptosis to exhibit the biphasic response. The biphasic response depends on GCDC dosage. The activation of Survivin regulates the biphasic response. NF-κB modulates Survivin signaling.
Keywords: Apoptosis; Glycochenodeoxycholate; Nuclear factor-kappaB; Hepatocyte; Survivin;

Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae by Silvia Bradamante; Alessandro Villa; Silvia Versari; Livia Barenghi; Ivan Orlandi; Marina Vai (1376-1385).
A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈ 40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).►Microgravity and hyperoxia (μg & O2) induce a huge GSH extracellular release in yeast ►μg & O2 upregulate GSH biosynthesis's gene transcription ►μg & O2 activate HOG & PKC pathways, oxidative stress response, and actin remodelling ►Actin remodelling activates volume-sensitive ion channels (chloride) ►Chloride channels are the main responsible for GSH extracellular release
Keywords: GSH; Oxidation; Actin; Yeast; Chloride channels; Microgravity;

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells by Kristine C. Nyhan; Noel Faherty; Gregg Murray; Laurence Berubé Cooey; Catherine Godson; John K. Crean; Derek P. Brazil (1386-1395).
The Jagged/Notch pathway has been implicated in TGFβ1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFβ1-stimulated gene responses in human kidney epithelial cells in vitro. TGFβ1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of γ-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFβ1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by γ-secretase inhibition, but other TGFβ1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFβ1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFβ1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFβ1, but did not affect α-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFβ1-responsive gene subset. Increased Jagged1 expression upon TGFβ1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFβ1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFβ1 and Notch cascades in kidney epithelia.►Jagged/Notch signalling is required for a subset of TGFβ1-regulated EMT genes in kidney epithelial cells. ►Upregulation of Jagged1 and Hes1 precedes changes in EMT genes such as E-cadherin. ►Inhibition of Jagged/Notch signalling prevents TGFβ1-induced changes in cell shape. ►Jagged1 upregulation by TGFβ1 is completely Smad3 dependent, and partially PI3K/ERK dependent.
Keywords: Kidney epithelial cell; Jagged Notch pathway; gamma-Secretase; Transforming growth factor beta;

Sigma-1 receptors amplify dopamine D1 receptor signaling at presynaptic sites in the prelimbic cortex by Yingmei Fu; Yanyan Zhao; Wenjie Luan; Lian-Yian Dong; Yi Dong; Bin Lai; Yanhua Zhu; Ping Zheng (1396-1408).
Sigma-1 receptors are highly expressed in the brain. The downstream signaling mechanisms associated with the sigma-1 receptor activation have been shown to involve the activation of protein kinase C (PKC), the control of Ca2 homoeostasis and the regulation of voltage- and ligand-gated ion channels. But few studies examined the regulatory effect of sigma-1 receptors on metabotropic receptor signaling. The present paper studied the regulatory effect of sigma-1 receptors on the signaling of dopamine D1 receptors, one of metabotropic receptors, by examining the effect of sigma-1 receptor agonists on the D1 receptor agonist-induced cAMP-dependent protein kinase (PKA) activation at presynaptic sites using the synaptosomes from the prelimbic cortex. The results showed that sigma-1 receptor agonists alone had no effects on the PKA activity, but could amplify the D1 receptor agonist-induced PKA activation. The sigma-1 receptor agonist also amplified the membrane-permeable analog of cAMP- and the adenylyl cyclase (AC) activator-induced PKA activation, but did not on the D1 receptor agonist-induced AC activation. The conventional PKC (cPKC), especially the PKCβI, and the extracellular Ca2+ influx through L-type Ca2+ channels might play key roles in the amplifying effect of the sigma-1 receptor agonists. The activation of PKC by sigma-1 receptor agonists was the upstream event of the increase in the intrasynaptosomal Ca2+ concentration. These results suggest that sigma-1 receptors may amplify the D1 receptor agonist-induced PKA activation by sigma-1 receptors - cPKC (especially the PKCβI) - L-type Ca2+ channels - Ca2+ - AC and/or cAMP signaling pathway.►Sigma-1 receptors amplify D1 receptor-induced PKA activation at presynaptic sites ►PKCβI plays an important role in the amplifying effect of the sigma-1 receptors ►Ca2+ influx via L-type Ca2+ channels plays an important role in the amplifying effect ►Activation of PKCβI by sigma-1 receptors is the upstream event of Ca2+ rise
Keywords: Sigma-1 receptors; Synaptosomes; Prelimbic cortex; D1 receptors; cAMP-dependent protein kinase; Ca2+ concentration;