BAG-1蛋白在衣霉素诱导人肺癌A549细胞内质网应激及提高顺铂敏感性中的作用

目的:体外观察低剂量衣霉素对人肺腺癌A549细胞内质网应激(endoplasmic reticulum stress,ERS)的诱导作用,以及此过程中Bcl-2结合抗凋亡基因1(Bcl-2 associated athanogene 1,BAG-1)表达的变化;同时,观察衣霉素作用后A549细胞对顺铂敏感性的变化以及此过程中BAG-1蛋白表达的变化.方法:采用蛋白质印迹法检测低剂量衣霉素作用A549细胞后ERS标志性蛋白GRP78及抗凋亡蛋白BAG-1的表达变化;MTT法测定衣霉素作用后顺铂对A549细胞的半数抑制浓度(half inhibitory concentration,IC50)变化;FCM法检测低剂量衣霉素作用后A549细胞对顺铂的敏感性变化;蛋白质印迹法检测衣霉素作用前后顺铂对A549细胞中BAG-1和procaspase-12蛋白表达的影响.结果:1.25 μg/mL衣霉素作用A549细胞8h后,能诱导细胞发生ERS,即ERS标志性蛋白GRP78表达上调,同时BAG-1蛋白表达也明显上调(P均＜0.05).衣霉素诱导A549细胞ERS能增加细胞对顺铂的敏感性(P＜0.05).细胞发生ERS前,1.25、2.5和5 μg/mL 3个质量浓度的顺铂均上调BAG-1蛋白的表达(P均＜ 0.05),但2.5和5μg/mL顺铂诱导BAG -1蛋白上调量均小于1.25 μg/mL组(P＜0.05);细胞发生ERS后,与单纯ERS未给予顺铂干预的细胞组比,1.25、2.5和5μg/mL 3个质量浓度的顺铂均下调BAG -1蛋白的表达(P均＜0.05),且下调量与顺铂浓度呈正比.2.5和5μg/mL顺铂能通过ERS途径诱导细胞发生凋亡,在此过程中BAG-1和procaspase-12蛋白表达均下调(P均＜0.05).结论:BAG-1蛋白可能是ERS和顺铂诱导肺癌细胞凋亡的一个重要调控因子之一,且ERS凋亡途径可能是顺铂诱导肺癌细胞凋亡的重要途径之一.     

Identification and functional characterization of mouse TPO1 as a myelin membrane protein

TPO1 is a member of the AIGP family, a unique group of proteins that contains 11 putative transmembrane domains. Expression of the rat TPO1 gene is upregulated in cultured oligodendrocytes (OLs) during development from pro-oligodendroblasts to postmitotic OLs. However, the distribution of native TPO1 protein in cultured OLs and in the brain has not been elucidated. We investigated the distribution and cellular function of TPO1 in myelinating cells of the nervous system. In mice, TPO1 gene expression was detected in the central (CNS) and peripheral (PNS) nervous systems and was markedly upregulated at postnatal days 10-20, an early phase of myelination in the mouse brain. To investigate TPO1 localization, we generated affinity-purified antibodies to synthetic peptides derived from mouse TPO1. Immunohistochemical analysis showed that TPO1 was expressed in OLs and Schwann cells but not in neurons and astrocytes. Schwann cells from trembler mice, which lack PNS myelin, had significantly decreased TPO1 expression and an altered localization pattern, suggesting that TPO1 is a functional myelin membrane protein. In OL lineage cell cultures, TPO1 was detected in A2B5+ bipolar early progenitors, A2B5+ multipolar Pro-OLs, GalC+ immature OLs and MBP+ mature OLs. The subcellular localization of TPO1 in OL lineage cells was mapped to the GM130+ Golgi in cell bodies and Fyn+ cell processes and myelin-like sheets. Furthermore, TPO1 selectively colocalized with non-phosphorylated Fyn and promoted Fyn autophosphorylation in COS7 cells, suggesting that TPO1 may play a role in myelin formation via Fyn kinase activation in the PNS and CNS.     

Characterization of endoplasmic reticulum-associated degradation of a protein S mutant identified in a family of quantitative protein S deficiency

INTRODUCTION: Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD).We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracellular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant. MATERIALS AND METHODS: We stably expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD. RESULTS AND CONCLUSIONS: Endoglycosidase H digestion and immunofluorescence staining revealed the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH(2)Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase I, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man(8)GlcNAc(2) targets the mutant for ERAD, however, the interaction with calnexin/calreticulin through monoglucosylated oligosaccharides may not be required for the degradation of the mutant.     

Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

内质网应激和肌萎缩侧索硬化

肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)最显著的病理改变是选择性的运动神经元损害。最近研究表明,运动神经元的缺失主要是由凋亡引起的。变异的过氧化物歧化酶1(superoxide dismutase1,SOD1)通过不确切的机制进入内质网形成蛋白聚集物,和其他不明确的原因一起引起内质网应激(endoplasmic reticulum stress,ERS)和未折叠蛋白反应(unfolded protein response,UPR),进而通过激活Caspase-12、凋亡信号调节激酶(apoptosis-signalregulating kinase1,ASK1)-c-Jun氨基末端激酶(c-Jun-NH2-terminal kinases,JNK)通路、CHOP表达升高、钙离子的释放,引起运动神经元的凋亡。最近研究表明,在罕见的家族性ALS患者的一个亚群中发现了空泡相关膜蛋白相关蛋白B(vesicle-associated membrane protein-associated protein B,VAPB)的显性遗传变异。VAPB在维持内质网稳态中起着一定的作用。...     

Factor XII Ofunato: Lys346Asn mutation associated with blood coagulation factor XII deficiency causes impaired secretion through a proteasome-mediated degradation

Introduction

Congenital blood coagulation factor XII (FXII) deficiency is a rare coagulation disease and an autosomal recessive trait. It is found by chance in many cases. We identified a novel mutation (Lys346Asn) in the FXII gene of a patient with FXII deficiency, designated as Factor XII Ofunato.

Methods

The proband was a 75-year-old Japanese woman with a prolonged activated partial thromboplastin time (52.8 s). The FXII activity and antigen were greatly reduced (activity, 5%; antigen, 4.5%). We analyzed FXII gene of this patient using a direct sequencing method and characterized mutant FXII through in vitro expression studies.

Results

Sequence analysis of the FXII gene revealed a G → C point mutation at nucleotide 9845, resulting in Lys346 (AAG) → Asn (AAC) replacement in the catalytic domain. Expression studies in Chinese hamster ovary cells demonstrated that mutant FXII (346 N-FXII) showed a lower level of accumulation in the cells than wild-type. Secretion of 346 N-FXII was greatly reduced in culture medium. We also investigated mRNA expression levels of wild-type and 346 N-FXII in transfected cells using quantitative RT-PCR. Both mRNA expressions were equivalent levels. Pulse-chase experiments showed that 346 N-FXII was extensively degraded intracellularly compared to wild-type. Using membrane-permeable inhibitors, we observed that degradation occurred in the pre-Golgi compartment and that proteasome apparently plays a central role in this process.

Conclusions

These results show that most 346 N-FXII is degraded intracellularly through endoplasmic reticulum-associated degradation as the protein quality control system, resulting in an insufficient secretion phenotype.     

二氧化硫对自发性高血压大鼠主动脉平滑肌细胞内质网应激的影响

目的探讨二氧化硫(sulfur dioxide,SO2)对自发性高血压大鼠(spontaneously hypertensive rat,SHR)主动脉平滑肌细胞内质网应激的调节作用。方法4周龄正常血压WKY(wistar-kyoto rat)大鼠7只作为WKY对照组。4周龄SHR大鼠12只分为SHR(spontaneously hypertensive rat)对照组及SHR+SO2供体(Na2SO3/NaHSO3)组,每组6只。5周后检测大鼠血压,采用高效液相色谱(HPLC)法测定血浆SO2水平,采用免疫组织化学方法检测主动脉平滑肌细胞GRP78和caspase-12的蛋白表达。结果9周时SHR对照组血压明显高于WKY对照组,血浆SO2水平显著低于WKY对照组,主动脉平滑肌细胞GRP78和caspase-12蛋白表达显著增高。与SHR对照组比较,SHR+SO2供体组大鼠血压明显降低,血浆SO2含量升高,主动脉平滑肌细胞GRP78和caspase-12蛋白表达降低。结论SO2可能抑制高血压大鼠主动脉血管平滑肌细胞内质网应激反应的激活。     

Inhibition of Cerebral Vasoconstriction by Dantrolene and Nimodipine

Introduction  Cerebral vasoconstriction is associated with increased cytosolic Ca²⁺ concentration in vascular smooth muscle, presumably due to Ca²⁺ influx and Ca²⁺ release from intracellular stores. We tested the hypothesis that dantrolene (a blocker of Ca²⁺-induced Ca²⁺ release from the ryanodine receptor channel on the sarco-endoplasmic reticulum) would potentiate the action of nimodipine (a voltage-dependent L-type Ca²⁺ channel blocker, considered standard therapy for SAH) in inhibiting the vasoconstriction of isolated cerebral arteries. Method  Sprague–Dawley rat basilar and femoral arteries were analyzed for ryanodine receptor expression by immunofluorescence and PCR. Vasoconstriction of basilar artery ex vivo was measured in a wire myograph while exposed to serotonin (5-HT) or endothelin-1 (ET-1) in the presence or absence of dantrolene (10–100 μM) and/or nimodipine (30 nM). Femoral artery was examined for comparison. Results  Basilar and femoral arteries express only the ryanodine receptor 3 (RyR3) isoform. In both basilar and femoral arteries, dantrolene significantly inhibited the constriction to 5-HT, whereas it poorly affected the constriction to ET-1. The inhibitory effect of dantrolene on 5-HT was substantially increased by nimodipine, inducing a 10-fold increase in the 50% effective concentration of 5-HT and a 46% reduction in maximum basilar constriction. In femoral artery, dantrolene modestly affected constriction to phenylephrine and there was no interaction with nimodipine. Conclusion  Dantrolene has synergistic effects with nimodipine against 5-HT-induced vasoconstriction in isolated cerebral arteries. Dantrolene–nimodipine interaction will require testing in a pathophysiological model but might provide treatment for reducing SAH-related vasospasm or other 5-HT-related vasospastic syndromes, such as Call-Fleming syndrome.     

Dantrolene: Mechanisms of Neuroprotection and Possible Clinical Applications in the Neurointensive Care Unit

Calcium plays a central role in neuronal function and injury. Dantrolene, an inhibitor of the ryanodine receptor, inhibits intracellular calcium release from the sarco-endoplasmic reticulum. We review the available data of dantrolene as a potential neuroprotective agent and briefly summarize its other pharmacologic effects that may have potential applications for patients in the neurointensive care unit (NICU). Areas with the need for continued research are identified.     

Signal transduction pathways of muscarinic receptor mediated activation in the newborn and adult mouse urinary bladder

OBJECTIVE

To study the role of M₂ and M₃ muscarinic receptor subtypes, sources of activator Ca²⁺, and mechanisms involved in increased force oscillations in muscarinic contractions in the bladders of newborn and adult mice, as in the adult bladder muscarinic M₃ receptors are considered to mediate the main part of bladder contraction, and this has not been established in the newborn bladder.

MATERIALS AND METHODS

Bladder preparations from newborn (0–2 days) and adult (10–12 weeks) mice were mounted for in vitro force registration and activated with carbachol and high‐K⁺ solution in the presence of M₃ (4‐DAMP 30 nm ) or M₂ (methoctramine, 100 nm ) receptor antagonists. Thapsigargin (1 µm ) or ryanodine (10 µm ) were used to inhibit sarcoplasmic reticulum Ca²⁺ release. L‐NAME (300 µm ) and 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 10 µm ) were used to inhibit nitric oxide synthase and guanylyl cyclase, respectively. Gap‐junction function was inhibited with by 18‐β‐glycyrrhetinic acid (18‐β‐GA; 0.1–100 µm ). Big‐conductance (BK) and small‐conductance (SK) K⁺ channels were inhibited by apamine and charybdotoxin (0.3 µm ), respectively.

RESULTS

Concentration–response relations for carbachol in the presence of 4‐DAMP and methoctramine showed that M₃ receptors are the main activating pathway also in the newborn bladder. Neither thapsigargin nor ryanodine influenced the muscarinic responses of the newborn and adult bladders. Carbachol‐induced contractions were not influenced by L‐NAME or ODQ. The 18‐β‐GA inhibited carbachol‐induced contractions in both newborn and adult tissue in a similar manner. Apamine and charybdotoxin slightly increased the amplitude of the contractile responses.

CONCLUSION

These results suggest that in the newborn mouse bladder, as in adult bladders, the M₃ muscarinic receptor subtype is mainly responsible for carbachol‐induced contractile responses. The main mechanism for muscarinic receptor‐induced activation is influx of Ca²⁺ from the extracellular medium, and there seems to be no major contribution of Ca²⁺ release from intracellular stores. The phasic contractile activity induced by carbachol in the newborn bladder is not influenced by gap junction inhibition and does not involve SK and BK channels.