Verticillium longisporum is a soil-borne vascular pathogen infecting cruciferous hosts such as oilseed rape. Quantitative disease resistance (QDR) is the major control means, but its molecular basis is poorly understood so far. Quantitative trait locus (QTL) mapping was performed using a new (Bur×Ler) recombinant inbred line (RIL) population of Arabidopsis thaliana. Phytohormone measurements and analyses in defined mutants and near-isogenic lines (NILs) were used to identify genes and signalling pathways that underlie different resistance QTL.V. longisporum-induced stunting, systemic colonization by the fungus and for V. longisporum-induced chlorosis were identified. Stunting resistance QTL were contributed by both parents. The strongest stunting resistance QTL was shown to be identical with Erecta. A functional Erecta pathway, which was present in Bur, conferred partial resistance to V. longisporum-induced stunting. Bur showed severe stunting susceptibility in winter. Three stunting resistance QTL of Ler origin, two co-localising with wall-associated kinase-like (Wakl)-genes, were detected in winter. Furthermore, Bur showed a much stronger induction of salicylic acid (SA) by V. longisporum than Ler. Systemic colonization was controlled independently of stunting. The vec1 QTL on chromosome 2 had the strongest effect on systemic colonization. The same chromosomal region controlled the level of abscisic acid (ABA) and jasmonic acid (JA) in response to V. longisporum: The level of ABA was higher in colonization-susceptible Ler than in colonization-resistant Bur after V. longisporum infection. JA was down-regulated in Bur after infection, but not in Ler. These differences were also demonstrated in NILs, varying only in the region containing vec1. All phytohormone responses were shown to be independent of Erecta.QTL for resistance to A. thaliana against V. longisporum were identified: Erecta mediated resistance against V. longisporum-induced stunting. Independent of Erecta, stunting was caused in a light-dependent manner with possible participation of SA and Wakl genes. ABA and JA showed a genotype-specific response that corresponded with systemic colonization by the fungus. Understanding the biological basis of phenotypic variation in A. thaliana with respect to V. longisporum resistance will provide new approaches for implementing durable resistance in cruciferous crops.Signalling systems with a hitherto unknown role in the QDR of  Verticillium longisporum is a soil-borne vascular pathogen of recent evolutionary origin with a host range centred on crucifers[V. longisporum causes significant and increasing yield losses on oilseed rape[V. longisporum induces early senescence[Quantitative disease resistance (QDR) is a complex phenomenon involving a plethora of molecular mechanisms,2. It isrucifers. QDR is eed rape-7. The feed rape,8,9. V. nescence,10 and cnescence. Since tV. longisporum has been described for various accessions of Brassica species[A. thaliana ecotypes[Brassica species[B. napus produced more phenolic substances in the xylem of the hypocotyl upon infection than a susceptible line[B. napus[A. thaliana defence against V. longisporum[Rfo1 has been shown to mediate resistance against V. longisporum-induced fresh weight loss in A. thaliana[Fusarium oxysporum[Quantitative resistance against  species-15  in the xylem of Brassica shoots[V. longisporum in A. thaliana roots[ahl19 gene acted as a positive regulator of defence to V. longisporum and other Verticillium species in A. thaliana[V. longisporum in Brassica and possibly play a role in defence[Recent research has revealed different processes that are involved in the host’s response to involved,22. V. la shoots. Previoua shoots,23, but a shoots. Whereasna roots, reactivna roots. Further thaliana. Several defence. The hos defence.V. longisporum is poorly understood so far.However, little is known about how these processes relate to QDR. It is unclear whether resistance QTL represent genes within regulatory systems that have already been shown to operate in the host-pathogen interaction, or whether they constitute new components, adding to the complexity of the pathosystem. Furthermore, the role of known defence signalling pathways in natural resistance to V. longisporum in A. thaliana. QTL for relevant resistance traits using a new (Bur×Ler) recombinant inbred line (RIL) population have been identified. It is shown that the Erecta gene corresponded to a strong QTL mediating stunting resistance and that a functional Erecta signalling pathway mediated resistance against V. longisporum-induced stunting. Evidence is provided that SA, abscisic acid (ABA) and JA contents responded to V. longisporum in a genotype-specific way, and that changes in ABA and JA content were controlled by the same QTL that also conferred resistance to systemic colonization by the fungus.The present study aims at identifying genes and signalling pathways that account for differences in QDR against A. thaliana ecotypes Bur-0, Col-0 and Ler-0 were originally obtained from the Arabidopsis Information Service (AIS) Frankfurt[A. thaliana genotypes were obtained from the Nottingham Arabidopsis Stock Centre (NASC). er-105, er-108, er-111, and er-118 were included as strong erecta mutants, whilst er-116 represented a weaker erecta mutant[agb1-1, which is defective in the β-subunit of the heteromeric G-protein, was included as a mutant of a signalling component acting downstream of Erecta[V. longisporum isolate ‘43’ (V43)[rankfurt and maina mutant,32. agb1of Erecta. The V. 3’ (V43) was useder-0 (♂). A total of 189 F2 plants were propagated via single-seed descent to the F6 generation. All plants were grown in pots of 5 cm diameter in a greenhouse under long-day conditions (16 h light/8 h dark) at 20°C. A total of 94 F6 plants were genotyped and phenotyped in F7. Information about the RIL population will be submitted to the The Arabidopsis Information Resource (TAIR) database[A. thaliana stock centres Arabidopsis Biological Resource Center (ABRC) and NASC.A total of 189 RILs were created originating from an F1 between the ecotypes Bur-0 (♀) and Ldatabase, and F8 er alleles.NILs were created by selfing RIL21, which was heterozygous for markers EH2-4 to nga361 on chromosome 2 and homozygous for all other marker loci investigated. NIL5 was selected from RIL21 offspring as homozygous for Bur alleles in the variable region, and NIL9 for Ler. Among them were 39 simple sequence repeat (SSR) markers, 21 sequence-characterized (SCAR) markers exploiting length polymorphisms between Bur and Ler, and 12 cleaved amplified polymorphic sequence (CAPS) markers developed on the basis of single nucleotide polymorphisms  has also been developed to differentiate between the Bur and the Ler allele of Erecta. Fifty-one markers have been published before[er were identified using the multiple SNP query tool (MSQT) database[The RILs were genotyped with 73 markers that were polymorphic between the parental ecotypes Bur and Ld before,34,35, adatabase. SNPs avdatabase,38 were erecta mutants were performed in two greenhouse experiments (Table er (Table er at 20 days post inoculation (dpi) and in Bur at 31 dpi. Stage II (mid- to late-flowering stage) was analysed at 27 dpi in Ler and at 38 dpi in Bur. Stage III (onset of silique maturity) was analysed at 31 dpi in Ler and at 48 dpi in Bur.Inoculation experiments were performed in the greenhouse or in a growth chamber  under long-day conditions (16 h light/8 h dark) on soil as described previously[inoculated/mean heightcontrol × 100) and “performance fresh weight” (mean FWinoculated/mean FWcontrol × 100). The performance parameters compensate for differences in plant height, and fresh weight between erecta mutants and WT. The height was measured between the hypocotyl and the apex of the longest shoot at the beginning of silique maturation when shoots were out-grown. Systemic colonization was determined in apical segments of the main shoot at the onset of silique maturity. To determine the percentage of colonized plants per replicate, one segment per inoculated plant was placed on a malt agar plate as described previously[Verticillium-specific primers was performed as described[V. longisporum-inoculated plants were showing chlorotic leaves. In order to determine the time-course of chlorosis, mock-inoculated and inoculated Ler and Bur plants were assessed every 3-4 days after inoculation in a growth chamber experiment  was added to each sample. Extraction was performed for 45 min at 4°C on a rotary shaker (160 rpm). Subsequently, each sample was spiked with 1.0 ml diethyl ether (DEE), shortly vortexed, and shaken  for an additional 30 min. The sample was centrifuged at 8500 g for 5 min. The upper DEE phase was transferred to a new 1.5 ml tube, and the remaining aqueous phase was re-extracted with 1.0 ml DEE exactly as in the previous step. Both DEE fractions were pooled and dried under vacuum at 30°C, re-dissolved in 200 μl HPLC (high-performance liquid chromatography) solvent (1:1 H2O:MeOH containing 7 mM CH3COOH), centrifuged once more at 8500 g for 5 min and transferred (180 μl) to an HPLC conical vial .Approximately 6 cm from the upper half of the stalk were sampled from 15-30 plants of the same treatment and pooled in a single sample. Material was shock-frozen in liquid nitrogen directly after harvest, subsequently lyophilized and ground to a fine powder in a ball mill. An aliquot of 50-100 mg (dry weight) was placed in 2.0 ml tubes and used for phytohormone extraction according to a modified protocol. One ml -1. Chromatography was performed on a Kinetex® C18 (100 mm × 2.10 mm with 2.6 μm particle size) column equipped with a C18 guard column purchased from Phenomex Inc. . The following programme was used for elution: 80% solvent A (water:acetonitrile 95:5) containing 7 mM CH3COOH and 20% solvent B (methanol containing 7 mM acetic acid) for 40 s; ramp to 98% B in 50 s; hold for 2 min 20 s followed by re-equilibration to 20% B.Extracted samples (10 μl) were injected into the HPLC and eluted at 40°C at a flow rate of 0.2 ml/minPhytohormones were detected in multiple reaction monitoring mode (MRM) in a triple quadrupole mass spectrometer (LC12000) equipped with an electrospray interface using settings described in. The fol-5. MapQTL® 6[Linkage groups were determined and allele frequencies were tested for segregation distortion with JoinMap. Map conapQTL® 6 was alsoapQTL® 6.All inoculation experiments were performed with batches of at least 15 plants per genotype and treatment. Most inoculation experiments were performed on 6 to 10 batch replicates. Some parameters, such as phytohormone contents, the performance parameters or the percentage of colonized shoot segments, were calculated on a batch basis, whereas some of the chlorosis parameters were calculated on a single-plant basis. Only one batch of 15-30 plants was inoculated per genotype to allow testing of 96 genotypes at the same time in the RIL inoculation experiments. The whole experiment was repeated to confirm the results. See Table er differed in important resistance traits against V. longisporum  RIL population. A genetic map was calculated and a physical map was produced using known positions of each marker on the Arabidopsis Genome Initiative (AGI) reference map[QTL controlling ence map (AdditioVerticilliumcolonization (vec) were detected on chromosomes 2 and 4, QTL for stuntingresistance (stre and r-stre) on chromosomes 1, 2 and 4, and QTL for resistance against V. longisporum-induced chlorosis(r-chl) also on chromosomes 1, 2 and 4. QTL were named according to the nomenclature used previously[QTL were detected for all three resistance traits Figure . QTL coneviously. QTL excvec QTL controlling systemic colonization consisted of arrays containing several LRS peaks depending on the parameter and the inoculation experiment  to a fragment between markers EH2-6, BLC2-2 and erecta, spanning approximately 1.6 cM.All  Figure . The map Figure . vec1 exstunting resistance could only be detected with the parameter “performance height” and not with “performance fresh weight”. Both QTL on chromosome 2 were detected in both inoculation experiments; the QTL on chromosomes 1 and 4 were only detected in experiment 1, which was performed in the winter. stre2 co-localised with marker BLC1-26, which is located in the Wakl9 gene, and explained up to 22.9% of the trait variance. stre1, explaining 16.8% variance, was also located in the vicinity of a Wakl gene (Wakl22/rfo1). r-stre1 near marker erecta on chromosome 2 explained approximately 30% of the trait variance in experiment 1  were only detected in experiment 1  RIL population.Epistatic interactions between marker loci could not be reproduced between the tests. Regarding the colonization data determined by the plating assay in experiment 1, a significant interaction between marker loci BLC2-7 on chromosome 2 and nga1111 on chromosome 4 was detected. Both loci lie within the confidence intervals of r-stre1 co-localised with the morphological marker erecta. Erecta encodes for a receptor-like kinase with many functions in plant development and also in QDR[er is disrupted[Erecta allele. Inoculation experiments were performed on erecta mutants and their respective WT ecotypes having a functional Erecta allele to investigate whether a functional Erecta pathway increases resistance. Ler was compared with the La-0 ecotype. Col-0 was compared with erecta mutants in the Col-0 background. The agb1-1 mutant in the Col-0 background that is defective in the β-subunit of heteromeric G-protein was included representing another component of the Erecta signalling pathway[erecta mutants and the agb1-1 mutant were significantly more stunted under V. longisporum challenge than their respective WT lines: The parameters “performance height” and “performance fresh weight” were decreased in all mutants compared to WT  was localized in the Erecta region, it was investigated whether loss of Erecta function influences chlorosis. The level of chlorosis in Col-0 was higher than in the erecta mutants with Col-0 background .It is hypothesized that SA induction and low light caused er differed in their ABA and JA responses to V. longisporum: ABA contents were much higher in Ler than in Bur upon infection  and 4 (r-stre3) depended on the season: They protected against stunting only during winter, which is consistent with prior observations for stre1 and stre2[A. thaliana was prolonged during winter (experiment 1) and developmental differences were more pronounced. Differences in light intensity and/or quality due to more natural illumination during summer are likely to cause these reactions. The strongest QTL on chromosome 1, stre2, co-localised with the Wakl9 gene, encoding a wall-associated kinase (WAK)-like protein[stre1 at the bottom of chromosome 1 is Rfo1 (Wakl22), encoding another WAK-like protein which has already been shown to play a role in V. longisporum resistance[V. longisporum interacts with the host plant primarily in the apoplast[Wak genes have been shown to be induced by SA[Wakl genes has not yet been shown, but it would correspond well with the early and strong SA induction detected in Bur. Increased SA levels have been observed to cause stunted growth also without disease[Verticillium-induced stunting mediated by the stre1 and stre2 alleles of Bur origin during winter. High levels of SA and its glucoside were also found in the xylem sap of V. longisporum-infected B. napus and were correlated to the degree of stunting and the amount of pathogen DNA[V. longisporum occurring already at the rosette stage, but is never seen in infected field crops. Light intensity and quality is a major difference between field and greenhouse. This suggests that V. longisporum-induced stunting under experimental conditions also depends to a certain extent on the combination of high SA levels with artificial lighting.The stunting resistance QTL on chromosomes 1  complicates an interpretation of these results.The QTL controlling chlorosis seemed to be less affected by genes controlling development. The opposite has been reported in the literature: A QTL dErecta has been shown to underlie the stunting resistance QTL r-stre1 on chromosome 2, which explained a large part of the trait variation observed. Receptor-like kinases (RLKs) are often involved in controlling developmental processes or mediating disease resistance reactions[Verticillium resistance gene identified so far, Ve1, also belongs to the RLK family[A. thaliana[erecta mutants show altered organ development resulting in compact growth[Erecta. Additionally, several resistance traits are reported to be controlled by Erecta in different pathosystems: Growth of the bacterial pathogen Ralstonia solanacearum was inhibited and wilt symptoms were reduced in A. thaliana plants with a functional Erecta gene compared to erecta mutants[Plectosphaerella cucumerina resulted in more chlorosis and necrosis in erecta mutants compared to the respective WT accessions[erecta mutants, leading to enlarged leaf lesions, has also been reported for infection with the oomycete Pythium irregulare[Agb1 are morphologically distinct from erecta mutants[agb1-1 mutants were at least as susceptible to V. longisporum-induced stunting as the erecta mutants. This supports the view that a functional ERECTA pathway mitigates the stunting effect of V. longisporum infection independently of its effect on morphology.In the present study, eactions. The onlK family,58 and hK family. The leu thaliana. erecta t growth,60. Cellt growth and stomt growth were als mutants. Infecticessions. Enhancerregulare. These r mutants, but defErecta gene in La-0 compared to Ler. This is strong evidence that Erecta, although it is located in vec1, is not involved in mediating resistance to systemic colonization by V. longisporum. A tendency towards stronger colonization of erecta mutants in the moderately susceptible Col-0 background compared to WT was never significant, and is interpreted as an indirect effect of the reduced plant height caused by erecta. V. longisporum may reach the apex of a shorter shoot more easily. This view is corroborated by the fact that the long stalks of the agb1-1 mutant were very poorly colonized, thus behaving very differently from the erecta mutants, despite the fact that both AGB1 and ERECTA act in the same signalling pathway.Resistance to systemic colonization was not enhanced by a functional Erecta has been proven to be a source of natural genetic variation in quantitative resistance not only to V. longisporum. Not much is known so far about the allelic variation of Erecta beyond the level of complete loss of function and its consequences for disease resistance in natural accessions. A comprehensive analysis of Erecta, its structural and regulatory variability and its homologues in cruciferous crop plants would, therefore, be desirable.Altogether, V. longisporum produces ABA itself, which could explain the high levels in heavily colonized plants. In B. napus, however, V. longisporum infection did not increase ABA levels in xylem sap[B. napus were taken at an earlier developmental stage. The results in B. napus make it more likely that different ABA levels depended on the host genotypes instead of fungal biomass.ABA and JA contents differed in a genotype-specific way that correlated with the rates of fungal colonization. It is not known whether ylem sap. The samV. longisporum resistance is complex. The ABA-deficient aba2-1 mutant was highly susceptible to V. longisporum-induced stunting, but other ABA signalling mutants were unaffected, indicating that a specific function of ABA2 was involved in the response observed that did not require ABA in general[Verticillium disease. ABA has been shown to influence plant defence reactions in various ways[V. longisporum on B. napus in the field is the induction of premature ripening, which can be recognized by chlorotic stems and which is leading to reduced seed size. Increased levels of ABA might support the deviation of mobilised nutrients to foster the growth of fungal biomass. Furthermore, it is possible that clogged vessels after V. longisporum-infection induce increased ABA levels as a result of drought stress. However, it has recently been shown that V. longisporum infection can even increase drought tolerance of the host as a consequence of de-novo xylem formation[The role of ABA signalling in  general. From thous ways. In mostous ways and/or aous ways. Howeverous ways. ABA hasous ways. ABA plaous ways. A majorormation. StudyinV. longisporum in Bur and the NIL that contained Bur alleles in the region of vec1. Plant-pathogenic Verticillium species are regarded as hemibiotrophs with a necrotrophic phase during the late stages of infection. Defence responses against necrotrophic pathogens are often induced by JA[Verticillium dahliae than WT plants[A. thaliana leaf senescence[V. longisporum possibly benefits from senescence processes induced by JA and ABA, which would suggest a stimulation of hormone production by the fungus. Alterations of senescence-like processes were also postulated to underlie increased resistance of the A. thaliana JA-receptor mutant coi1 against V. longisporum colonization[Coi1 has been shown to be JA-independent. These results confirm that fine-tuning of a resistance reaction by cross-talk of phytohormone signalling pathways is highly individual for each pathosystem[JA contents decreased after infection with ed by JA. AccordiT plants. In the nescence, V. longnization; howeverhosystem. In addiV. longisporum in natural A. thaliana accessions. ERECTA, SA, ABA, and JA signalling has been shown to mediate an ecotype-specific response of A. thaliana to V. longisporum infection. The ecotype-specific differences for ABA and JA contents were mediated by the same genomic region on chromosome 2 that also controlled systemic colonization by V. longisporum. This region contains the major QTL controlling systemic colonization, vec1, and also Erecta. As colonization resistance was shown to be independent of Erecta, this region should contain other gene(s) that control the degree of fungal colonization. This type of resistance is likely to depend on ABA and JA signalling, as both NILs differed for these hormone contents in the same way as the parental lines that contributed the respective alleles in the polymorphic region. Ongoing studies on differential gene expression depending on vec1 should allow one to draw a more comprehensive picture of the defence reactions leading to colonization resistance. Furthermore, cloning of a gene that controls this reaction should be feasible by combining map-based confinement of the QTL region with expression analysis. It was demonstrated that different resistance traits were controlled independently on a physiological basis, but were still genetically linked. Understanding the biological basis of phenotypic variation in A. thaliana with respect to V. longisporum resistance will provide new approaches for implementing durable resistance in cruciferous crops.Phytohormone signalling processes have been demonstrated to be subject to allelic variation and underlie QDR against The authors declare that there are no competing interests.EH planned all the plant experiments, supervised experiment 3, carried out all other plant experiments, analysed the data, and drafted the manuscript. PK provided the qPCR data on fungal biomass and helped to draft the manuscript. RS provided all data on phytohormone contents and helped to draft the manuscript. AT carried out experiment 3, collected the data and helped with their analysis. ED conceived of the study, participated in its design and coordination, participated in QTL analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.Marker information. Lists all markers that have been used for genotyping the (Bur×Ler) RIL population. It consists of two tables. Table S1 is an overview of all markers used, their type, chromosomal location, and their source. In Table S2, the technical information of all new markers is listed. This includes primer information, melting temperature of primers, information on the polymorphism, and the PCR protocol.Click here for fileGenetic and physical map. Shows the genetic map and the physical map of the (Bur×Ler) RIL population. The genetic map shows marker distances for all chromosomes in cM, the physical map shows the location of markers in kilobases (kb) according to the AGI map[ AGI map.Click here for fileQTL information. Lists information for all QTL detected in the present study with 94 (Bur×Ler) RILs in two separate experiments. QTL for the traits systemic colonization, stunting resistance and resistance to chlorosis detected with different parameters are listed in a table. The chromosomal location, the LRS peak position(s), the LOD score, the additive component, the explained trait variance, and allelic means are given for each QTL.Click here for fileFrequency distribution of phenotypic data. Shows the frequency distribution histograms for the phenotypic data used in QTL mapping. Histograms are shown for the parameters “% colonized shoot segments”, “pg Verticillium DNA/mg fresh weight”, “performance height”, “Mean number of yellow leaves in V. longisporum-inoculated plants”, “Mean number of yellow leaves related to total rosette leaf number”, and “Mean difference in yellow leaves between inoculated and mock-inoculated plants”.Click here for fileSystemic colonization of Erecta signalling mutants and corresponding WT-lines. Contains a bar chart visualizing systemic colonization of erecta mutants, agb1-1 mutant and the corresponding WT genotypes. It provides evidence that Erecta is not involved in mediating resistance to systemic colonization by V. longisporum.Click here for fileSA contents in Bur, Ler and NILs. Contains a bar chart visualizing SA contents in Bur, Ler and two NILs differing in the vec1 region. The data provide evidence that SA hyperinduction, which is characteristic for Bur, is not associated with the vec1 locus.Click here for filePhytohormone contents of erecta mutants, corresponding WT-lines and Bur. Contains three bar charts visualizing SA, ABA and JA contents of Erecta signalling mutants and corresponding WT lines after mock-inoculation and V. longisporum-infection. Additional fileV. longisporum between Bur and Ler are not caused by Erecta.Click here for file


CCN1 plays distinct roles in various tumor types, but little is known regarding the role of CCN1 in leukemia.We analyzed CCN1 protein expression in leukemia cell lines and in AML bone marrow samples. We also evaluated the effects of antibody- or siRNA-mediated inhibition of CCN1 on the growth of two AML cell lines (U937 and Kasumi-1 cells) and on the MEK/ERK pathway, β-catenin and other related genes.U937 and Kasumi-1 cells had markedly higher CCN1 expression than the 5 other leukemia cell lines, and CCN1 protein expression was higher in the AML bone marrow samples than in the normal bone marrow samples. Blocking CCN1 with an antibody in U937 and Kasumi-1 cells suppressed proliferation, increased apoptosis, down-regulated Bcl-xL and c-Myc expression, up-regulated Bax expression, and had no effect on Survivin. siRNA-mediated down-regulation of CCN1 inhibited the proliferation and colony formation of U937 and Kasumi-1 cells and increased cytarabine-induced apoptosis. Furthermore, CCN1 siRNA reduced MEK and ERK phosphorylation without affecting β-catenin; the CCN1 antibody similarly affected MEK and ERK phosphorylation. These changes in phosphorylation could influence the expression of Bcl-xL, c-Myc and Bax in AML cells.The data suggested that CCN1 is a tumor promoter in AML that acts through the MEK/ERK pathway to up-regulate c-Myc and Bcl-xL and to down-regulate Bax. CCN1 , a member of the CCN protein family, is an extracellular matrix-associated protein .CCN1 plays unique roles in different tumor types: it is an oncogenic factor for cancers of the breast , prostatNumerous signaling pathways have been implicated in acute myeloid leukemia (AML), including the Wnt/β-catenin , RAF/MEKHere, we show that CCN1 is expressed in two AML cell lines (U937 and Kasumi-1) and in AML bone marrow samples but not in other leukemia cell lines, such as Jurkat (T-ALL), K562 (CML), CEM (T-ALL) or HL-60 (AML) cells. Antibody-mediated CCN1 inhibition increased the apoptosis of both U937 and Kasumi-1 cells; siRNA-mediated CCN1 down-regulation inhibited U937 and Kasumi-1 cell growth and increased cytarabine-induced apoptosis. We demonstrated that CCN1 influences the MEK/ERK pathway in AML cells, potentially regulating c-Myc, Bcl-xL and Bax. These findings suggest that CCN1 is elevated in AML and promotes survival through the MEK/ERK pathway by up-regulating c-Myc and Bcl-xL and by down-regulating Bax. CCN1 could be a diagnostic marker and a therapeutic target for AML.We analyzed CCN1 expression in seven leukemia cell lines and found the highest levels in two AML lines, U937 and Kasumi-1. CCN1 was undetectable in another AML cell line, HL-60   to further study the role of CCN1 in AML cells.CCN1 siRNA suppressed the proliferation and colony formation of both AML cell lines Figure A-C to a CCN1 regulates β-catenin in esophageal squamous carcinoma and lung cancer cells ,25]. We. We25]. Previous studies have shown that CCN1 activates ERK in ovarian carcinoma cells  and osteOur results showed that CCN1 was overexpressed in two AML cell lines (U937 and Kasumi-1) and in AML bone marrow samples. However, CCN1 was undetectable in certain AML samples and in one AML cell line (HL-60 cells). These data suggested that CCN1 is not the only factor necessary for AML cell survival. CCN1 may be a tumor promoting factor in AML and could potentially be a diagnostic marker for AML. Our data also showed that CCN1 acts through the MEK/ERK pathway, not the β-catenin/Survivin pathway Figure D.CCN1 has distinct effects on apoptosis in different cells. CCN1 enhances the Fas-mediated apoptosis of skin fibroblasts  and the Previous studies have shown that c-Myc is overexpressed  and funcβ-catenin, which is overexpressed in AML, is required for the self-renewal of AML stem cells -43], an, an43], As mentioned in the introduction, ERK is activated in AML and is a potential target for AML therapy. ERK is regulated by CCN1 in breast cancer and during osteoblast differentiation ,32]. We. We32]. We demonstrated that CCN1 is up-regulated in AML bone marrow samples and that CCN1 promotes AML cell growth. These functions of CCN1 involve the activation of the MEK/ERK pathway, but not the β-catenin/Survivin pathway, with the consequent up-regulation of c-Myc and Bcl-xL and down-regulation of Bax. Our study furthers our understanding of the role of CCN1 in AML and provides a potential alternative therapeutic target and/or diagnostic marker for AML.The U937 and Kasumi-1 AML cell lines, the Jurkat and CEM T-ALL cell lines, and the K562 CML cell line were maintained in RPMI-1640 medium (Hyclone) with 10% fetal bovine serum . The KU812 CML cell line and the HL-60 AML cell line were maintained in IMDM medium (Hyclone) with 20% FBS, and HEK293 cells were maintained in DMEM (Hyclone) with 10% FBS.Bone marrow samples from sixteen newly diagnosed and untreated AML patients and from 8 healthy donors were obtained from Chongqing Xinqiao Hospital. The patients and healthy donors provided informed consent, and the study was approved by the Ethical Committee of Third Military Medical University. Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque .The ERK inhibitor PD98059 (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich). Cytarabine was purchased from Sigma-Aldrich.The CCN1 siRNA targeted the sequence 5′-GGGAAAGTTTCCAGCCCAA-3′, and the control sequence did not target any genes. The sequences were cloned into the pSEB-hus plasmid, and this plasmid was transfected into HEK293 cells along with the pCL-Ampho plasmid to package retrovirus. Retroviral supernatants were used to infect U937 and Kasumi-1 cells. The infected cells were maintained under selection using Blasticidin S . The cells infected with CCN1 siRNA were termed U937/siCCN1 or Kasumi-1/siCCN1; the control cells were denoted U937/siControl or Kasumi-1/siControl.Apoptosis assay: Cells were washed 3 times with ice-cold PBS and stained with Annexin V-APC  and propidium iodide . Apoptotic cells were identified by flow cytometry (BD Biosciences) based on positive staining for Annexin V-APC and PI negativity.Proliferation assay: Ten microliters of CCK-8 reagent  was added to the wells of 96-well plates in which cells were cultured (100 μl/well). After a 90-minute incubation at 37°C, the OD values were measured using a Bio-Rad Microplate Reader.3) were plated in 6-well plates in a methylcellulose (Sigma-Aldrich) semi-solid medium . After seven days, colonies containing more than 40 cells were counted.Cells , and an anti-Histone H1 antibody (Santa Cruz Biotechnology) was used as the loading control.The experiments were conducted three times, and the data are presented as the means ± SD. Significance was determined by Student’s t-test using SPSS software. p < 0.05 was considered to be statistically significant.The authors declare that they have no competing interests.Chang-Chun Niu, Wei-Ke Si: the conception and design of the study; Chang-Chun Niu, Chen Zhao, Xiao-Li Zhang, Jing Pan, Chen Zhao, Jing Pan: acquisition of data, or analysis and interpretation of data; Chang-Chun Niu, Zhong Yang, Wei-Ke Si : drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript.


Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca2+ uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn2+ to the cells.Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca Saccharomyces cerevisiae, large increases in the cytosolic calcium concentration have been observed in response to hypo- or hypertonic shock, sugar refeeding, mating pheromone α factor, or Ca2+-depletion of the secretory pathway2+ influx system2+ influx system (HACS), composed of three interacting proteins, Cch1p, Mid1p, and Ecm72+ channel Yvc1p2+ out of the cytosol.As a closed compartment, the cell must adapt to environmental changes and has therefore developed intracellular signaling systems that can identify these stimuli and implement cellular responses to counteract the stress. In eukaryotic cells, calcium ions play a key role in the transduction of external signals into the cytosol. Upon stimulation, the signal is generated by a sudden, transient, and massive calcium influx into the cytosol from the external medium and/or internal stores. In the yeast 2+-binding proteins, of which calmodulin (CaM) is the best studied. The Ca2+-CaM complex activates several Ca2+-responsive signaling pathways, including the serine/threonine protein phosphatase calcineurin pathway. Calcineurin inhibits the vacuolar Ca2+/H+ exchanger Vcx1p through a, as yet, poorly understood post-translational regulatory mechanismPMR1 and PMC1 via dephosphorylation of the transcription factor Crz1p and its subsequent mobilization into the nucleusPMR1 encodes a high affinity, low capacity, P-type Ca2+/Mn2+-ATPase primarily required for maintaining a suitable calcium concentration in the Golgi apparatus (around 200 μM) and, indirectly, in the endoplasmic reticulum (ER) (around 10 μM)82+-ATPase Pmc1p, Pmr1p also plays a crucial role in detoxifying the cytosol when high calcium concentrations are encountered in the environment, allowing the maintenance of low [Ca2+]cyt levelspmr1 and pmc1 mutants therefore show increased sensitivity to high external Ca2+ Accumulation of calcium in the cytosol is directly sensed by different Ca) around  μM82+-ATet al.2+/H+ exchanger that, in the absence of VCX1, is activated upon Ca2+ stress . Other well-known examples are the putative transporters X and M responsible for the influx of external Ca2+ through the plasma membrane which have not yet been identifiedAlthough the calcium transport system has been intensively studied, the molecular identity of some transporters remains unknown. For instance, Miseta 2+/cation antiporter in yeast2+ (CaCA) exchanger superfamily142+/Mn2+-ATPase Pmr1p, Gdt1p is involved in tolerance to high external Ca2+ concentrations. We previously showed that a strain lacking either of these transporters is sensitive to an increase in the external Ca2+ and that this sensitivity is increased by the loss of both transporters2+ stress tolerance by two distinct pathways and that one pathway can compensate for the absence of the other. Interestingly, the Ca2+ sensitivity of the gdt1 mutant was subsequently shown to be suppressed by the expression of bacterial orthologs or the human ortholog TMEM165132+/cation antiporters regulating Ca2+ homeostasis132+ concentration in organelles involved in the secretory pathway.We recently suggested that Gdt1p is a novel putative Golgi-localized Cain vivo transport assay in Lactococcus lactis cells expressing Gdt1p, we observed that Gdt1p promoted Ca2+ influx into the cytosol. Interestingly, Ca2+ influx was enhanced as the external pH increased, suggesting that Gdt1p couples calcium transport to proton transport and probably acts as a Ca2+/H+ antiporter. Furthermore, we showed in yeast that Gdt1p is involved in the Ca2+ response to environmental osmotic stress when Pmr1p, the major Ca2+ pump under normal conditions, is absent. The amplitude of the Ca2+ response was also found to increase with an increase in the cellular calcium stores. Importantly, we also showed that GDT1 is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p, probably by maintaining an appropriate Ca2+ concentration in organelles involved in protein glycosylation. Strikingly we found that this defect was restored by the addition of Mn2+ in the external medium.In this report, we present direct evidence that the budding yeast family member Gdt1p transports calcium. Using an 2+ transport, we developed an in vivo functional transport assay based on the heterologous expression of Gdt1p in L. lactis, an organism that has been shown to be a valuable host for expressing eukaryotic membrane proteins17L. lactis. As shown in Δ23GDT1, a tagged version of Gdt1p lacking the first 23 amino acids predicted to be a signal peptide, could be expressed under the control of the nisin-inducible promoter in the wild type (WT) NZ9000 strain of L. lactis. We previously observed that the signal peptide of Gdt1p is not essential for the function of the protein and that this tagged version of Gdt1p is functional in yeast (data not shown). Nisin concentration and induction time were optimized to obtain the highest yield of Gdt1p  in L. lactis DML1 cells expressing GDT1 or containing the empty vector. As shown in 2 to DML1 cells expressing GDT1 resulted in a marked increase in the [Ca2+]cyt, whereas no increase was seen in control (C) cells lacking GDT1. Moreover, we observed that the [Ca2+]cyt increased as the extracellular Ca2+ concentration was increased from 0.1 to 2 mM (GDT1 (data not shown). These results demonstrate that Gdt1p mediates Ca2+ transport across the plasma membrane when expressed in L. lactis. As mentioned above, Gdt1p shows striking similarities to members of the CaCA exchanger superfamily which transport Ca2+ across membranes against their electrochemical gradient by utilizing the downhill gradient of other cations, such as H+ or Na+ 2+/H+ antiporters, using the proton gradient as the driving force for Ca2+ influx across the Golgi membrane. To test this hypothesis, Fura-2-loaded L. lactis cells expressing tagged-Δ23Gdt1p were resuspended in assay buffer at different pH values (7.0 and 8.0) and Ca2+ accumulation was measured after addition of 0.5 mM CaCl2. As shown in 2+ influx activity that was dependent on the extracellular pH, with Ca2+ transport increasing as the external pH was increased from 7.0 to 8.0. This could be explained by the fact that proton extrusion from the cell is energetically more favorable at a higher pH and Gdt1p can use this energetically favorable condition to couple Ca2+ influx to the H+ efflux. No marked difference in the cell density of the Fura-2-loaded L. lactis cells was seen before and after CaCl2 addition, indicating that CaCl2 addition did not cause cell lysis, regardless of the external pH . Loss of the Golgi Ca2+-ATPase Pmr1p is known to induce Ca2+-depletion in the secretory compartments that is compensated by a higher Cch1p/Mid1p-mediated Ca2+ influx and a net increase in the resting [Ca2+]cyt is observed in cells grown under normal conditionspmr1Δ cells led to a higher calcium peak than in WT cells, suggesting either a higher rate of Ca2+ influx into the cytosol or a reduced reabsorption of Ca2+ into the organelles. We then tested whether Gdt1p was involved in the Ca2+ response in the absence of PMR1 by monitoring the [Ca2+]cyt in pmr1Δ cells lacking or overexpressing GDT1. The relative amount of Gdt1p detected in WT, pmr1Δ and pmr1Δ + GDT1 strains is shown in pmr1Δ cells compared to WT cells while overexpression of GDT1 in pmr1Δ cells increases its levelGDT1 in pmr1Δ cells led to a smaller increase in the [Ca2+]cyt than in pmr1Δ cells, while its overexpression (indicated as “ +GDT1”) resulted in a greater increase than in pmr1Δ cells. Similar calcium responses were observed after exposure of the cells to sorbitol  was assessed in the WT and the 3 M NaCl sorbitol . TogethePMR1-deleted strains . This result confirms the findings of Marchi et al.PMC1 gene due to the loss of PMR1. This higher level of PMC1 expression in pmr1Δ cells was unaffected by the level of expression of GDT1 (lanes 6–8). Finally, Vcx1p levels were higher in the gdt1Δ cells (lane 4) than in the WT cells (lane 1) and were further increased in pmc1Δ cells (lane 5) and pmr1Δ cells (lane 6). Note that, in the pmr1Δ strain, Vcx1p levels were further increased after deletion of GDT1 (lane 7) and decreased when GDT1 was overexpressed (lane 8). These results rule out an indirect effect of Yvc1p, Pmr1p, and Pmc1p levels on the Gdt1p-dependent modulation of the Ca2+ response to osmotic stress, as their levels were not modified by deletion or overexpression of GDT1. In addition, the observed changes in Vcx1p levels cannot be linked to the Ca2+ responses, as, regardless of its level of expression in the pmr1Δ strains, Vcx1p should be inhibited as a result of calcineurin activationPMR1-deleted strains is suggested by the overexpression of PMC1  when treated with FK506, an inhibitor of calcineurin  to measure Ca2+ levels in the WT and the four mutants that we used in the aequorin-based assay. As shown in 2+ content between the WT and the gdt1Δ strain, whereas, in line with previous findings2+ content of the pmr1Δ strain was significantly higher than that in the WT . In a pmr1Δ mutant, Ca2+ depletion of the secretory pathway occurs and is compensated by entry of calcium into the cell. As a result, the [Ca2+]cyt increases, resulting in activation of the calcineurin signaling pathway, leading to overexpression of Pmc1p and, therefore, higher sequestration of Ca2+ into the vacuole2+ concentration in the pmr1Δ mutant was dependent on GDT1 expression, showing a decrease to 8.5 mmole/kg dry weight when GDT1 was deleted and an increase to 13.2 mmole/kg dry weight when Gdt1p was overproduced. These results demonstrate that Gdt1p controls Ca2+ stores in yeast. As the yeast vacuole accumulates over 95% of the total cellular Ca2+ 2+ content in pmr1Δ mutants expressing different levels of GDT1 might explain the Ca2+ responses to osmotic shock as a result of modification of the amount of Ca2+ released through the vacuolar calcium channel Yvc1p.To assess the effect of Gdt1p on the total Ca2+ concentration is essential for the activity of many ER- and Golgi-resident enzymes involved in membrane trafficking and protein folding and glycosylation825Maintenance of a suitable intraluminal CaGDT1 and/or PMR1 grown either in YD medium alone or in YD medium containing 500 mM Ca2+. As shown in pmr1Δ mutant grown in YD medium migrated more rapidly on SDS gels (lane 3) than those in WT lysates (lane 1), but this difference was not seen when CaCl2 was added to the growth medium (lane 11) , but did in Ca2+-supplemented YD medium (lane 10). It is likely that these smaller mature forms reflect defects in glycosylation probably resulting from deregulation of the Ca2+ concentration in the Golgi caused by GDT1 deletion and a high external Ca2+ concentration. As shown in gdt1Δ cells grown in presence of smaller Ca2+ concentrations (50 mM) and appeared to increase when the Ca2+ concentration is rising. The size differences observed for the double deletant gdt1Δ/pmr1Δ arise from impaired glycosylation in YD medium due to the absence of PMR1 (lane 4) and in Ca2+-containing medium due to the absence of GDT1 (lane 12). Note that the mature form of Gas1p in the gdt1Δ/pmr1Δ mutant is even smaller (lane12), suggesting that both N- and O- glycosylations are impaired.In order to assess the involvement of Gdt1p in protein glycosylation, we used Western blotting to examine glycosylation of CPY and Gas1p in the WT and mutants deleted for lane 11) . This is2+ (lanes 13–16). These results confirm that N-linked glycosylation of CPY was impaired in pmr1Δ grown in YD medium and highlight the requirement for Gdt1p in order for N-glycosylation to take place in the presence of high calcium. As shown in pmr1Δ mutant than in the WT and gdt1Δ mutant in YD medium (lane 7) and in the gdt1Δ mutant than in the other two strains in Ca2+-supplemented YD medium (lane 14). Note that, depending on the growth medium, after Endo H digestion, the gdt1Δ/pmr1Δ strain showed a phenotype comparable to either the pmr1Δ strain (lane 8) or the gdt1Δ strain (lane 16). As Gas1p is subjected to both N-linked and O-linked glycosylation2+ is a new observation.To analyze whether these mobility shifts were due to alterations in the N-glycosylation, the proteins were treated with endoglycosidase H (Endo H) before Western blotting. As shown in Thus, our results demonstrate, for the first time, that Gdt1p is required for both N-linked and O-linked protein glycosylation at a high external calcium concentration and are consistent with the fact that mutation in the human ortholog, TMEM165, is linked to a genetic disease  that is caused by defects in glycosylation2+ is an important cation required as cofactor for many glycosyltransferases and glycosidases involved in glycosylation292+ alone (Mn2+) or combined with 500 mM Ca2+ (Ca2+Mn2+). A similar glycosylation profile to that found in cells grown in YD medium was observed for CPY and Gas1p in presence of Mn2+ (2+ in the growth medium did not affect the glycosylation in the wild type (lane 17) and gdt1Δ strain (lane 18) and did not restore N-linked and O-linked glycosylation defects in pmr1Δ (lane 19) and gdt1Δ/pmr1Δ (lane 20) since changes in mobility were still observable. Note that the mobility shift observed for CPY seems less important in presence of Mn2+ compared to YD suggesting a possible partial restoration of the N-glycosylation. Strikingly no glycosylation defect could be observed in Ca2+Mn2+ medium (2+ to Ca2+-containing medium restores the glycosylation defects of gdt1Δ (lane 26) and gdt1Δ/pmr1Δ (lane 28) strains. Interestingly, no mobility shift could be observed after removal of N-glycans by Endo H treatment (lanes 29–32), confirming that the addition of Mn2+ to Ca2+-containing medium restores both N- and O-glycosylation pathways for Gas1p in the gdt1Δ mutants.Mn of Mn2+ . Additio+ medium , highlig2+ transport system involving members of the highly-conserved UPF0016 family and showed that the yeast protein Gdt1 and its human ortholog, TMEM165, are functionally related and involved in Ca2+ and pH homeostasis2+ transport across membranes. When direct transport assays were carried out in L. lactis, Gdt1p-dependent Ca2+ influx through the plasma membrane was observed upon addition of calcium to the external medium  concentration in the organelles involved in the secretory pathway. Secondly, it is responsible for the detoxification of the cytosol when the external Ca2+ concentration rises. The pmr1Δ mutant shows a growth defect on medium containing a low (~ 3 μM) or high (~400 mM) Ca2+ concentration reflecting, respectively, Ca2+ starvation of the secretory pathway organelles or toxic accumulation of Ca2+ in the cytosol9PMR1 deletion induces depletion of the secretory Ca2+ pools. Then the cell integrity-related mitogen-activated protein kinase Slt2p becomes activated and stimulates Ca2+ influx by activating the high-affinity Ca2+ uptake system (HACS) in order to refill Ca2+ stores in organelles involved in the secretory pathway2+ influx is increased in a pmr1Δ mutant, the rate of Ca2+ efflux is unaffected, leading to an elevated [Ca2+]cyt2+/calcineurin-dependent pathway2+ uptake via the HACS channel by a negative feedback mechanism involving direct dephosphorylation of Cch1pPMC1 expression via Crz1p2+ uptake into the vacuole via Pmc1p is then increased, which results in higher cellular calcium storespmr1Δ mutant are consistent with those described in the literature. In the absence of PMR1, we observed an increase in the resting [Ca2+]cyt  and an inverse correlation was found between Vcx1p levels and the Ca2+ response. This observation is compatible with the proposed role of Vcx1p in Ca2+ reabsorption after a transient increase in its cytosolic concentrationpmr1Δ strains, the calcineurin pathway is activated and Vcx1p should be inhibitedWe also investigated the involvement of Gdt1p in Cae stress  or osmote stress  and on t+ stocks . When th2+ homeostasis. One important question that requires answering is the direction of Ca2+ transport. From a thermodynamic point of view, both directions can be considered. In the hypothesis of a Ca2+/H+ exchange, and applying the Gibbs equation to the concentration values reported in literature, the transport of Ca2+ against its gradient (from the cytosol to the Golgi apparatus) would be thermodynamically feasible in exchange for 3 H+. On the other hand, Gdt1p could acidify the Golgi apparatus by transporting H+ against its gradient in a stoichiometry 1:1 (1 H+ for 1 Ca2+) or 2:1 (2 H+ for 1 Ca2+). To date, our current data do not provide the answer, but this point could be resolved, for instance, if an adapted Golgi-localized luminal calcium sensor could be engineered. Note that we cannot exclude the possibility that Gdt1p transport would be reversible, adapting the direction of transport according to conditions.Together, our data clearly demonstrate that Gdt1p plays an important role in Ca2+-containing medium, we previously demonstrated that the human ortholog TMEM165 is able to restore the growth defect observed in gdt1Δ on Ca2+-containing mediumet al.2+ concentration, Gdt1p was required for N-linked and O-linked protein glycosylation in yeast. Interestingly, Gdt1p and Pmr1p affected glycosylation in different ways, as we showed that CPY and Gas1p glycosylation defects occurred in the pmr1Δ strain in YD medium and were overcome by addition of Ca2+, whereas impaired glycosylation in the gdt1Δ strain was only observed in the presence of added Ca2+  may be explained by two ways. First, if we consider that Ca2+ concentration is too high in gdt1Δ mutant, Mn2+ could compete with Ca2+ to enter into the Golgi apparatus via the Mn2+/Ca2+-ATPase Pmr1p and therefore reduce the total intraluminal Ca2+ content. Alternatively, Ca2+ could compete with Mn2+ ions within the ER/Golgi lumen and alter glycosylation process. In this case, adding back more Mn2+ could restore the defect. Measuring the ions concentration in the organelle lumen would help to answer this question.Using a complementation assay on Caded Ca2+ . GlycosyIn conclusion, we have demonstrated that Gdt1p is a calcium transporter localized in the Golgi apparatus and plays a crucial role in calcium homeostasis and protein glycosylation. Our results provide new insights into the molecular causes of the defect in glycosylation described in TMEM165-deficient patients.Saccharomyces cerevisiae strains used are listed in Lactococcus lactis NZ9000 wild type strain and its derivative, the evolved DML1 strain, were kindly provided by B. Poolman 600 ~0.4–0.5) and harvesting the cells 3 hours later.The GDT1, was obtained previously and has been described by Demaegd et al.et al.L. lactis, we used the pNZ8048 plasmid expressing a tagged version of GDT1 lacking the 23 first amino acids corresponding to the predicted signal peptide (pNZ8048-10His-strep-TEV-Δ23GDT1) under the control of the nisin-inducible promoter. This plasmid was constructed as follows. The yeast pRS416 vector containing the sequence coding for the 10His-strep-TEV tagged Δ23GDT1 was used as the DNA template for PCR amplification, then the amplified PCR products were digested with Pst1/SacI and inserted into the pNZ8048 vector carrying the nisin-inducible promoter. Bacterial transformations were performed by electrotransformation (Bio-Rad Laboratories) as described previously by Holo et al.Yeast and bacterial plasmids were obtained following standard molecular biology protocols, and the authenticity of all genetic constructs was validated by sequencing. pRS416-pTPI-GDT1, the yeast plasmid overexpressing L. lactis as described above, then the cells were harvested  and washed once with washing buffer , and centrifuged as above, then the pellet was resuspended in one volume of ice-cold lysis buffer [50 mM Tris/HCl pH 7.6, 500 mM NaCl, 10% glycerol, 1 mM Tris(2-carboxyethyl) phosphine (TCEP), 1 mM PMSF, 2 mg/ml lysozyme, and protease inhibitor cocktail . After 30 min incubation at 28 °C, the cells were lysed with glass beads (cell pellet/ice-cold lysis buffer/glass beads at a weight ratio of 1:1:1) using a Precellys cell disrupter (Bertin Technologies) at 5,000 RPM for 5 × 30 sec. Cells debris were removed by centrifugation at 1,700 g for 12 min at 4 °C and the supernatant centrifuged at 112,000 g for 60 min at 4 °C to pellet the total membrane fraction, which was resuspended in ice-cold resuspension buffer  and stored at −80 °C.Recombinant Gdt1p proteins were expressed in 600 of 1.2 as described previously by Morsomme et al.et al.Yeast total membrane extracts were prepared from 100 ml of culture at an ODL. lactis samples were incubated at 37 °C for 30 min. Membrane proteins were separated on a 10% SDS/PAGE gel and Western blotting performed as described previouslyRoutinely, 15–20 μg of membrane proteins was mixed with 4× concentrated non-reducing sample loading buffer . Yeast samples were used as such, while et al.Samples (17 μg) of total membrane protein were precipitated using chloroform/methanol as described by Wessel 2+ concentrations in L. lactis were measured using the fluorescent calcium dye Fura-2/AM, following the method previously described by Chang et al.L. lactis cells were resuspended in solution A containing a final concentration of 0.1 mM EGTA and 50 mM Tris-HCl  and fluorescence measurements performed on 2 ml aliquots at 25 °C with constant stirring. The intracellular Ca2+ concentration was monitored as the change in the ratio of the fluorescence intensities (510 nm) at the excitation wavelengths of 340 nm and 380 nm using a JASCO FP8500 fluorimeter controlled by Spectra Manager softwareTM. The baseline fluorescence was routinely recorded every 10 sec for 2 min before addition of the indicated concentration of Ca2+. The fluorescence intensity ratio was converted into the Ca2+ concentration using the equation described by Liao et al. (Kd = 315 nM)Intracellular Caet al.600 of 1.2 by adding a final concentration of 1.33 M NaCl or 2.66 M. sorbitol.Aequorin-based experiments were performed as described by Demaegd 600 of 3, then were collected by vacuum filtration using membrane filters  and washed successively with 1 mM ethylene glycol tetraacetic acid disodium salt solution and water. The cells were then resuspended in 10 ml of water and dried in an oven at 70 °C for 24 h, then in a desiccator for 24 h. The dried matter was weighted and mineralized by heating at 500 °C overnight, then the ashed sample was dissolved in 65% HNO3 and used for inductively couple plasma atomic emission spectroscopy (ICP-AES) analysis. Ca2+ measurements were performed using a ICAP 6500 spectrometer (Thermo Scientific) and the cellular Ca2+ concentration calculated based on the dry weight of the samples and the dilution factor.Yeast cultures were grown in 50 ml of synthetic medium at 28 °C to a final ODHow to cite this article: Colinet, A.-S. et al. Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation. Sci. Rep.6, 24282; doi: 10.1038/srep24282 (2016).


We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca2+ responses at fertilisation. To test this hypothesis we performed the first examination of Ca2+ dynamics in eggs from young and naturally-aged mice. Strikingly, we find that Ca2+ stores and resting [Ca2+]i are unchanged with age. Although eggs from aged mice feature a reduced ability to replenish intracellular Ca2+ stores following depletion, this difference had no effect on the duration, number, or amplitude of Ca2+ oscillations following intracytoplasmic sperm injection or expression of phospholipase C zeta. In contrast, we describe a substantial reduction in the frequency and duration of oscillations in aged eggs upon parthenogenetic activation with SrCl2. We conclude that the ability to mount and respond to an appropriate Ca2+ signal at fertilisation is largely unchanged by advancing maternal age, but subtle changes in Ca2+ handling occur that may have more substantial impacts upon commonly used means of parthenogenetic activation.The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca Oocyte aging is a complex multifactorial process resulting in deterioration of oocyte viability with advancing maternal age. Perhaps the best known aspect of oocyte aging is oocyte aneuploidy, in which an age–related increase in chromosome segregation errors during meiosis is associated with a decline in female fertility1252+ concentration ([Ca2+]i) within the egg cytoplasm known as Ca2+ oscillations. Ca2+ oscillations are initiated by a sperm-borne soluble protein, most probably phospholipase C zeta (PLCζ)2+ oscillation is generated by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release from the endoplasmic reticulum (ER), the main intracellular Ca2+ store in the egg, followed by a influx of extracellular Ca2+ to replenish stores in time for the next oscillation2+ oscillations are not only necessary and sufficient for egg activation102+ entry into the egg2+ dynamics is not known.At the time of ovulation mammalian oocytes become arrested at metaphase-II (MII) of meiosis, at which point the oocyte can be referred to as an egg. In mammals, liberation from MII arrest and initiation of the embryonic developmental program, commonly termed egg activation, occurs at fertilisation as a result of a spatiotemporal series of increases in intracellular Ca2+ handling in various somatic cell types with age. This so-called calcium-hypothesis of aging is considered a major mechanism of age-related somatic cell dysfunction, and has particularly been studied with respect to the pathological pathways underlying Alzheimer’s disease14152+ dysregulation with advancing maternal age, and that this would provide a mechanistic explanation for the reduced ability of eggs from older women to resume development after insemination or ICSI. Although some studies show that the latency period between ovulation and fertilisation either in vivo or in vitro, sometimes referred to as “post-ovulatory aging”, perturbs Ca2+ oscillations at fertilisation17182+ response is unknown. Therefore, using live fluorescence imaging, we performed the first examination of Ca2+ dynamics in eggs from young and naturally-aged mice. Perhaps surprisingly we find that, in contrast to somatic cells, [Ca2+]i homeostasis remains relatively stable with advancing age, with naturally-aged eggs capable of mounting and responding to an appropriate Ca2+ signal at fertilisation. Instead, our experiments suggest mammalian eggs are adapted to avoid age-related Ca2+ signalling defects that might jeopardise reproductive capacity.A growing body of evidence, largely from naturally-aged mice, reveal changes in Cain vivo aging, and have been extensively used to identify age-related changes in numerous cell types, including eggs. Here we used CD1 mice at 12–15 months old, which have been well characterised as a model for maternal age-related egg defects, as measured by aneuploidy levels202122Naturally-aged mice are an established model of 2+]i. MII eggs from young and naturally-aged mice were collected, microinjected with Calcium GreenTM-1 dextran and Rhodamine B dextran as a ratiometric imaging system, and imaged contemporaneously in a side-by-side manner using time-lapse epifluorescence microscopy in normal Ca2+ -containing media . The calculated area under the curve for young and aged eggs was 252.8 ± 14.4 and 224.8 ± 16.1 (P = 0.11), respectively, and peak fold-change fluorescence levels of young and aged eggs were 1.69 ± 0.02 and 1.65 ± 0.03 (P = 0.34), respectively, revealing no significant difference in [Ca2+]ER stores with egg age , which triggers an influx of Ca2+ into the egg and is an indication of store refilling  in the first hour (2+ spikes (P = 0.46) in the first two hours, respectively (2+]i between young and aged eggs were 2.19 ± 0.02 and 2.16 ± 0.02 (P = 0.50), respectively  in the first two hours , respectively , respectively, indicating that age does not affect the sensitivity of Ca2+ oscillations initiated by PLCζ expression    . NotablyP = 0.7) , compare 0.0001) . Thus ag2+ transients2+ -induced oscillations observed in aged eggs affected the temporal sequence of egg activation, we recorded the timings of second polar body (Pb2) extrusion and pronucleus (PN) formation in young and aged eggs, and correlated the timing of these events with total oscillation number on an individual egg-by-egg basis  on egg Ca2+ regulation has previously been studied17182+ influx capacity is markedly impaired, the ability to mount a normal sperm- and PLCζ-induced Ca2+ oscillatory signature is largely unchanged. Interestingly, on the other hand, we find that eggs of advanced age elicit an abnormal oscillatory pattern in response to artificial activation with SrCl2. The discussion that follows will therefore first focus on the effect of aging on egg Ca2+ handling, and then comment on the effect of aging upon egg activation in a clinical context.Egg activation failure is a prominent barrier to the success of ICSI in the clinic52+ influx occurs in eggs. Reductions in store-operated Ca2+ entry (SOCE) activity and expression of key SOCE-specific proteins, Stim1 and Orai1, are a characteristic feature of naturally-aged mitotic cells482+ oscillations remain robust and unchanged in eggs from older females in the face of substantially reduced store-operated Ca2+ influx. Moreover no obvious difference in the pacemaker potential was observed, though more detailed analysis of this aspect of the Ca2+ oscillation would benefit from more rapid sampling. Whilst fertilisation in nominally Ca2+ -free media results in oscillation termination2+ is essential for ongoing long-term oscillations, our data suggest that Ca2+ oscillations can persist provided a threshold amount of Ca2+ influx is available. Consistent with this, SOCE inhibition at fertilisation has no effect on the Ca2+ oscillatory response2+ oscillation phenotype of the aged eggs in this study. Nonetheless an unavoidable conclusion of our data is that, in contrast to other cell types in which an age-related dysregulation of Ca2+ homeostasis is common142+ transients is apparently safeguarded. This difference in the pathogenesis of aging between somatic cells and oocytes may be unique to Ca2+ , as aged oocytes feature other hallmarks of somatic cell aging, such an accumulation of reactive oxygen species2+ aging, as a mechanism of avoiding endangering the germline.Whilst it is well established that oocyte aneuploidy is a major consequence of oocyte aging and a leading cause of age-related infertility144462+ oscillations, our data reveal that aged eggs exhibit a substantially different oscillatory response following activation with SrCl2 compared to young. Perhaps surprisingly, given its widespread use in mouse eggs as a parthenogenetic agent, exactly how SrCl2 initiates a sustained train of oscillations remains poorly understood. Having entered the egg, SrCl2 is thought to stimulate oscillations by potentiating the InsP3 receptors (InsP3R)2 entry into mouse oocytes, with TrpV3−/− eggs failing to mount an oscillatory response, presumably as a result of failed access to the InsP3R2-induced oscillations, and for the subtle defects in Ca2+ influx. It is also plausible that defects in downstream signalling such as possible activation of oocyte-resident PLCs2-induced oscillation competence.Whereas we found little effect of natural maternal aging upon sperm- or PLCζ-induced Ca2+ ionophores such as A231872+ that does not reflect the series of Ca2+ oscillations seen at fertilisation, and may be suboptimal for development2+ oscillations in mouse and human eggs similar to that of spermOur results have important implications for our appreciation of the effect of maternal aging on oocyte health in clinical settings. In cases where egg activation fails following ICSI, many clinics attempt artificial egg activation using Ca2+ dynamics are largely maintained with advancing maternal age and therefore do not afford a simple explanation for age-related failed activation following assisted reproductive procedures. One possible alternative is that whilst the gross Ca2+ oscillatory signature is unchanged, the downstream molecular messengers decoding the Ca2+ signal could be compromised. For example, the Ca2+ signal is instrumental in inactivation of cytostatic factor (CSF), the cytoplasmic activity that maintains MII arrest. CSF inactivation occurs via Ca2+ -dependent calmodulin-dependent kinase II activity, and is entirely dependent upon Ca2+ oscillations2+ -sensing appears unlikely, since egg activation was unaffected by maternal age. This was even the case for SrCl2, where oscillation frequency decreased with age, but careful analysis revealed no difference in the temporal dynamics of polar body and pronucleus formation, though we cannot exclude the possibility of more subtle effects of aging on the subsequent kinetics of preimplantation embryonic development. Thus, whilst in our experiments Ca2+ dysregulation does not explain failed activation, the question of what causes egg activation failure following ICSI in maternally aged eggs remains unanswered. The role of paternal age in ICSI failure is often hard to analyse in clinical studies45562+ oscillations2+ dysregulation is minor in a well established model of mammalian oocyte aging, we cannot formally exclude that Ca2+ perturbations may be more severe in human eggs from aged patients in suboptimal culture conditions, where fertilisations are performed at various times after egg collection (post ovulatory aging). Future studies of Ca2+ responsiveness in failed ICSI cases under controlled conditions in the clinic will thus be invaluable.Our data show that egg Ca2+ from the extracellular environment, however this has no effect on overall physiological output of Ca2+ oscillations. We speculate that, unlike some somatic cells, oocytes may have adapted a defence mechanism to prevent Ca2+ dysregulation in the germline, to avoid jeopardising reproductive capacity.In conclusion, our data show advanced maternal age leads to a deterioration in the oocyte’s ability to replenish CaMII eggs were collected from the oviducts of 3 month old (referred to as ‘young’ eggs) and 12–15 month old  female Swiss CD1 mice (Harlan and Charles River Laboratories) following stimulation with pregnant mare’s serum gonadotrophin  and superovulation with human chorionic gonadotropin (hCG)  at 48 hour intervals. Naturally-aged mice were acquired as retired breeders at 7–9 months of age, and housed for a further 5–7 months. Mice were sacrificed 14 hours post-hCG and cumulus masses were released into M2 media containing hyaluronidase (0.3 mg/ml) by rupture of the oviduct with a 27-gauge needle. Cumulus-free eggs were washed through three drops of M2 under mineral oil at 37 °C. All experiments were performed with young and naturally-aged eggs collected contemporaneously. All animal experiments were approved by the Comité Institutionnel de Protection des Animaux du CHUM (CIPA) or the UK Home Office. All animal experiments were performed in accordance with relevant guidelines and regulations of CIPA or the UK Home Office.Eggs were microinjected using Narishige manipulators mounted on a Leica DMI4000 inverted microscope, as described previously2+]i, eggs were co-microinjected with Calcium GreenTM-1 dextran (1 mM) and Rhodamine B dextran (1 mM), or incubated in M2 containing Cal-520 AM (5 μM) for 30 minutes at 37 °C before the experiment. Where necessary, the zona was removed with brief exposure to acidified Tyrode’s solution. Imaging was performed in a glass bottom petri dish heated to 37 °C on a Leica DMI4000 inverted epifluorescence microscope. Images were acquired every 10 seconds and captured for up to 3.5 hours. Ratiometric calculations were performed by dividing Calcium GreenTM-1 dextran values by Rhodamine B after background subtraction. Cal-520 AM fluorescence and the luminescent signal from firefly luciferase were imaged concurrently in the same eggs using a Zeiss Axiovert S100TV microscope. Cal-520 AM was excited from 450–490 nm. The fluorescent and luminescent emission light was collected through the same filter set at 515 nm. The luminescence values presented represent the number of measured photon counts per second.To measure [Ca2+]ER content, thapsigargin, an inhibitor of the ER Ca2+ -ATPase, was pipetted directly into the petri dish . After [Ca2+]i returned to baseline, CaCl2 (1.7 mM) was added to measure Ca2+ influx. ICSI was performed as described360 (w/v) (Sigma) to immobilise for Piezo-pulsed decapitation. PLCζ cRNA tagged with firefly luciferase2-containing (10 mM) Ca2+ -free M2 media.To examine [Cahttp://fiji.sc/Fiji) and fluorescence intensity from each egg was plotted against time. All changes in [Ca2+]i were statistically analysed using GraphPad Prism Software version 6. The normality of all data sets was assessed using the Shapiro-Wilk test. Data were then analysed using either a Student’s two-tailed unpaired t test (parametric data) or a Mann-Whitney test (nonparametric data) as appropriate. Statistical significance was defined as P < 0.05. Actual P values are presented except where P < 0.0001. Data is presented as mean ± standard error (s.e.m.).Ratiometric images were analysed using Fiji software (Image J) .


We have studied Ca2+ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca2+ could substitute for Mg2+ at M3, with maximal activity for Ca2+/Zn2+-TNAP around 40% that of Mg2+/Zn2+-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca2+ occurred faster than by Mg2+. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca2+ and Mg2+ for M3 similarly, excluding a catalytic role for Ca2+ in the TNAP M4 site. At pH 9.8, Ca2+ competed with soluble Zn2+ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn2+, forming Ca2+/Ca2+-TNAP with a catalytic activity only 4–6% that of Mg2+/Zn2+-TNAP. At pH 7.4, competition with Zn2+ and its displacement from M1 and M2 required >10-fold higher Ca2+ concentrations, to generate weakly active Ca2+/Ca2+-TNAP. Thus, in a Ca2+-rich environment, such as during skeletal mineralization at pH 7.4, Ca2+ adequately activates Zn2+-TNAP at M3, but very high Ca2+ concentrations compete with available Zn2+ for binding to M1 and M2 and ultimately displace Zn2+ from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca2+ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca2+ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca2+ gradient first activates TNAP, but gradually inactivates it at high Ca2+ concentrations, toward completion of mineralization.The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn In vitro, APs are quite promiscuous in their substrate specificity, being able to catalyze both the hydrolysis of monoesters of phosphoric acid and a transphosphorylation reaction in the presence of large concentrations of phosphate acceptors   to upload bound enzyme, for 2 h. Microtiter plates were then incubated for 60–90 min with the substrate pNPP (1 or 10 mM), dissolved in 1 M Tris-HCL buffer, pH 7.4 . [Mg2+]/[TNAP●Mg2+],Binding of Mga and kd represent the association and dissociation rate constant of this reaction, respectively and Kd is the dissociation constant.In which kapp for the binding was calculated from the time to reach 50% of the maximal TNAP activity, t1/2 (kapp = ln2/t1/2) and was then fitted to the equationappvs. [Mg2+] were therefore constructed, to derive kd and ka, enabling calculation of Kd and comparison to directly determine the Kd from dose-response studies. These dose-response curves for TNAP activation vs. metal ion concentration were calculated after a steady-state was reached, i.e. from the “reference” interval from 60–90 min, by fitting the data to a one-site binding model (GraphPad Prism), from which plots the Kd, maximal activity (plateau) and Hill coefficient were derived.The apparent first order rate constant kAll experiments were carried out at least three times and were confirmed at different enzyme dilutions. When executed in identical conditions, data were averaged and represented as the mean values ± SD; when repeated with different concentrations, one representative example is shown. Dissociation constants are expressed with their SD, calculated form the fitted lines (GraphPad Prism). Groups and dissociation constants were compared using unpaired Student t tests, calculating two-tailed p-values, defined in the text or figure legends, as required.2 (yielding Zn2+-TNAP with Zn2+ in M1 and M2 but free M3). Prior to investigating TNAP activation as a result of binding of Ca2+ to M3, we verified whether human TNAP activity complies with the model described for bovine kidney AP  (1 mM), binding was almost immediate (constant slope for ΔA405nm/time). The first derivative of these curves (i.e. the plot of the slopes vs time) describing the formation of Mg2+/Zn2+-TNAP  allowed estimating the kinetic constants of Mg2+ binding (kd = 4.4 ± 0.2 x 10−4 s−1 and ka = 23 M−1s−1), resulting in Kd = kd/ka = 17 μM , consistent with previously determined values for bovine kidney AP at pH 8 (ka = 7 M−1s−1 and kd = 4 x 10–4 s-1). In other words, the allosteric effect of [Mg2+] on human TNAP is consistent with that reported for bovine kidney AP  also dose-dependently stimulated Zn2+-TNAP  inhibited Ca2+/Zn2+-TNAP activity to a level equating 13 mA405nm/min (at 10 mM), i.e. below that of the baseline (22 mA405nm/min). This activity, representing 8% of that of Ca2+/Zn2+-TNAP , thus abrogated the role of Zn2+ in the baseline activity of Zn2+-TNAP , with the ascending limb describing a one-site saturation, with an estimated Kd = 220 ± 26 μM and a pseudo-plateau at 8.3 mA405nm/min, i.e. 3.7-fold lower than the Mg2+ plateau. The descending limb revealed potent TNAP inactivation with an IC50 = 5.4 mM CaCl2 and a Hill coefficient = −2.2, compatible with binding to M1 and M2  and dissociated phosphate ions in pNPP at pH 9.8  inhibited TNAP activity between 1–10 mM  in a peripheral location  (10–20 mM) was compatible with some Ca2+-induced TNAP inactivation. To enable proper study of the interaction of Ca2+ and M1 and M2, we have prepared holo-TNAP, by overnight incubating AbM2-bound TNAP with 250 μM EDTA, at room temperature. This procedure fully stripped TNAP from its bound metal ions  at higher [Ca2] unexpectedly triggered holo-TNAP activation dose-dependently, with an apparent Kd = 509 ± 49 μM. To quench this non-specific TNAP activation, triggered by divalent metal ion contaminants [2+ (0.2–20 μM). This procedure shifted Ca-saturation curves to lower [CaCl2] with Kd = 144 ± 49 μM for 0.2 μM and Kd = 137 ± 24 μM for 2 μM, i.e. in fair agreement with Kd = 217 ± 50 μM, for the binding of CaCl2 to Zn2+-TNAP at M3 . 2 competes with ZnCl2 for binding to M1 and M2 at [CaCl2]>10 mM, more efficiently when [ZnCl2] is low (2 μM), with an apparent IC50 around 250 mM. On the contrary, when AbM2-bound TNAP was fully loaded overnight with 20 μM ZnCl2, CaCl2 only displaced bound Zn2+ at concentrations as high as 100 mM, independently of the presence of soluble ZnCl2 (0–20 μM).Therefore, to analyze the competition between Ca2+ binding to M1 and M2, and allosterically activated by Mg2+ binding to M3   , 27. InsCl2]  is not a matter of concern, since transported Ca2+ is capable of adequately substituting for Mg2+. Indeed, at physiological pH, the affinities of Ca2+ and Mg2+ for M3 only differ 2–3 fold and the maximal activity is only 2.5 fold weaker for Ca2+/Zn2+-TNAP than for Mg2+/Zn2+-TNAP.In humans, baseline plasma Znnd 12 μM  and free1–1.3 mM . The pre2+ can also easily be replaced at M3 by Mn2+, Co2+ and Ni2+  could overcome this limitation, allowing proper competition and displacement studies between ZnCl2 and high [CaCl2] at pH 7.4.We have previously described APs as allosteric enzymes in which asymmetry between monomers generates activity patterns which differ from the expected weighed properties of the two monomers . In partctivator . Inclusi2+ at all metal ion sites. Our work also suggests that TNAP in other Ca2+-rich environments can act as Ca2+/Zn2+-TNAP, e.g. regulating the hydrolysis of phospholamban in the sarcoplasmic reticulum of cardiomyocytes and in skeletal muscle [2+ levels [2]-dependent conversion of Ca2+/Zn2+-TNAP into Ca2+/Ca2+-TNAP.Medial vascular calcification is associated with chondrocyte transdifferentiation and expression of TNAP . It is tl muscle . The inj+ levels . The str+ levels  can also2+-rich milieus, until very high concentrations of Ca2+ occupy M1 and M2 leading to greatly reduced enzymatic activity.In conclusion, our work has identified that ionized calcium supports TNAP activity in CaS1 Fig2+-TNAP formation, measured from the increase of A405 nm vs. time during pNPP hydrolysis, after incubation of AbM2-bound TNAP with 1 mM EDTA (2 h) followed by addition of the indicated [Zn2+] (2 h), dissolved in Chelex-treated TBS, followed by addition of Chelex-treated pNPP (10 mM) at pH 9.8; b. Slope (first derivative) to the line for 40 μM ZnCl2 in 2+-TNAP formation, from plots of initial AP activity (ΔA405 nm/20 min) vs. the indicated [Zn2+]; the red line represents the corresponding AP activity for native non-EDTA treated AbM2-boundTNAP. Results are representative of 3 independent experiments.a. Progressive Zn(TIF)Click here for additional data file.S2 Fig2, measured from its activity at A405 nm vs. time in Chelex-treated pNPP (10 mM) at pH 9.8; Note the curvi-linearity at low [CaCl2] and linearity at 5 and 10 mM CaCl2 respectively (left panel); the maximal activity (“max”) represents activity of fully metalated TNAP in pNPP, pH 9.8, measured in the presence of 20 μM ZnCl2 and 1 mM MgCl2; b. Dose-response of TNAP activation and inhibition, from plots of AP activity  vs. the indicated [CaCl2] (left panel) or [MgCl2] (right panel); ●: absence of ZnCl2; ■: 2 μM ZnCl2 mixed with CaCl2; μ: 2 μM ZnCl2 mixed with MgCl2. Insert: one-site binding model fit for the ascending limb, without added Zn2+. Results are representative of 3 independent experiments.a. Progressive activation and inhibition of AbM2-bound EDTA-treated TNAP in the absence (left panel) and presence (right panel) of 2 μM ZnCl(TIF)Click here for additional data file.S3 Figa. Western blots of TNAP, PLAP and their mutants, after purification from COS-1 cellular medium, via AbM2 detection; b. Heat inactivation curves of PLAP and the indicated mutants, plotted as residual activity after 10 min incubation at the indicated temperature; c. Heat inactivation curves of TNAP and the indicated mutants, plotted as residual activity after incubation for the indicated time interval at 56°C in TBS. Results are representative of 3 independent experiments.(TIF)Click here for additional data file.S4 Figa. Dose-response of Zn2+-TNAP inhibition by high [MgCl2] (0–20 mM) at pH 7.4; AP activity was measured as mean mA405nm/min in steady-state (between 60–90 min) (left panel); tracings of TNAP activity vs. time, in the presence of the indicated medium to high [MgCl2] at pH 7.4, illustrating a slight deflection in hydrolysis rate after 60 min (solid line vs. dotted line) (right panel); b. Similar tracings, in the presence of the indicated medium to high [CaCl2], illustrating clear deflection in hydrolysis rate after 50 min (left panel); kinetics of TNAP inactivation by high [CaCl2], at pH 9.8, reaching steady-state after 10 min (right panel). Activities were measured in Chelex-treated pNPP (1 mM); results represent mean ± SD for 3 identical experiments.(TIF)Click here for additional data file.S5 Fig2] (a) and [CaCl2] (b) at identical AbM2-bound [Zn2+-TNAP]; activities were measured in Chelex-treated pNPP (1 mM or 10 mM as indicated) at pH 7.4. Results represent mean ± SD for 3 identical experiments.Dose-response of generated AP activity (mean mA405nm/min) in steady-state (between 60–90 min) for increasing [MgCl(TIF)Click here for additional data file.S1 FileSequence of the primers used for site-directed mutagenesis and presentation of the results and discussion of the data reported in the 5 supplemental figures.(DOCX)Click here for additional data file.


Corneal endothelium engineering is focused in producing transplantable cell sheets to overcome the shortage of corneal graft tissue donors for the treatment of corneal blindness. For this purpose, the use of a proper animal model plays a key role. Corneal parameters of White New Zealand rabbits such as endothelial cell density, central corneal thickness, and corneal diameter decrease with age, similarly as in humans. However, as opposed to humans, they retain the ability to restore their corneal endothelium after injury. Therefore, they are considered as an inappropriate corneal endothelial wound healing model.Here we analyze the corneal endothelium mitotic ability of White New Zealand rabbits aged 3, 6, 12 and 18 months, 36 and 72 hours after thermal injury. The highest mitotic activity was observed in the 3-month rabbits 36 h after wounding. Rabbits of 12 months registered decreased mitotic activity and those of 18 months did not show mitotic activity 72 h after injury.These results propose that rabbits of 18 months represent a suitable model for human corneal endothelium engineering research. This occurs because human CE cells are arrested in the G1-phase of the cell cycle [The corneal endothelium (CE) is the monolayer of polygonal cells that forms the posterior surface of the cornea facing the aqueous humor ,2. Its mll cycle . When inll cycle -6.Currently, engineered corneas are being tested in animal models in order to develop new strategies for CE regeneration . The rabn was determined to be an appropriate size by using the resource equation method [A total of 16 New Zealand rabbits were used in accordance with the standards set in the Guide for the Care and Use of Laboratory Animals and with the ethical approval of the Internal Committee for the Use and Care of Laboratory Animals from the School of Medicine, Tecnologico de Monterrey. Four age groups were formed: 3, 6, 12 and 18 months. Rabbits were studied with whole cornea histological preparation. All animals were anesthetized with an intramuscular injection of ketamine HCL (30 mg/kg) and xylazine (5 mg/kg), and each animal received two drops of proparacaine prior to wounding. Rabbits were injured in the central cornea for 15 sec with a 2-mm brass cryoprobe cooled to -60°C. Local analgesia with pranoprofen (0.01%) was administered three times per day in each animal after wounding. One-half of the animals were sacrificed at 36 h post-wounding and the other half at 72 h post-wounding. The experimental groups were designated 3/36, 6/36, 12/36, 18/36, 3/72, 6/72, 12/72 and 18/72  was characterized by nuclei dispersing and no mitotic figures. In the adjacent zone (regenerative zone) an increase in the number of nuclei and mitotic figures was found. The innermost zone , showed scattering of the nuclei, cell migration, and spaces between cells Figure .Figure 1p < 0.05). Mitotic figures found among the different age groups at 36 h decreased considerably from an average of 190 to 40. The highest mitotic activity was found in the 3/36 group with a mean of 190 mitotic figures . Group 18/72 registered no mitotic figures.For all ages, the highest number of mitoses was observed at 36 h, with a significant decrease occurring at 72 h. This was most apparent in the 3/36 vs. 3/72 group ; meanwhile, in older groups (6–18/36), mitotic activity was relatively constant.Previous reports have shown that rabbit corneal endothelium repairs by cell division and migration. In a study where the regenerative capacity between young cats and rabbits was compared, it was shown that after 24 h post-wounding, the cells of the rabbit corneal endothelium at the margin of the wound were more likely to divide and migrate than those at the peripheral zone . Later, et al. [In previous studies, it has been shown that parameters such as CECs density, central corneal thickness, and corneal diameter decrease with age in rabbits as in humans. Morita et al.  found thet al.  concludeIn summary, our results complement the previous knowledge regarding the similarities the rabbit CE shares with that of human. Our findings showed that the replicative ability of the corneal endothelium found in rabbits demonstrates an inverse relationship with age.