Expression of FAM20C in the Osteogenesis and Odontogenesis of Mouse

Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth. (J Histochem Cytochem 58:957–967, 2010)     

Structural and Functional Analyses of the FAM46C/Plk4 Complex

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Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.     

Evidence for the involvement of FAM110C protein in cell spreading and migration

A number of factors, including protein kinases, Rho GTPases and actin and microtubule cytoskeletons play a crucial role in cell migration and spreading. We have recently shown that ectopic expression of FAM110C can alter cellular morphology by mechanisms yet to be determined. In this study, a FAM110C antiserum has been developed and used to study endogenously expressed FAM110C. Our data show that FAM110C is expressed by different cell lines and it can be detected throughout the cell. Interestingly, depletion of FAM110C by short interfering RNA reduced integrin-mediated filopodia formation, hepatocyte growth factor-induced migration, and phosphorylation of the Akt1 kinase in the epithelial cell line HepG2. Furthermore, co-immunoprecipitation and co-localization studies show that both ectopically and endogenously expressed FAM110C interact, or is part of a protein complex, with the Akt1 kinase. This interaction is transient and follows the activation of Akt1. In addition, we show that alpha-tubulin co-precipitates with FAM110C which further supports an interaction with the microtubule cytoskeleton. Collectively, these findings suggest a new function for FAM110C in the regulation of cell spreading, migration and filopodia induction.     

FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma

FAM46C, frequently mutated in multiple myeloma (MM), has recently been shown to encode a non-canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain mostly unknown. Using CRISPR-Cas9 technology and gene expression analysis, we found that the inactivation of FAM46C in MM down-regulates immunoglobulins (Igs) and several mRNAs encoding ER-resident proteins, including some involved in unfolded protein response and others that affect glycosylation. Interestingly, we show that FAM46C expression is induced during plasma cell (PC) differentiation and that Ig mRNAs encoding heavy and light chains are substrates of the ncPAP, as revealed by poly(A) tail-length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail-shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C up-regulates metastasis-associated lncRNA MALAT1 and results in a sharp increase in the migration ability. This phenotype depends mainly on the activation of PI3K/Rac1 signalling, which might have significant therapeutic implications. In conclusion, our results identify Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production and suggest that its role as a tumour suppressor might be related to the inhibition of myeloma cell migration.     

Evidence that the Modulation of Membrane-Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E-115 Murine Neuroblastoma Cell Differentiation

Abstract: Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.     

Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.     

V170 Area Plays an Essential Role in the Biological Activity of Human Ciliary Neurotrophic Factor

Abstract: The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.     

Appl1 Is Dispensable for Mouse Development, and Loss of Appl1 Has Growth Factor-selective Effects on Akt Signaling in Murine Embryonic Fibroblasts

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1^−/− murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1^−/− MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1^−/− MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.     

第20位脯氨酸对人肿瘤坏死因子的活性和结构的作用

采用寡聚核苷酸指导的体外基因定点突变方法,将重组人肿瘤坏死因子第２０位脯氨酸变为精氨酸。突变体在大肠杆菌中的表达量是ＴＮＦＰｒｏ２０的６０％。表达产物经一系列离子交换层析分离纯化以后,突变体的比活是ＴＮＦＰｒｏ２０的千分之。一突变体和ＴＮＦＰｒｏ２０在非变性非还原的条件下进行聚丙烯酰胺凝胶电泳,两者显示相同的区带,这表明突变体也可以形成三聚体活性形式,ＴＮＦＰｒｏ２０和突变体的荧光光谱分析揭示,Ａ     

Dentin Sialophosphoprotein (DSPP) Plays an Essential Role in the Postnatal Development and Maintenance of Mouse Mandibular Condylar Cartilage

Recently, dentin sialophosphoprotein (DSPP) was found to be expressed in the mandibular condylar cartilage (MCC), but the possible roles of this molecule in the formation, growth, and maintenance of the cartilage are largely unclear. To analyze such roles, we examined Dspp null mice compared with wild-type mice to assess the consequences of Dspp deletion on the morphology and structure of the MCC. Our data showed that DSPP is expressed in the prechondroblastic, chondroblastic, hypertrophic layers of the MCC. Dspp null mice exhibited decreases in the amount of MCC, with reduced formation of articular and prechondroblastic layers in which progenitor cell proliferation levels were distinctly affected. The expression of extracellular matrix molecules, including biglycan and collagen II, IX, and X, was remarkably altered. The findings in this study indicate that continuous DSPP action is required for the growth and/or maintenance of the MCC.     

异源nifA^C对根癌土壤杆菌nif基因表达的调节作用

用三亲水交配方法分别将载有褐球固氮菌（Ａｚｏｔｏｂａｃｔｅｒｃｈｒｏｏｃｏｃｃｕｍ）呈组成型表达的ｎｉｆＡＣ的质粒ｐＣＫ５和肺炎克氏杆菌（Ｋｌｅｂｓｉｅｌｌａｐｎｅｕｍｏｎｉａｅ）含有ｎｉｆＡ＾Ｃ和ｎｉｆＡ－ｎｔｒＣ基因的质粒ｐＣＫ３,ｐＳＺ３６和ｐＳＺ２３－ＣＡ导入根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｎｅｆａｃｉｅｎｓ）Ｃ５８／ｐＧＶ３８５０所得转移接合子的生长速率和野生型相似。在１０ｍ     

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C- and C-I were present in all tissues examined, whereas the C-II isoform was absent in the lung and the liver. Protein kinase C- was identified in brain, spinal ganglia, and adrenal gland. The C- isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C- and C- isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C- isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-, C-I, C-II, C-, C- and C- were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals. Both qualitative and quantitative differences were seen in comparison with the same organs from a 17-day fetus.     

The Nitric Oxide-Cyclic GMP Pathway Plays an Essential Role in Both Promoting Cell Survival of Cerebellar Granule Cells in Culture and Protecting the Cells Against Ethanol Neurotoxicity

Abstract: NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N ^G-nitro- l -arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono)bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.     

非小细胞肺癌组织KIF2A、KIF2C、KIF20A mRNA表达与临床病理特征和预后的关系

摘要 目的：探讨非小细胞肺癌（NSCLC）组织驱动蛋白超家族成员2A（KIF2A）、驱动蛋白超家族成员2C（KIF2C）、驱动蛋白超家族成员20A（KIF20A）信使核糖核酸（mRNA）表达与临床病理特征和预后的关系。方法：选择2016年9月至2019年9月天津医科大学总医院手术切除的NSCLC患者106例,取其癌组织及其对应的癌旁组织,应用荧光定量聚合酶链式反应（RT-qPCR）检测组织中KIF2A、KIF2C、KIF20A mRNA表达,分析其与临床病理特征的关系。应用 Pearson相关性分析NSCLC组织中KIF2A、KIF2C、KIF20A mRNA表达间的关系。随访3年,应用Kaplan-Meier生存曲线分析KIF2A、KIF2C、KIF20A mRNA表达与患者预后关系。结果：NSCLC癌组织中KIF2A、KIF2C、KIF20A mRNA表达水平显著高于癌旁组织（P<0.05）。低分化、淋巴结转移、临床分期Ⅲ A 期NSCLC癌组织中KIF2A、KIF2C、KIF20A mRNA表达水平显著高于中高分化、无淋巴结转移及临床分期I、II期NSCLC癌组织（P<0.05）。Pearson相关分析显示,NSCLC癌组织中KIF2A mRNA表达与KIF2CmRNA、KIF20A mRNA表达呈正相关,KIF2C mRNA表达与KIF20A mRNA表达呈正相关（P<0.05）。Kaplan-Meier法分析显示KIF2A mRNA低表达组、KIF2C mRNA低表达组、KIF20A mRNA低表达组3年生存率分别为（84.78%,86.27%,81.48%）显著高于KIF2A mRNA高表达组、KIF2C mRNA高表达组、KIF20A mRNA高表达组（59.62%,55.32%,59.09%）（P<0.05）。结论：KIF2A、KIF2C、KIF20A mRNA在NSCLC组织中存在高表达,且与低分化、淋巴结转移、临床分期及预后有关。