Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.β-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre floxed β-catenin animals were viable but demonstrated small body size and shortened median survival. The pancreata from knockout mice were hypoplastic and histologically demonstrated a striking paucity of exocrine pancreas, acinar to duct metaplasia, but generally intact pancreatic islets containing all lineages of endocrine cells. In animals with extensive acinar hypoplasia, putative hepatocyte transdifferention was occasionally observed. Obvious and uniform pancreatic hypoplasia was observed by embryonic day E16.5. Transcriptional profiling of Pdx1-cre floxed β-catenin embryonic pancreata at E14.5, before there was a morphological phenotype, revealed significant decreases in the β-catenin target gene N-myc, and the basic HLH transcription factor PTF1, and an increase of several pancreatic zymogens compared to control animals. By E16.5, there was a dramatic loss of exocrine markers and an increase in Hoxb4, which is normally expressed anterior to the pancreas.β-catenin mutations in carcinomas of acinar cell lineage seen in humans.We conclude that β-catenin expression is required for development of the exocrine pancreas, but is not required for development of the endocrine compartment. In contrast, β-catenin/Wnt signaling appears to be critical for proliferation of PTF1+ nascent acinar cells and may also function, in part, to maintain an undifferentiated state in exocrine/acinar cell precursors. Finally, β-catenin may be required to maintain positional identity of the pancreatic endoderm along the anterior-posterior axis. This data is consistent with the findings of frequent  Wnt1 under control of the Pdx-1 promoter was associated with murine pancreatic agenesis  in PBS, prior to embedding and freezing in OCT.Dewaxed sections were subjected to antigen unmasking with Citrate solution pH 6. . Slides were incubated in 0.3% hydrogen peroxide/methanol and then blocked with Beat Block, from HistoMouse max kit [Invitrogen Inc.] Primary antibody Anti-Active-β-Catenin [anti-ABC] clone 8E7  at 1:100 was applied O/N, 4°C. Secondary antibody, chromagen and counterstain were provided and the manufacturer's protocol was used. Primary antibodies to albumin  and amylase  were incubated with slides at 1:3000 and 1:500, respectively, 1 hr R/T after citrate epitope retrieval, avidin and biotin blocking [Invitrogen Inc.]. Nonspecific binding was blocked with appropriate serum incubations for 30 minutes. Biotin conjugated secondary antibodies  were applied 30 minutes R/T followed by incubation with HRP Streptavidin 15 minutes and visualized with DAB . They were counterstained with Mayer hematoxylin. Apoptotic cells were detected by flourescein in situ tunnel method, TACS TdT kit . Proteinase K was used for epitope retrieval and all steps were performed according to manufacturer's protocol.For immunolabeling, tissues were sectioned and fixed in 4% PFA for 15 minutes at room temperature, washed in PBS and stained as previously described . The following antibodies were used at the indicated dilutions; rat monoclonal anti-E-cadherin , goat polyclonal anti-amylase , goat polyclonal anti-β-catenin , guinea pig polyclonal anti-insulin , rabbit polyclonal anti-Pdx1  rabbit polyclonal anti-PTF1a-p48 , rabbit anti-phosphohistone H3 . The following reagents were purchased from Jackson ImmunoResearch Laboratories: Biotin-conjugated anti-goat, anti-guinea pig , Cy3-conjugated anti-rabbit [1:300], Cy2-conjugated anti-rat [1:300], and Cy2-, Cy3-, C5-conjugated streptavidin .® filters for 7 days in culture medium  in the presence or absence of 20 nM Wnt-pathway inhibitor compound PKF118–310 [ref]. Culture medium was changed every day and by the end of the culture period, explants were either embedded in OCT and frozen in liquid nitrogen for immunostaining analysis or put in TRIzol® Reagent for total RNA extraction.Isolation and culture of pancreatic rudiments were carried out essentially as previously described . Intact Pdx1-cre floxed β-catenin mice and immediately submerged in RNAlater . Total RNA was purified using RNeasy columns  according to the manufacturer's directions and quality was assessed using an Agilent 2100 Bioanalyzer . 120 ng of total RNA from 2 wild type [WT] and 4 β-catenin knock-out [KO] pancreata at the 14.5 and 16.5 time points was amplified twice with the Arcturus RiboAmp kit  and cRNA was labeled with biotin-UTP, CTP using T7 RNA polymerase . Biotinylated cRNA from each embryonic pancreas was purified with RNeasy columns and hybridized to an Affymetrix GeneChip mouse genome 430 v2.0 array and stained using standard procedures . The 430 v2.0 array contains 45,000 probe sets representing over 34,000 mouse genes.Pancreata were dissected from 12.5, 14.5 and 16.5 day embryos taken from timed matings of We sought to identify genes whose expression was lost or gained as a result of loss of β-catenin gene expression. To do this, microarrays were generated from RNAs isolated from independent wildtype and knockout embryos as previously described. Affymetrix MOE430plus2 microarray Cel files generated from GCOS 5.0 were subjected to RMA normalization as implemented in GeneSpring 7.1. All E14.5 genechip RMA values were converted to the ratio relative the average of the corresponding day wildtype values. In this fashion one does not see the effect of increased or decreased expression in WT E16.5 relative to WT E14.5. Probe sets were first filtered for those that are overexpressed or underexpressed in knockout animals at E14.5 , and the same for E16.5 . These two lists were combined [967] and then subjected to statistical analysis differentially expression in knockouts with p < 0.05 using the Students T-test assumption of equal distribution and Benjamini-Hochberg false discovery corrected p-values. This procedure generated a list of 770 probe sets. Log2 gene expression ratios were then subjected to hierachical clustering using the standard correlation distance metric as implemented in GeneSpring. Individual clusters were then analyzed for functional relatedness of genes within the cluster using David 2.0.For quantitative RT-PCR, total RNA was purified and characterized as described above. 500 to 1000 ng of RNA was converted to cDNA using random hexamers and Superscript III . Amplification was carried out with an ABI 7300 real time PCR system  using SYBR green and the standard protocol. The reference gene was β-glucuronidase for all genes except Ptf1, for which Gapdh was used. Data was normalized using the delta-delta-Ct method. Whenever possible, primers were designed to span an intronic sequence and were validated by PCR and gel analysis.Primer sequences were as follows:β-glucuronidase5'-TTGAGAACTGGTATAAGACGCATCAG-3' forward5'-TCTGGTACTCCTCACTGAACATGC-3' reverseGapdh5'-AATGGTGAAGGTCGGTGTG-3' forward5' GAAGATGGTGATGGGCTTCC 3' reverseAmylase5'-GGTTCTCCCAAGGAAGCAG-3' forward5'-TGTCACACGGCCATTTCC-3' reverseElastase5'-CATCCAGACAGCTTGCCTC-3' forward5'-CTCAGGGTGTCAGGACTGTTC-3' reverseFgfr25'-AAGCAGGAGCATCGCATTGG-3' forward5'-TGACGGGACCACACTTTCCATA-3' reverseN-myc5'-CTCAAGTCAGTGCAGGCGAG-3' forward5'-CCACCGTTACGACATCAATCTC-3' reversePtf15'-CAGGCCCAGAAGGTTATCATCTG-3' forward5'-AGGAAAGAGAGTGCCCTGCAAG-3' reversePdx1-cre strain developed in our laboratory with a strain containing a floxed β-catenin allele in the second and sixth intron [Pdx1-cre mice heterozygous for the floxed β-catenin allele displayed no apparent abnormalities in either the embryonic or adult pancreas. Mice with conditional knockout of both β-catenin alleles in the pancreas [Pdx1-cre floxed β-catenin] were viable, but demonstrated small body size at birth that persisted into adulthood. For simplicity, these animals are referred to as β-catenin KO in the remainder of the text. β-catenin KO mice showed no obvious defects in their ability to nurse or wean, but had a significantly shortened lifespan, with a median survival of 29 days . Endocrine cells were further characterized by immunostaining for insulin and glucagon. These studies revealed that despite the absence of β-catenin expression, insulin and glucagon cells were present and normally distributed within the islets Fig . Three sWe next analyzed pancreata from β-catenin KO mice beginning at ED 12.5 to determine when pancreatic development was perturbed. We noted no obvious difference in the size of pancreata until E16.5, at which point loss of β-catenin results in an obvious and consistent decrease in pancreatic mass, corresponding to a decrease in the number of recognizable nascent acini Figure .This finding led us to hypothesize that in normal pancreatic development, the presence of Wnt signaling would be critical prior to E16.5. To explore this idea, we examined embryonic pancreas for expression of the dephosphorylated, active form of β-catenin using a phospho-specific antibody. We detected activated β-catenin protein expression at E12.5 that remained at E14.5 but then rapidly disappeared by E16.5 Figure . DephospGiven that the β-catenin KO was based on conditional activation of the cre recombinase expressed from a transgenic allele, it is likely that cre expression was mosaic. While the majority of the acinar pancreas was absent, it was striking that there were clusters of normal appearing acini. Therefore, we hypothesized that the presence of preserved acini would correspond to the presence of β-catenin expression. This proved to be the case as essentially all acini were found to express β-catenin. Co-staining for β-catenin/carboxypeptidase A in E 12.5 pancreata Figure  revealedWe next sought to evaluate whether deletion of β-catenin influenced the development of pancreatic progenitors expressing Pdx1. In other genetically modified mice, pancreatic hypoplasia has been associated with a reduction in the Pdx1-expressing progenitor cells . We analIn order to determine if the loss of acinar cells in the β-catenin knockout was related to a decrease in proliferation of β-catenin-deficient cells, we performed immunostaining of β-catenin KO and wildtype pancreata at ED 14.5 using an antiphosphohistone H3 antibody Figure . These sThus far, we have demonstrated that deletion of β-catenin is associated with a defect in exocrine development, possibly related to decreased proliferation of exocrine progenitors. It is probable that such a defect relates to a decrease in the Wnt signaling function of β-catenin, given the temporal expression of dephosphorylated β-catenin at this stage in the developing pancreas. However, β-catenin also functions in the E-cadherin-catenin cell adhesion complex and it is therefore possible that part of the phenotype we have observed is related to this function of β-catenin . To invePdx1 transcript levels did not differ between wildtype and β-catenin KO. Expression of PTF1 transcript was reduced in the β-catenin KO at E14.5 by microarray and as early as E12.5 by QRT-PCR . Consistent with this, N-myc was only recently shown to be a direct target of Wnt signaling in the lung , an RNA binding protein implicated in maintaining the identity of progenitor cell fates in the brain [Hhip] [1.9 fold] suggesting a potential role for this signaling pathways in exocrine pancreatic development. A transcription factor of unknown function, the leucine zipper-like transcription factor1 (Lzltf1), was among the most reduced genes at E14.5, with its expression being decreased by an average of 5.9 fold. Hox b4 was significantly increased in E16.5 β-catenin mutant pancreata suggesting that loss of Wnt signaling results in a partial homeotic transformation , and a slight increase in glucagon (Gcg) [1.4 fold] at ED14.5. By 16.5, however, none of these genes were differentially expressed compared to wildtype mice. "Raw and normalized microarray data from this study can be obtained from the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database [Among the transcripts most down regulated in E16.5 β-catenin KO mutants included acinar cell specific genes such as pancreatic elastase  and amylase 2 (Amy2). Ela3b was represented 5 times on the chip and was highly up regulated at e14.5 in each. If we relax the stringency of our clustering analysis, numerous other proteinases were also upregulated at E14.5 in 3 out of 4 samples. These include elastase2 (Ela2), trypsinogen 16 (Trygn16), trypsin 4 (Try4), carboxypeptidase A3 and B1 , and serine protease 2 (Prss2). By E16.5, when there is obvious exocrine hypoplasia, all of these exocrine markers are down regulated, demonstrating that their increased expression at E14.5 is only transient.Consistent with our observed exocrine deficiency, we observed reduced mRNA levels of numerous exocrine markers in β-catenin mutants. However at E14.5, prior to any visible exocrine hypoplasia, we were surprised to see a striking increase in mRNA levels of several markers of exocrine differentiation including pancreatic elastase3b  and exocrine gene expression was observed even though pancreas development was largely arrested. In addition, although p48/PTF1 is necessary for exocrine pancreas development, it is unlikely that all exocrine genes are direct targets of p48/PTF1. It is also possible that an increase in exocrine markers at E14.5 via microarray analysis is because some remaining β-catenin + cells increase exocrine gene expression in attempts to compensate for the global exocrine loss of differentiation of exocrine progenitor cells. This is consistent with the concept that loss of a mitogenic signal can cause perturbations in proliferation, cell death or differentiation. Our immunostaining does not support this since the remaining β-catenin + cells did not show increased levels of pancreatic enzyme expression. Taken together our data suggests that proliferation and differentiation, but not apoptosis are effected in β-catenin mutants. Moreover, the fact that some exocrine markers are reduced whereas others are elevated suggesting that β-catenin signaling is key at E14.5 to maintain a proper balance between proliferation and differentiation of the exocrine lineage.In adult mice that survived past 30 days, we observed several instances wherein the pancreatic parenchyma appeared to be partially replaced by tissue that histologically resembles the liver and expresses albumin. This suggests the possibility of hepatic transdifferentiation suggesting that β-catenin function is required to maintain a pancreatic phenotype. Our findings cannot exclude the possibility that disruption of normal pancreatic development caused by loss of β-catenin could have induced nascent hepatocytes to migrate to this area. The phenomenon of hepatic transdifferentiation has been described in other models of severe acinar cell loss including copper or methionine depletion and subsequent repletion but the molecular mechanisms underlying transdifferentation in adults are not well known ,39. A hiFgfr2 is concordant with this [The FGF pathway has been shown to regulate exocrine pancreatic development and our finding of down regulation of ith this ,43. Compith this ,45. Specith this .In summary, our studies support a model wherein β-catenin regulates proliferation and differentiation of the developing exocrine pancreas through its role in the Wnt signaling pathway. This observation is consistent with the findings in human exocrine cancers wherein β-catenin/Wnt signaling is inappropriately active and likely supports uncontrolled proliferation. Further studies are required to precisely define the Wnt target genes critical to proliferation of developing acini and to understand the signals that regulate Wnt signaling functions. Our findings also raise the question of whether modulation of β-catenin/Wnt signaling in the adult pancreas could enhance recovery from injury following diseases such as acute pancreatitis, and/or alter the abnormal balance between proliferation and differentiation observed in pancreatic cancer.The author(s) declare that they have no competing interests.JMW carried out pancreata harvest, helped conceive the study, analyze data and draft the manuscript. FE performed the dorsal explant studies and immunohistochemistry, designed figures and assisted with data analysis. GB analyzed all histopathology analyses and assisted with apoptosis assays. BJA performed microarray experiments and bioinformatics. WS performed genotyping, harvested pancreata, prepared samples for microarray analyses and performed quantitative RT-PCR. CC performed genotyping and immunohistochemistry. AS performed immunohistochemistry and apoptosis assays. SDL helped conceive portions of the study, analyze data, and draft the manuscript. AML conceived the study, reviewed and analyzed all data and drafted the manuscript. All authors read and approved the final manuscript.


In this study, we investigated the expression pattern of β-catenin and cyclin D1 using immunohistochemistry and searched for mutations in exon 3 of the β-catenin gene and Axin gene in esophageal squamous cell carcinoma.Samples were obtained from 50 esophageal cancer patients. Immunohistochemical staining for β-catenin and cyclin D1 was done. Mutational analyses of the exon3 of the β-catenin gene and Axin gene were performed on tumors with nuclear β-catenin expression.catenin exon 3 in the four cases with nuclear β-catenin staining. Sequencing analysis of the Axin cDNA revealed only a splicing variant  which was present in the paired normal mucosa.Four (8%) esophageal cancer tissues showed high nuclear β-catenin staining. Overexpression of cyclin D1 was observed in 27 out of 50 (54%) patients. All four cases that showed nuclear β-catenin staining overexpressed cyclin D1. No relationship was observed between the expression pattern of β-catenin and cyclin D1 and age, sex, tumor size, stage, differentiation grade, lymph node metastasis, response to chemotherapy, or survival. No mutational change was found in β-A fraction of esophageal squamous cell carcinomas have abnormal nuclear accumulation of β-catenin accompanied with increased cyclin D1 expression. Mutations in β-catenin or axin genes are not responsible for this abnormal localization of β-catenin. In addition to its function in cell-cell adhesion, β-catenin plays an important role in signal transduction; it is involved in the Wnt signaling pathway that regulates cellular differentiation and proliferation .catenin gene [The level of free β-catenin is low in normal cells, since the protein is sequestered in a complex, which includes the adenomatous polyposis coli (APC) protein, a serine threonine glycogen kinase (GSK-3β) and conductin or Axin, leading to degradation of β-catenin by proteasome. The binding of β-catenin by APC requires phosphorylation of β-catenin by GSK-3β on 3 serine and 1 threonine residues, all of which are encoded by exon 3 of the β-nin gene -4.cyclin-D1, c-myc, c-jun, fra-1, uPAR, ZO-1, MMP7, NBL4, DRCTNNB1A, MCP-3 [In colorectal cancers, mutations of APC or β-catenin result in stabilization of β-catenin and a significant accumulation of this protein within the cytoplasm . FurtherA, MCP-3 -12. HoweA, MCP-3 ,13,14.Recently, cyclin D1 has been identified as a target of the β-catenin/T-cell factor/lymphoid enhancer factor complex ,15. CyclRecent experiments demonstrated that Axin functions as a tumor suppressor in hepatocellular carcinoma . The difEsophageal squamous cell carcinoma is an aggressive disease with a poor prognosis, and the genetic mechanism of its carcinogenesis remains to be solved. The progression of this tumor is associated with multiple genetic alterations, including loss of heterozygosity in chromosomes 3p, 5q, 9p, 9q, 13q, 17p, 17q and 18q, and amplification of epidermal growth factor receptor (EGFR), HER-2, c-myc, and cyclin D1 . The moscatenin gene (CTNNB1) and Axin gene was studied in these samples.In this paper, we investigated the expression pattern of β-catenin and cyclin D1 in esophageal squamous cell carcinoma. We found aberrant localization of β-catenin in a minor proportion of the tumor and mutation in exon 3 of the β-Samples were obtained from 50 esophageal cancer patients who had undergone operations at the Department Surgery II, Nagoya City University Medical School between 1996 and 2000. They were classified according to the guideline for the clinical and pathologic studies on carcinoma of the esophagus . All samImmunohistochemical staining was done with anti-β-catenin monoclonal antibody (Transduction Lab.) and anti-cyclin D1 monoclonal antibody (Oncogene Research Product). A formalin-fixed paraffin-embedded tissue block was cut and de-waxed. Each section was treated for 15 min in citric-acid buffer (pH 6.0) with autoclave at 120°C for antigen retrieval, and was immunostained with the standard indirect ABC technique. Counterstaining was done with hematoxylin. When more than 20% of the cells were stained, the staining was scored as positive.Total RNA was extracted from the esophageal cancer tissue, and normal esophageal mucosa taken from the mucosa as far apart from the tumor as possible, using Isogen kit . The concentration of the RNA was adjusted to 200 ng/ml, using spectrophotometer. Reverse transcriptional reaction was carried out at 42°C for 90 min and 95°C for 5 min followed by incubation at 72°C for 15 min, using 1 μg RNA, 0.5 mg oligo(dTi) primer, Superscript II enzyme .Polymerase chain reaction (PCR) and direct sequencing analysis were performed on the four tumors with nuclear β-catenin expression. Four microliter of cDNA mixture was subjected to amplification in 80 μl containing 0.8 U of Taq polymerase, 10×PCR buffer, 25 μM each of dATP, dCTP, dGTP, dTTP, 25 μM of MgCl2 and 80 pmol each of 5' and 3' primers. PCR conditions for β-catenin were initial denaturation for 5 min at 95°C, followed by 38 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec and a final extension at 72°C for 5 min. PCR conditions for axin were initial denaturation for 5 min at 95°C, followed by 38 cycles of denaturation at 94°C for 60 sec, annealing at 54°C for 60 sec, extension at 72°C for 60 sec and a final extension at 72°C for 5 min. The primer pair used for amplification of the entire exon 3 of the β-catenin gene and four sets of primers for axin gene are shown in the Table χ-square test was used to analyze the relationship between the two variables. A p value of less than 0.05 was considered significant.Fifty esophageal cancer tissues and paired normal esophageal mucosa were stained for β-catenin using immunohistochemistry. In all the normal esophageal mucosa, β-catenin staining was restricted to the plasma membrane. The intensity of the staining did not vary very much among individuals. In most of the 50 esophageal cancer tissues, the β-catenin staining was at the plasma membrane. However, four cases (8%) of esophageal cancer tissue were judged as showing widespread nuclear staining. And in these cases, β-catenin was not expressed on the plasma membrane Fig. . Normal We then analyzed the expression of cyclin D1, one of the possible downstream targets of the Wnt signal pathway, using immunohistochemistry. In all the normal esophageal mucosa, cyclin D1 staining was negative Fig. . OverexpTo investigate the mechanism of β-catenin nuclear localization, we amplified the β-catenin gene by PCR using the primer pair flanking the entire exon 3. All four cases showed the single band of 502 bp, and none showed an aberrant PCR product within the amplified region. Then we determined the sequence of each band by direct sequencing. No mutational change was found in any of these cases (data not shown).We then searched for mutational changes in the Axin gene. Axin gene mutation may have caused the abnormal distribution of β-catenin. We examined the Axin cDNA in four cases showing nuclear localization of β-catenin protein. Sequencing analysis of the Axin cDNA revealed a splicing variant  and a normal cDNA in two of the cases tested. We sequenced the cDNA from the normal mucosa in these cases, and the same variant was found. This deletion affected the whole exon 9  out of the 50 esophageal cancer samples. The aberrant location of this protein was probably function suggested by the increased expression of cyclin D1 in all 4 of these cases. However, we were unable to find any genetic alterations responsible for this aberrant β-catenin distribution.et al. have reported that overexpression of β-catenin was observed in 3 of 22 cases studied (13.6%). Although no mutation of the β-catenin gene was observed, the common silent mutation of the APC gene was found in all the cases [et al. have reported that 18% cases of the esophageal squamous cell carcinoma showed overexpression of β-catenin in the cytoplasm and nuclei of tumor cells. They found no genetic alteration of the β-catenin gene [catenin gene was found in any of the cases we studied, as reported by others [Ninomiya he cases . De Cast studied .6%. Alth studied .6%. Althet al. observed a correlation between reduced expression of β-catenin and poor prognosis [In esophageal adenocarcinomas, Krishnadath rognosis . Furtherrognosis . In anotrognosis . In otherognosis .et al. have reported that the overexpression of cyclin D1 may be a useful prognostic indicator in patients with squamous cell carcinomas of the esophagus [et al. have reported that overexpression of cyclin D1 was highly associated with nuclear accumulation of β-catenin in ovarian endometrioid adenocarcinomas [et al. all four cases in our study showing nuclear localization of β-catenin protein overexpressed cyclin D1.It has been reported that immunohistochemical examination of cyclin D1 expression may provide important prognostic information for esophageal cancer . Takeuchsophagus . And Zharcinomas . In our A number of studies have shown that Axin is critical for mediating the down regulation of β-catenin ,35. One In conclusion, the nuclear localization of β-catenin occurs in a fraction of esophageal carcinomas. All the cases showing nuclear localization of β-catenin protein overexpressed cyclin D1 which may have contributed to the oncogenesis of the esophageal carcinoma. The abnormal localization of β-catenin apparently did not result from the genetic alterations of either the β-catenin or Axin gene.The author(s) declare that they have no competing interests.YF and JK planned the experiment. JK performed most of the experiments with the help and supervision of NH and HI. TN, YS, YT HS, NS, MK, and YK provided the surgical materials. JK and YF wrote the paper.All authors read and approved final version of manuscript.


Here we report that β-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase–AKT signaling. AKT phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated level of control of β-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of β-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase–AKT signaling and β-catenin accumulation. During mammalian development and adulthood, β-catenin regulates the transcription of a family of genes with multiple essential roles in cell proliferation and differentiation. β-catenin also plays a role in cancer when it carries mutations that result in uncontrolled β-catenin function. Here, we report that the lifetime of the β-catenin–encoding transcript is under regulatory control. We show that specific cellular signals relevant to proper mammalian development and implicated in tumor formation can prolong β-catenin transcript half-life, leading to the accumulation of β-catenin protein. We identify a molecular mechanism for this prolongation by showing that a protein factor responsible for β-catenin transcript instability (and thus degradation) is impaired by phosphorylation, a chemical modification. When this factor is impaired, β-catenin mRNA and protein accumulate. Our results point to an unanticipated control of β-catenin levels through regulation of its transcript half-life in response to signals related to proliferation and differentiation. The authors show that the half-life of β-catenin mRNA is prolonged by PI3K-AKT signaling, revealing a new level of control on β-catenin. Whereas transcripts containing the AU-rich element (ARE) are labile, activation of signal transduction pathways induces their stabilization [trans-acting factors responsible for the decay control of labile mRNAs [The half-life , a mimicker of Wnt signaling , or AKT In this report, we describe that β-catenin mRNA is labile due to destabilizing elements located in its 3′ UTR and that KSRP is a major molecular determinant of β-catenin mRNA instability. β-catenin transcript can be stabilized by either Wnt or PI3K-AKT signaling activation. AKT phosphorylates KSRP at a unique serine residue favoring its interaction with the adaptor protein 14-3-3. As a consequence, KSRP fails to interact with the exosome and, in turn, β-catenin mRNA is stabilized.½ is prolonged in αT3-1 cells in response to LiCl treatment, a large-scale analysis using the ARE-cDNA microarray system was performed [½ of approximately 45 min, whereas the control β2-microglobulin (β2-MG) was stable. In order to verify whether β-catenin AREs were responsible for the rapid decay of the endogenous transcript, we transfected αT3-1 cells with a reporter plasmid containing the entire β-catenin 3′ UTR placed at the 3′ end of chloramphenicol acetyltransferase (CAT) sequence. As shown in ½ while control CAT mRNA was stable.We previously reported that the treatment of mouse pituitary αT3-1 cells with LiCl  stabilierformed . The mic½ of β-catenin mRNA while the t½ of β2-MG was unaffected. Furthermore, treatment of αT3-1 cells with recombinant mouse Wnt-3A increased β-catenin mRNA steady-state levels  of the human KSRP primary sequence performed at medium stringency indicated serine 193 (bold in the following peptide: GLPERSVSLTGAPES) as a potential AKT phosphorylation site , and the phosphorylated serine is conserved, in a corresponding position, also in nonmammalian species  (unpublished data). S193 was mutated to alanine in KH1-4 and KH1-2, and the mutant proteins were expressed in bacteria. As shown in We hypothesized that AKT activation stabilizes β-catenin transcript by targeting the mRNA decay machinery. Among ARE-BPs known to affect mRNA turnover, only KSRP  and HuR La cells D. In ordLa cells E. The enOverall, these data suggest that AKT phosphorylates human KSRP at the unique site S193.The results presented above indicated that KSRP was phosphorylated by AKT and led us to hypothesize that KSRP could be involved in AKT-induced stabilization of β-catenin mRNA. We and others demonstrated that KSRP regulates the stability of select mRNAs in response to different stimuli ,36. ThusWe ruled out the possibility that AKT activation could change KSRP expression levels affecting its protein stability. As shown in Altogether, these results indicate that KSRP is crucial in controlling β-catenin mRNA decay and, in turn, β-catenin expression.To investigate the functional consequences of KSRP phosphorylation by AKT, we performed in vitro reconstitution experiments. Either nonphosphorylated or AKT2-phosphorylated KSRP was added to S100 extracts from αT3-1–shKSRP in typical in vitro degradation assays. As presented in The sequence of the peptide including S193 closely resembles the phosphoserine-containing motif that is recognized by members of the 14-3-3 protein family . Indeed,Altogether, our results indicate that AKT-phosphorylated KSRP interacts with 14-3-3 and that this interaction impairs the decay-promoting activity of KSRP in vitro.We investigated whether the impairment of the mRNA decay-promoting activity of KSRP upon phosphorylation by AKT2 see A was dueCoimmunoprecipitation experiments performed in 293T cells showed that expression of myrAKT1 impaired the interaction of KSRP with core exosome components including hRrp4p and hRrp41 and with the exosome-associated factor hMtr4p  Figure D. DifopeThe evidence that myrAKT1 expression does not affect the interaction of KSRP with PARN prompted us to investigate whether β-catenin mRNA deadenylation was affected by myrAKT1 expression in αT3-1 cells. As shown in in vitro degradation experiments presented in Our results suggest that phosphorylation by AKT and interaction with 14-3-3 affect the ability of KSRP to interact with the exosome.½ is prolonged by Wnt and PI3K-AKT signaling. The mRNA decay-promoting factor KSRP is required to ensure rapid degradation of β-catenin transcript in unstimulated cells. AKT phosphorylates KSRP at a unique serine residue, creating a functional binding site for the molecular chaperone 14-3-3. As a consequence, AKT activation impairs KSRP ability to interact with the exoribonucleolytic complex exosome and, in turn, to promote rapid mRNA decay.Here we report that β-catenin is encoded by a labile transcript whose t½ and the steady-state levels of β-catenin mRNA, thus increasing β-catenin protein levels and enhancing the β-catenin–dependent TOP-FLASH– and c-myc promoter–dependent reporter transcription. Notably, KSRP knock-down did not affect β-catenin protein degradation rate. While this manuscript was in preparation, a report from Thiele et al. [With its involvement in the Wnt signal transduction pathway and tumorigenesis, β-catenin is an extensively studied protein. However, the vast majority of studies focused on the control of β-catenin protein degradation upon its signal-induced phosphorylation –19. Lopee et al.  describee et al.  reportede et al. ).½ in αT3-1 cells (unpublished data). However, our results indicate that PI3K-AKT consistently induces β-catenin mRNA stabilization and protein accumulation in both cell lines. Even though our data indicate that PI3K-AKT activation, regulating the mRNA decay machinery, can lead to β-catenin protein accumulation in the absence of changes in its protein degradation rate, it is conceivable that an integrated control of both mRNA and protein stability could be required to ensure rapid and sustained changes of β-catenin expression in response to certain proliferative and differentiative cues. Notably, AKT-induced interaction of β-catenin protein with 14-3-3 has been shown to increase both β-catenin levels and its transcriptional activity [Our data suggest that PI3K-AKT signaling stabilizes β-catenin transcript targeting the mRNA decay machinery. In the past, conflicting results on PI3K-AKT–induced control of β-catenin protein degradation have been provided –45. The activity . Therefo½ of select myogenic transcripts inhibiting KSRP function [½ of β-catenin mRNA (unpublished data). How the decay of distinct sets of transcripts can be specifically regulated by different signaling pathways that target the same ARE-BP is still an unsolved issue.KSRP, a major regulator of β-catenin mRNA decay, is phosphorylated by AKT at a unique residue S193). It is a general concept that destabilizing ARE-BPs, including BRF1, KSRP, and TTP, are responsible for rapid decay of labile mRNAs in unstimulated cells recruiting the mRNA decay machinery –6. We an3. It is Our data indicate that KSRP phosphorylation by PI3K-AKT creates a functional 14-3-3 binding site. Our current and previous findings, together with existing literature ,47,48, sPhosphorylation by AKT, followed by interaction with 14-3-3, impairs the ability of KSRP to promote β-catenin mRNA decay, reducing KSRP interaction with the 3′-5′ exoribonucleolytic complex exosome while leaving unaffected the ability of KSRP to interact with the mRNA. The results obtained using the high-affinity 14-3-3 competitor peptide difopein suggest that KSRP–14-3-3 interaction is implicated in this process. Unexpectedly, we found that AKT activation does not affect KSRP interaction with the deadenylase PARN and that deadenylated β-catenin mRNA accumulates when incubated with S100 from αT3-1-myrAKT1 cells in in vitro degradation assays. We previously demonstrated that KSRP associates with mRNA decay enzymes, including PARN and the exosome components . HoweverThe four KH domains that constitute the central core of the KSRP are all necessary to ensure its interaction with the entire decay-promoting machinery . S193 li½ and, consequently, of its expression. PI3K-AKT signaling activation, with consequent KSRP phosphorylation and functional deactivation, might contribute to sustain β-catenin accumulation and, as a result, activation of target genes potentially able to accelerate tumor development.In conclusion, the expression levels of β-catenin have to be tightly regulated. As the amount of β-catenin rises, it accumulates in the nucleus, where it interacts with specific transcription factors, leading to regulation of target genes. Inappropriate activation of the β-catenin pathway is linked to a wide range of cancers, including colorectal cancer and melanoma. On the other hand, AKT has emerged as a crucial regulator of widely divergent cellular processes including apoptosis, differentiation, and metabolism. Disruption of normal AKT signaling has now been documented as a frequent occurrence in several human cancers, and it appears to play an important role in their progression. The results we obtained point to KSRP phosphorylation as a link between PI3K-AKT signaling and the control of β-catenin mRNA thttp://www.roche.com).LY and insulin were obtained from Sigma ; triciribine, from Calbiochem/EMD Biosciences (http://www.emdbiosciences.com); Wnt-3A, from R&D Systems (http://www.rndsystems.com); and histone 2B, from Roche (http://www.stratagene.com) in frame with the FLAG sequence to obtain a FLAG-KSRP plasmid. Mutagenesis was performed using FLAG-KSRP and the QuickChange Site-Directed Mutagenesis kit (Stratagene) to obtain FLAG-KSRP(S193A). HA-myrAKT1 cassette was excised from the plasmid previously described by Andjelkovic et al. [http://www.invitrogen.com) to obtain pCMV-myr-AKT1, AKT1(K179M) [http://www.upstate.com). Murine c-myc promoter  fused with Luciferase was previously described [cDNA fragments containing mouse β-catenin cDNA fragments   in pUSEaescribed . To geneescribed .Affinity-purified human KSRP, expressed using the Baculovirus system, was described in Briata et al. . GST-KSRhttp://www.bdbiosciences.com); anti-KSRP antibody [Mouse monoclonal anti–14-3-3β , anti–14-3-3zeta , anti-HuR monoclonal antibody 3A2, and anti-HDAC2 were obtained from Santa Cruz Biotechnology (http://www.scbt.com); mouse anti-GST was from Chemicon (http://www.chemicon.com); rabbit anti-AKT was from Cell Signaling ; mouse anti–β-catenin was from BD Transduction Laboratories , and human HeLa cells were cultured in DMEM plus 10% FBS, and murine C2C12 myoblasts were cultured in DMEM plus 20% FBS. Cell transfections were performed using LipofectAMINE Plus (Invitrogen), and G418 (Invitrogen) was used at 500 to 800 μg/ml (depending on the cell line) for selection. Cell pools of transfectants were used for experiments. αT3-1 cells were starved in DMEM plus 0.5% FBS for 16 h prior to LiCl treatment, and αT3-1–myrAKT1 cells were starved in DMEM plus 0.5% FBS for 16 h prior to experiments. HIRc-B cells were starved in DMEM plus 0.1% FBS for 16 h prior to experiments or further treatments. Transient transfections of αT3-1 with either pcDNA3-CAT or pcDNA3-CAT–β-catenin plasmids were performed as described in [http://www.qiagen.com) and treated with DNase I  according to the manufacturer's instructions. First-strand cDNA was obtained with Transcriptor Reverse Transcriptase (Roche).Cells under different culture conditions were treated with 5 μg/ml actinomycin D and harvested at the indicated times, and total RNA was isolated using RNeasy Mini Kit  and the MiniOpticon Real-Time PCR Detection System (Bio-Rad). The sequence-specific primers used for PCRs are listed in For quantitative RT-PCR, 150 ng of DNase I–treated total RNA was retrotranscribed using random examers and PCRs were performed using the IQ Sybr Green Mix Super  was from Amersham Biosciences (http://www.amersham.com). In vivo 32P orthophosphate metabolic labeling of transiently transfected HeLa cells was performed as previously described [AKT (1 and 2) kinase assays were performed using preactivated enzymes purchased from Upstate Biotechnology (50 ng of the active enzyme/reaction) as recommended by manufacturer. [γ-escribed , incubat3 spectrum) as previously described in detail [http://www.matrixscience.com) against the publicly available human database.Purified recombinant KSRP was phosphorylated by AKT2 in standard kinase assays. The reactions were analyzed by gel electrophoresis; bands were excised, digested with trypsin, and enriched for phosphopeptides using titanium dioxide microcolumns; and the peptides were analyzed by automated nanoflow LC-MS/MS with a method where the neutral loss of the phosphate group activate the acquisition of a second fragment ion spectrum (an MSn detail . All MS/pSUPER-Puro-shKSRP was previously described . αT3-1 cTransient transfections of either mock αT3-1 or αT3-1–shKSRP were carried out with LipofectAMINE Plus (Invitrogen). Luciferase activity was determined after 48 h with the Dual Luciferase System (Promega) following the indications of the manufacturer and using pRL-SV40 as a normalizing vector.Figure S132P-labeled and capped RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods.(A) In vitro RNA degradation assays using S100s from either mock C2C12 or C2C12 cells expressing myrAKT1. Internally (B) Semiquantitative RT-PCR analysis of β-catenin mRNA in either mock C2C12 cells or C2C12 cells expressing myrAKT1. Total RNA was isolated as indicated after the addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicate.(442 KB TIF)Click here for additional data file.Figure S2(A) In vitro RNA degradation assays using S100s from either mock-transfected (empty pSUPER-Puro vector) αT3-1 (mock αT3-1) or pSUPER-Puro-shKSRP–transfected αT3-1 cells (αT3-1–shKSRP).32P-labeled and capped RNA substrates were incubated with S100s for the indicated times, and their decay was analyzed as described in Materials and Methods.(B) In vitro RNA degradation assays using S100s from either LiCl-treated mock-transfected αT3-1 cells or LiCl-treated FLAG-KSR–transfected αT3-1 cells. Internally (792 KB TIF)Click here for additional data file.Figure S3(A) Immunoblot analysis of total extracts from the indicated cell lines with anti-KSRP and α-tubulin antibodies.(B) Either mock αT3-1 or αT3-1–myrAKT1 cells were treated with cycloheximide (50 μg/ml) for the indicated times. Total cell extracts were prepared and the levels of KSRP were quantitated by immunoblotting and densitometric scanning. Results are the average (±SEM) of three experiments. α-Tubulin immunoblotting was used to verify the equal protein loading.(341 KB TIF)Click here for additional data file.Figure S4RT-PCR analysis of total RNA from either mock αT3-1 cells (untreated or treated as indicated) or αT3-1–myrAKT1. The primers used are listed in (467 KB TIF)Click here for additional data file.Table S1The primers used for RT-PCRs were m-β-catenin (mouse β-catenin), m-AKT1 (mouse AKT1), r-β-catenin (rat β-catenin), b-C (the entire 3′ UTR of mouse β-catenin), m-β2-MG (mouse β2-MG), r-β2-MG (rat β2-MG), CAT; CAT-β-catenin (the entire 3′ UTR of mouse β-catenin placed at the 3′ of CAT), m-β-cat Q-PCR .(35 KB DOC)Click here for additional data file.


Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The β-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of β-catenin signaling in bone repair.Western blot analysis showed significant up-regulation of β-catenin during the bone healing process. Using a β-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that β-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/β-catenin pathway) inhibited β-catenin signaling and the healing process, suggesting that WNT ligands regulate β-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized β-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific β-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of β-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated β-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture.These results demonstrate that β-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of β-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, β-catenin positively regulates osteoblasts. This is a different function for β-catenin than has previously been reported during development. Activation of β-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage. In a study in mice Benjamin Alman and colleagues show that β-catenin functions differently in different stages of fracture repair; moreover, activation of β-catenin by lithium improves fracture healing when used in later phases of repair. Most people break at least one bone during their life. If the damaged bone is immobilized with a plaster cast or with metal plates and pins, most fractures heal naturally and quickly. Soon after a bone is damaged, cells called pluripotent mesenchymal cells collect at the injury site. Here, they multiply and change (differentiate) into osteoblasts (cells that make bone) and chondrocytes . Osteoblasts and chondrocytes mend the fracture by making new bone, a process called ossification. Bone healing involves two types of ossification. In intramembranous ossification, mesenchymal cells and osteoblast progenitor cells make bone directly, forming a hard “callus” within the fracture. In endochondral ossification, mesenchymal cells differentiate into chondrocytes and make cartilage at the fracture site, which osteoblasts turn into bone. Finally, the bone made by both types of ossification is remodeled so that it closely resembles the damaged bone's original shape and strength.Unfortunately, fractures do not always heal efficiently. If healing is delayed, additional surgery may be needed to repair the break. But surgery can be risky, so drug-based ways of encouraging bone repair would be very useful. To develop such treatments, researchers need to understand what controls the differentiation and activity of osteoblasts and chondrocytes during normal healing. In this study, the researchers have investigated the role of the β-catenin signaling pathway in bone repair. This pathway regulates bone formation during embryonic development, a process that closely resembles bone healing. β-catenin is usually degraded rapidly in cells. However, if a member of a particular family of proteins known as the WNT family binds to a WNT receptor on the surface of a cell, β-catenin moves into the cell's nucleus where it interacts with a protein called T cell factor (TCF). This interaction activates the transcription  of numerous genes and alters the behavior of the cell.The researchers first measured β-catenin levels in mouse and human bones. In both species, much more β-catenin was made in bones undergoing repair than in intact bones. Then they studied TCF reporter mice—animals in which TCF controls the expression of a marker gene. β-catenin-mediated TCF-dependent transcription, they report, was activated during both bone and cartilage formation after a fracture in these mice. Next, the researchers made mice that could be induced to express an inactive form of β-catenin or a stabilized (permanently active) form of β-catenin in all the cells in a bone fracture. Expression of inactive β-catenin slowed the rate of healing but, unexpectedly, so did expression of stabilized β-catenin. Osteoblast-specific expression of inactive β-catenin also delayed bone healing, whereas osteoblast-specific expression of stabilized β-catenin enhanced the process. Finally, treatment of wild-type mice with lithium (which prevents the degradation of β-catenin) enhanced bone healing if given after a fracture, but interfered with it if given before.These findings indicate that β-catenin signaling  has different effects at different stages of bone repair. Early in the process, it controls the ratio of osteoblasts and chondrocytes made from the pluripotent mesenchymal cells. Consequently, too much or too little β-catenin interferes with bone healing at this stage. Later on, β-catenin promotes the differentiation of osteoblasts and enhances their ability to make bone, and so too little β-catenin at this stage prevents healing, whereas increased β-catenin levels stimulate healing. These findings need to be confirmed in people before testing agents that affect β-catenin signaling for their effects on human bone healing. Nevertheless, the researchers' final discovery that lithium improves bone healing if given at the right time is particularly encouraging; lithium is widely used to treat one form of depression so could be readily tested in clinical trials.http://dx.doi.org/10.1371/journal.pmed.0040249.Please access these Web sites via the online version of this summary at broken bones and on bone fracture repair (in English and Spanish)MedlinePlus encyclopedia contains pages on bone fracture and on bone healing Wikipedia has pages on broken bonesThe UK National Health Service Direct encyclopedia provides pages on intramembranous and endochondral ossification are available from the Ministry of Advanced Education, Training and Technology, Province of British Columbia, CanadaAnimations of informative discussion of fracturesThe American Academy of Orthopedic Surgeons has an SickKids, which contains a page on child physiology, including diagrams of bone developmentThe Hospital for Sick Children in Toronto (where the authors of this study are affiliated) has a Web site called  Fracture healing is a complex regenerative process initiated in response to injury, which in the optimal case results in restoration of skeletal function. In the initial phase of fracture repair, undifferentiated mesenchymal cells aggregate at the site of injury, proliferate, and differentiate, presumably in response to growth factors produced by the injured tissues . This prβ-catenin signaling has emerged as a key regulator of embryonic bone development ,8. SecreWNT1-induced secreted protein 1 (WISP1), a WNT target gene, is also expressed during fracture healing, and overexpression of this gene increases proliferation but decreases the differentiation in a chondrocytic cell line  or β-catenin stabilized alleles in osteoblasts [stabα1(I)-Catnb]. A significant increase in bone mass was found in stabα1(I)-Catnb mutant mice, as previously reported [To determine the function of β-catenin in osteoblasts, we observed fracture healing in mice in which β-catenin loss-of-function or gain-of-function was restricted to cells committed to the osteoblast lineage. Mice expressing conditional alleles were crossed with eoblasts ), resultreported . Westernnullα1(I)-Catnb mice exhibited a fracture callus mainly composed of cartilage matrix, while chondrogenesis was not evident in stabα1(I)-Catnb mice (unpublished data). Histomorphometric assay confirmed these histology changes (nullα1(I)-Catnb mice showed that, although there was some calcified callus present, the newly formed bone had not completely bridged the fracture gap, as compared to wild-type mice (nullα1(I)-Catnb mice, as compared to wild-type mice (nullα1(I)-Catnb mice (nullα1(I)-Catnb mutant mice -Catnb mice showed enhanced fracture healing, characterized by a calcified callus bridging the fracture gap as well as an increase in bone density (stabα1(I)-Catnb mutant mice showed a larger volume of regenerated bone tissues within the fracture site -Catnb mutant mice. However, SOX9 was down-regulated in stabα1(I)-Catnb mutant animals. Expression of RUNX2 in these two mice, however, was regulated in the opposite manner -Catnb mice may be confounded by an increased bone density prior to the fracture, the results from lithium treatment shows that stabilization of β-catenin through a pharmacologic approach initiated after the fracture results in a similar enhancement in bone healing. This suggests that the enhanced bone repair in stabα1(I)-Catnb mice is due to β-catenin levels in the fracture healing cells committed to the osteoblast phenotype in the healing fracture, rather than to the underlying increased bone mass alone.Lithium can have other cellular effects in addition to inhibiting GSK3β. To determine if lithium is indeed acting through β-catenin, we gave lithium treatment to t Ad-Cre , suggestTaken together, our results confirm that lithium increases bone healing through its activation of β-catenin signaling pathway. These data, therefore, not only support our notion that β-catenin signaling functions differently at different stages of fracture repair, but more importantly, raises the possibility that lithium treatment can be used to enhance bone repair.In this study, we demonstrated that β-catenin signaling plays a crucial role in fracture healing. We observed that precise regulation of β-catenin is important in the early phases of fracture healing to allow differentiation of mesenchymal cells into osteoblast and chondrocyte lineages. Our findings also indicate that β-catenin plays a disparate role in undifferentiated mesenchymal cells and in committed osteoblasts, and as such acts differently during different phases of fracture repair. These findings are important not only for understanding the role of β-catenin in different cell types, but also has a practical implication for therapy—pre-fracture lithium treatment inhibits the repair process, but post-fracture treatment enhanced bone healing. As such, lithium will enhance fracture healing, but only if started after cells have become committed to the osteoblast lineage.During fracture repair, β-catenin-mediated TCF-dependent transcription is not activated in the earliest mesenchymal tissues present at the fracture site. Once cells that begin to show phenotypic features of either chondrocyte or osteoblast precursors, they exhibit β-catenin-mediated TCF-dependent transcription activity, suggesting a role in the initial phases of differentiation. In osteoblast lineage cells, β-catenin signaling activity decreased in later phases of differentiation, as osteoblasts matured into osteocytes. This finding is in agreement with a previous in vitro study, in which van der Horst and colleagues also observed a down-regulation of β-catenin signaling during the formation of a mineralized bone matrix in KS483 cells undergoing late phase osteoblast differentiation , suggest2+ pathway, which regulates Ca2+ flux and Ca2+-sensitive protein kinases and transcription factors, such as PKCα and calcium/calmodulin-dependent protein kinase II [Our results showed that many WNT ligands and receptors are selectively up-regulated during bone healing, several of which  are known to activate the canonical WNT pathway ,37. Moreinase II ,46. WNT1inase II ,48. Indelox(ex3)Catnb mice whose β-catenin-encoding gene had been stabilized by treatment with Cre recombinase. The fracture site in this case was also composed of immature mesenchymal cells, suggesting a block to differentiation. This observation was in stark contrast to the results from stabα1(I)-Catnb mice, which expressed osteoblast-specific stabilized β-catenin alleles; these mice exhibited significantly enhanced bone healing. Thus, during fracture healing, Cre-mediated conditional β-catenin stabilization represses differentiation of these mesenchymal cells, perhaps maintaining these cells in an undifferentiated proliferative state. Previous in vitro studies and studies of bone development provide evidence to support this notion, as WNT signaling activation maintains mesenchymal stem cells in a less differentiated state during osteogenic differentiation, and stabilization of β-catenin can interfere with the differentiation of precursors into chondrocytes as well as osteoblasts during development [We observed that β-catenin-mediated TCF-dependent transcription is activated during both chondrogenesis and osteogenesis in fracture repair. Mice expressing conditional β-catenin null alleles after treatment of Cre recombinase failed to heal, and there was a lack of bone and cartilage at the fracture site. The histology studies showed immature mesenchymal cells at the fracture sites. This finding is not unexpected given the role of β-catenin during fetal bone development, in which conditional deficiency in early osteoblast progenitors results in a blockade of osteoblast differentiation, with immature mesenchymal cells persisting ,50. Howeelopment –51. In cnullα1(I)-Catnb mice healed with abundant cartilage matrix at the fracture site, while tm2KemCatnb mice in which conditional β-catenin null alleles were regulated by Ad-Cre mainly exhibited undifferentiated mesenchymal-like cells at the fracture site. These data imply that mesenchymal precursors that have already become committed to an osteoblast phenotype will adopt a chondroblastic phenotype instead if β-catenin is absent. This result agrees with data from bone development, which show that osteoblast precursors lacking β-catenin are blocked in differentiation and develop into chondrocytes instead [Interestingly, fractures in  instead ,50.Our findings raise the possibility that therapy to activate β-catenin could be used to enhance fracture repair. However, since activation of β-catenin signaling inhibits differentiation of mesenchymal cells not yet committed to the osteoblastic phenotype, such treatment should be utilized only after cells are committed to the osteoblast lineage. One potential therapeutic agent is lithium, which activates the canonical WNT signaling through inhibition of GSK3β –43, and Alternative Language Abstract S1(144 KB PDF)Click here for additional data file.Figure S1t-test. p < 0.001 was considered statistically significant (*p < 0.001).For histomorphometric analysis, callus tissues at 3 wk after fracture were fixed in 4% paraformaldehyde, decalcified in 20% EDTA (pH 7.4), and embedded in paraffin. 10 μm sections were prepared and stained with HE. For each callus, an average of ten tissue sections was used to determine callus parameters, including trabecular thickness (μm), trabecular number (per mm), and trabecular separation (μm). Four animals were analyzed for each group. Data were expressed as mean ± standard deviation. Statistical differences were calculated by using Student tm2Kem, Catnblox(ex3),Catnb and Ad-GFP-treated control mice.(A) Trabecular number assay from Ad-Cre-treated tm2Kem, Catnblox(ex3),Catnb and Ad-GFP-treated control mice.(B) Trabecular thickness assay from Ad-Cre-treated tm2Kem, Catnblox(ex3),Catnb and Ad-GFP-treated control mice.(C) Trabecular separation assay from Ad-Cre-treated (D) Trabecular number assay from Ad-DKK1- and Ad-GFP-treated wild-type mice.(E) Trabecular thickness assay from Ad-DKK1- and Ad-GFP-treated wild-type mice.(F) Trabecular separation assay from Ad-DKK1- and Ad-GFP-treated wild-type mice.null, α1(I)-Catnbstab,α1(I)-Catnb and wild-type mice.(G) Trabecular number assay from null, α1(I)-Catnbstab,α1(I)-Catnb and wild-type mice.(H) Trabecular thickness assay from null, α1(I)-Catnbstab,α1(I)-Catnb and wild-type mice.(I) Trabecular separation assay from (781 KB PDF)Click here for additional data file.Figure S2At different time points after fracture, calluses were harvested and total RNA was isolated. Expression of mRNA was compared to that of the housekeeping control β-2 macroglobin. All experiments were performed in triplicate. Protein extracts were also isolated, and Western blot analysis was performed to determine protein expression. Protein expression was also normalized to GAPDH as a loading control.(A) Several WNT ligands  and receptors  were activated at mRNA level during fracture healing.(B) Both WNT5a and its signaling mediator PKCα were up-regulated during fracture repair.(737 KB PDF)Click here for additional data file.Figure S3Bone healing was observed 3 wk following generation of a tibia fracture in mice treated with lithium or control started after creation of the fracture. There were five mice in each group. In mice expressing β-catenin null alleles activated by treatment with Ad-Cre there was reduced bone mass compared to mice in which Ad-Cre was not administered or mice in which neither Ad-Cre nor lithium was administered. An asterisk above the mean indicates a statistically significant difference between β-catenin null and wild-type mice treated with lithium. Control mice (not treated with lithium) were assigned an average relative bone density of 1. These data show that the effect of lithium is at least partially mediated by β-catenin.(2.6 MB PDF)Click here for additional data file.Table S1(781 KB PDF)Click here for additional data file.Table S2(781 KB PDF)Click here for additional data file.


Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its α kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's α kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its α kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to  Shugoshin helps to keep newly replicated chromosomes together by protecting cohesins from phosphorylation, until the moment for the chromosomes to separate has arrived Cohesion between sister chromatids ensures that traction of sister chromatids towards opposite poles (known as bi-orientation) generates a tug-of-war between microtubules attempting to pull sisters apart and cohesion between them resisting this. The resulting tension is thought to stabilize the attachment of kinetochores to microtubules . Only whSister chromatid cohesion depends on a multisubunit complex called cohesin, which is composed of a heterodimer of Smc1 and Smc3 proteins associated with an α kleisin protein called Scc1 ,3, that In yeast, cohesin persists at centromeres and along chromosome arms until the onset of anaphase, whereupon it is removed by the APC/C-separase pathway . In animDrosophila melanogaster . Ce. Ce16]. To investigate whether the loss of cohesin from centromeres is due to precocious activation of separase, we used in situ immunofluorescence to assess cyclin B1 levels and localization of Mad2 in mock-treated and Sgo1-depleted cells 11 h after their release from the second thymidine block. In mock-treated cells, cyclin B1 was concentrated within the nuclei of prophase cells and on the mitotic spindles of all prometaphase and most metaphase cells, although also throughout their cytoplasm, and was absent from anaphase or telophase cells A. To quaAnother event that normally occurs at the onset of anaphase is the disappearance of Aurora B from inner centromeres and its accumulation at the spindle midzone . Aurora Removal of cohesin from chromosome arms during prophase and prometaphase depends on the activity of mitotic kinases, including Plk1. Might this Plk1-dependent process in normal cells be prevented from attacking centromeric cohesin by Sgo1? To test this idea, we analysed whether synchronised cells depleted for both Sgo1 and Plk1 also lose sister chromatid cohesion when they enter and arrest in mitosis. Mock-treated, Plk1-depleted, Sgo1-depleted, and Sgo1- and Plk1-depleted cells were harvested at 7, 9, and 11 h after release from the thymidine block, and the state of spread chromosomes was analysed by Giemsa staining  that Plk1 is not necessary for the precocious separation of sister chromatids induced by Sgo1 depletion. Interestingly, Sgo1 depletion permitted not only sister centromere separation but also that along chromosome arms in cells supposedly lacking Plk1. This raises the possibility that the tight cohesion between sister chromatid arms that persists in Plk1-depleted cells depends on Sgo1.Aurora B kinase activity has also been implicated in the removal of cohesin from chromosome arms. To address whether this kinase is responsible for the precocious loss of sister centromere cohesion in Sgo1-depleted cells, we examined whether inhibition of Aurora B by the small molecule inhibitor Hesperadin can suppress their sister chromatid separation. Because Aurora B and its yeast equivalent Ipl1 are also required to prevent cell cycle arrest of cells with defective sister chromatid cohesion  or cellsInterestingly, treatment with nocodazole had a clear effect on the arrangement of the separated sisters in Sgo1-depleted cells. By 9 h, up to 73% of mitotically arrested cells resembled the image shown in We conclude that Hesperadin cannot prevent the precocious separation of sister chromatids induced by Sgo1 depletion, at least when such cells are prevented from exiting mitosis by addition of nocodazole. In a very similar experiment in which nocodazole was omitted, Hesperadin did indeed reduce the number of mitotic cells whose sisters had separated precociously (unpublished data). We believe that this effect is probably a statistical artefact caused by the failure of Sgo1-depleted cells treated with Hesperadin to arrest in mitosis.In a related paper, Hauf et al.  describeWe therefore analysed the effects of Sgo1 depletion in cell lines in which either wild-type SA2 or SA2 12xA protein (both tagged with nine myc epitopes) is expressed from a doxycycline-inducible promoter at levels (when induced) that are comparable to endogenous SA2 protein see A and S5.It is nevertheless possible that nonphosphorylatable Scc3-SA2 suppresses a mitotic defect of Sgo1-depleted cells responsible for their cell cycle arrest, and as a consequence these cells might not have long enough to lose sister chromatid cohesion. To address this possibility, nocodazole was added to doxycycline-induced cells 4 h after transfection , which caused both mock-treated and Sgo1-depleted cells to accumulate in mitosis C. In theTo examine more carefully whether expression of nonphosphorylatable Scc3-SA2 permits cells lacking Sgo1 to undergo anaphase, we created a new cell line that expressed histone H2B tagged with EGFP as well as doxycycline-inducible Scc3-SA2 12xA-myc. Sgo1 was depleted in induced and uninduced cells from the same cell line and the behaviour of chromosomes followed by time-lapse video microscopy D. Three Finally, we addressed whether the loss of mitotic phosphorylation sites on Scc3-SA2 enables cohesin to persist at centromeres as well as along chromosome arms in mitotic Sgo1-depleted cells. To do this, we analysed mitotic chromosomes from Sgo1-depleted cells expressing either Scc3-SA2-myc or Scc3-SA2 12xA-myc. Wild-type SA2 myc-tagged protein was associated with chromosomes in very few cells, while nonphosphorylatable SA2 myc tagged protein was found along the axes of all chromosomes in nearly 40% of cells E and 8F.It has long been recognized that cohesion between sister chromatids in the vicinity of centromeres has an especially important role during both meiosis and mitosis. Centromeric cohesion is special during meiosis because it completely resists destruction at the first meiotic division when dissolution of cohesion along chromosome arms triggers the resolution of chiasmata. It is special during mitosis because, unlike cohesion along chromosome arms, centromeric cohesion persists even when cells are arrested in mitosis for prolonged periods by spindle poisons. The work described in this paper suggests, somewhat surprisingly, that both of these special attributes of centromeric cohesion might be conferred by the same protein, namely Sgo1.S. cerevisiae and its probable orthologue in D. melanogaster MEI-S332 are associated with centromeres during mitosis as well as meiosis. We show here that a related protein in human cells concentrates at centromeres during mitosis and disappears from chromosomes during anaphase. Remarkably, depletion of human Sgo1 by RNAi prevents HeLa cells from completing mitosis. Though most chromosomes congress to a metaphase plate in Sgo1-depleted cells, cells never undergo anaphase and instead arrest for a prolonged period in a prometaphase-like state with high cyclin B1 levels. Our data suggest that this is caused by a catastrophic separation of sister chromatids at around the time cells should have normally undergone anaphase, and that this event occurs in the absence of APC/C activation or dissociation of Aurora B from centromeres. This precocious separation is accompanied by loss of cohesin from centromeres as well as chromosome arms and activation of the mitotic spindle checkpoint, as documented by persistent recruitment of Mad2 to kinetochores. Remarkably, expression of a version of cohesin's Scc3-SA2 subunit whose C-terminal domain can no longer be phosphorylated as cells enter mitosis (Scc3-SA2 12xA-myc) not only alleviates the precocious loss of sister chromatid cohesion of Sgo1-depleted cells but also permits a large fraction of them to complete what appears, at least superficially, to be a normal mitosis.Recent work suggests that, in both budding and fission yeast, Sgo1 protects centromeric cohesion at meiosis I by preventing cleavage of cohesin's α kleisin subunit by separase. Surprisingly, both Sgo1 in Our findings together with those described by Hauf et al.  suggest The suppression of the mitotic arrest induced by Sgo1 depletion by Scc3-SA2 12xA-myc implies that it is the precocious loss of sister chromatid cohesion and not the lack of some other function of Sgo1 that is largely responsible for the pathological mitotic arrest of Sgo1-depleted cells. Our findings demonstrate for the first time that Sgo1-like proteins promote sister chromatid cohesion by regulating the activity of cohesin. This is inconsistent with the suggestion that Sgo1/MEI-S332 proteins confer a form of sister chromatid cohesion during mitosis that is distinct from that conferred by cohesin ,35.Salic et al.  have alsHow might Sgo1 regulate cohesin? One possibility is that Sgo1 enables cohesin to be refractory to the effects of Scc3-SA2 phosphorylation. If cohesin embraces chromatid fibres within its ring structure, then phosphorylation of Scc3-SA2's C-terminal tail might induce the cohesin ring to open, and this process might be blocked by Sgo1. Alternatively, Sgo1 might prevent cohesin's dissociation by blocking Scc3-SA2's phosphorylation in the first place or by recruiting a phosphatase that reverses its phosphorylation B. AntiboSeveral lines of evidence suggest that the process against which Sgo1 protects centromeric cohesin does not involve separase-mediated Scc1 cleavage. First, Scc3-SA2 12xA-myc's ability to suppress cohesin's dissociation both from arms in wild-type cells and from centromeres in Sgo1-depleted cells suggests that these two phenomena have a common cause. If the former is separase-independent, then so, presumably, is the latter. Second, the persistence of cyclin B1 throughout the period during which centromeric cohesion is lost in Sgo1-depleted cells implies that the APC/C is not activated and separase must therefore remain associated with its inhibitor chaperone securin. Third, cells expressing Scc3-SA2 12xA-myc proliferate fairly normally, and their timely destruction of sister chromatid cohesion at the onset of anaphase presumably involves the activation of separase by the APC/C. If separase deregulation were responsible for cohesin's precocious disappearance from centromeres in Sgo1-depleted cells, then Scc3-SA2 12xA-myc would have to suppress this defect by delaying Scc1 cleavage, which is difficult to reconcile with the timely onset of anaphase in cells expressing Scc3-SA2 12xA-myc. It is therefore likely that Sgo1 protects centromeric cohesin from different processes during mammalian mitosis and yeast meiosis, from chromosomal dissociation induced by Scc3-SA2 phosphorylation during mitosis, and from Rec8 or Scc1 cleavage during meiosis. Although different, both processes may have a key property in common, namely regulation by Plk1-mediated phosphorylation.It is noteworthy that many shugoshins, including human Sgo1, disappear from centromeres during anaphase. This event might be triggered by their destruction at the hands of the APC/C along with securin and cyclin B . This evThe identity of the mitotic kinases responsible for phosphorylation of Scc3-SA2 remains unclear. Because Plk1 is required for dissociation of cohesin from chromosome arms in HeLa cells containing Sgo1 , it was D. melanogaster, MEI-S332, is not apparently essential for somatic cell divisions, which contrasts with Sgo1's essential function in HeLa cells. It is conceivable that small changes in the kinetics with which cohesin is dissociated from centromeres in Sgo1/MEI-S332-depleted cells is key to whether a cell is able to undergo anaphase before precocious loss of sister chromatid cohesion triggers the mitotic spindle checkpoint.Our study of human Sgo1 has hitherto been confined to HeLa cells. It will be important in the future to analyse its function in other cell types, especially in untransformed cells, and ultimately in real tissues from mice whose Sgo1 gene could be deleted by homologous recombination. It is curious, for example, that Sgo1's likely orthologue in One of the most surprising implications of our work is that centromeric sister chromatid cohesion, which has long appeared to be a very stable state and to be the cornerstone  of mitotic chromosomes, only exists because shugoshins protect centromeric cohesin from mitotic protein kinases that maraud mitotic chromosomes and threaten to destroy their integrity. This may have important implications for meiotic cells where protection of cohesin by Sgo1-like proteins  is essential for the second meiotic division. Chromosome mis-segregation during meiosis is responsible for trisomies such as Down's syndrome and possibly for age-related infertility and a large fraction of spontaneous abortions . Our wor10) ; rat anti-HP-1β ; and rabbit anti-SA2  with the sequence YKEPTLASKLRRGDPFTDL (aa 474–492) from the C terminus of a mouse cDNA  and a second (antibody 95) with the sequence QKRSFQDTLEDOKNRMKEKRNKN (aa 7–29) from the N terminus of a mouse cDNA (the corresponding sequence in human protein contains just four substitutions). Each peptide was conjugated with KLH and injected into rabbits. Sera were taken by a standard scheme, and specific antibodies were obtained by affinity purification using antigenic peptide-conjugated columns. For Western blotting, blots were incubated with the primary antibodies overnight at 4 °C at a 1:500 dilution. For immunofluorescence, cells were incubated with the primary antibody overnight at 4 °C at a dilution of 1:100. Other antibodies used in this study were: mouse anti-Aurora B antibody ; CREST serum ; mouse anti-cyclin B1 antibody ; rabbit anti-Mad2 serum ; mouse a466; see ).2, and 200 μg/ml RNase A] for 20 min at 37 °C.HeLa cells were cultured in DMEM supplemented with 10% FCS, 0.2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. For synchronisation, HeLa cells were grown in the presence of 1 mM thymidine  for 12 h, washed with PBS, and grown in fresh medium for 8 h. Cells were then transfected (see below) in serum-free medium  supplemented with 0.3% FCS for 4 h, followed by addition of 1 mM thymidine and FCS to a final concentration of 20%. After a further 12 h, cells were again washed in PBS and transferred to fresh medium. Samples were harvested at various time points up to 15 h after the second release, as described below and in the figure legends. Nocodazole and Hesperadin were each used, as indicated, at a final concentration of 100 ng/ml and 100 nM, respectively. Doxycycline-inducible nonphosphorylatable SA2 HeLa cells were established as described in the accompanying paper . For genSgo1 (oligo Sgo1.1A) was 5′-CAGUAGAACCUGCUCAGAA-3′. The oligonucleotide sequence used to target human Plk1 cDNA was 5′-CGAGCUGCUUAAUGACGAG-3′  and cloned them into the pGEM-T vector . Multiple clones were sequenced and PCR error-free sequences hsSgo1 cDNA cloning, we had found two potential splicing variants in the ENSEMBL database (BC001339 and BC017867) that have different 3′ sequences. When primers 5′-CTGGAGAGCTTCGAAGAGCCTTGA-3′ and 5′-CCTCTCCTGAAGCAACAGAAAGAG-3′ (designed to amplify products from BC001339) were used, 0.9- and 1.0-kb bands were detected (lane 3), and hsSgo1A–hsSgo1D cDNAs were obtained. When primers 5′-CTGGAGAGCTTCGAAGAGCCTTGA-3′ and 5′-CTGAGTGAAACAGACTGTCAACAC-3′ (designed to amplify products from BC017867) were used, six bands between 0.9 and 2.0 kb were detected (lane 4), and cDNA clones obtained from those bands were named hsSgo1E–hsSgo1H and hsSgo1J–hsSgo1L. We could not obtain a clone that encodes the identical open reading frame encoded by BC001339.For Sgo1 in HeLa cells and various other human and mouse tissues, we performed Northern analysis. Total HeLa RNA was prepared as described in (A), and 20 μg was run on a formaldehyde agarose gel and RNA blots were prepared. A blot was probed with a [32P]-labelled probe (probe H1) generated against the common N terminus region of hsSgo1 [nucleotides (nt) 1–647 of hsSgo1E). Many bands, although faint and ambiguous, were seen (lane 1). We could not assign each signal to a specific cloned cDNA variant; however, these data support the notion that many species of splicing variant are expressed in HeLa cells. When the blot was probed by another probe (probe H2) that is designed to hybridize to the central region specific for hsSgo1E and hsSgo1F cDNAs (nt 665–1454 of hsSgo1E), a signal at ∼2.2 kb was seen (lane 2). Using two probes of hsSgo1, we could confirm that the two relatively long transcripts, hsSgo1E and hsSgo1F, that possess the central region, and the other shorter transcripts that lack the central region, are expressed in HeLa cells.(B) To examine the expression of hsSgo1 mRNA was studied using a commercially available Multi Tissue Northern blot . Multiple, relatively strong, signals were detected in testis, among other organs, on both blots probed by H1 and H2 . This higher expression of Sgo1 in testis might reflect the fact that testis contains a higher fraction of proliferating cells. Expression analysis of mouse Sgo1 was also performed in parallel. Both a probe against the common N-terminal region of mmSgo1  and an mmSgo1A-specific probe  detected potential mmSgo1 messages in spleen and testis . Mouse Sgo1 messages in spleen were more abundant than in testis, but human Sgo1 message was not detected in human spleen. The biological significance of the different levels of Sgo1 expression observed between human and mouse spleen is unclear. The tested polyA+ RNAs are from spleen (SP), thymus (TH), prostate (PR), testis (TE), ovary (OV), small intestine (SI), colon (CO), peripheral blood leukocyte (PL), heart (HE), brain (BR), lung (LU), liver (LI), skeletal muscle (SM) and kidney (KI). In conclusion, we cloned multiple splicing variants of hsSgo1s and mmSgo1s from HeLa cells and mouse organs. The presence of a variety of messages was also confirmed. However, the functional difference between and biological meaning of such variation remains unknown and requires further study.(C) The tissue distribution of hsSgo1A–hsSgo1H and hsSgo1J–hsSgo1L.(D) Structures of cDNAs for (E) Predicted structures of proteins depicted as cDNAs in (D).mmSgo1A and mmSgo1B.(F) Structures of cDNAs for (G) Predicted structures of proteins depicted as cDNAs in (F).(2.7 MB EPS).Click here for additional data file.Figure S2Identical Sgo1 localisation patterns were obtained by staining cells with two peptide antibodies raised against different regions of the protein (see (A) HeLa cells grown on glass coverslips were paraformaldehyde-fixed and costained with Sgo1 antibody 94  and CREST antiserum (shown in red) to label kinetochores. Five different stages of mitosis are illustrated as indicated.(B) As (A), except cells were costained with Aurora B antibody (shown in red) to label inner centromere.(C) As (B), except Sgo1 was stained with antibody 95 .(10 MB EPS).Click here for additional data file.Figure S3Synchronised HeLa cells were transfected as described in (1.3 MB EPS).Click here for additional data file.Figure S4HeLa cells expressing Scc1-myc were mock-treated or Sgo1 siRNA-transfected and harvested as described for (583 KB EPS).Click here for additional data file.Figure S5A sample of the cells from the experiment described in (4.7 MB TIF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) and ENSEMBL (http://www.ensembl.org/) accession numbers of the genes discussed in this paper are hsSgo1  and mmSgo1 . The pending GenBank/EMBL/DDBJ accession numbers of the cDNA sequences discussed in hsSgo1A (AB193056), hsSgo1B (AB193057), hsSgo1C (AB193058), hsSgo1D (AB193059), hsSgo1E (AB193060), hsSgo1F (AB193061), hsSgo1G (AB193062), hsSgo1H (AB193063), hsSgo1J (AB193064), hsSgo1K (AB193065), hsSgo1L (AB193066), mmSgo1A (AB193067), and mmSgo1B (AB193068).The NCBI GeneID (


Accurate chromosome segregation during meiosis and mitosis is essential for the maintenance of genomic stability. Defects in the regulation of chromosome segregation during division predispose cells to undergo mitotic catastrophe or neoplastic transformation. Cohesin, a molecular glue holding sister chromatids together, is removed from chromosomes in a stepwise fashion during mitosis and meiosis. Cohesin at centromeres but not on chromosome arm remains intact until anaphase onset during early mitosis and the initiation of anaphase II during meiosis. Several recent studies indicate that the activity of protein phosphatase 2A is essential for maintaining the integrity of centromeric cohesin. Shugoshin, a guardian for sister chromatid segregation, may cooperate with and/or mediate PP2A function by suppressing the phosphorylation status of centromeric proteins including cohesin. At the molecular level, cohesin mediates sister chromatid cohesion through formation of a proposed "ring" structure that entraps chromosomes [Sister chromatid cohesion is established during DNA replication and maintained throughout Gomosomes . The cohomosomes . Upon miomosomes  that is omosomes ,5.During the past several years, the mechanism preventing centromeric cohesin from undergoing cleavage/dissociation during early mitosis as well as meiosis I and early meiosis II has been a subject of intensive investigation ,7. ThrouNature and Developmental Cell have convincingly demonstrated that enhanced dephosphorylation of centromeric proteins including cohesin may be a key mechanism responsible for centromeric cohesion of sister chromatids during meiosis and mitosis [A recent study by the Nasmyth group showed that expression of SA2 mutant resistant to Plk1 phosphorylation results in suppression of premature separation of sister chromatids in Sgo1-depleted cells . This su mitosis -17.Par1B', Ppa2C) are important for accurate chromosome segregation in fission yeast as their deletions results in moderate to severe missegregation of chromosomes; a detailed analysis revealed that chromosome missegregation occurs in meiosis II, but not in meiosis I, in fission yeast with either Par1B' or Ppa2C deletion [Sgo1 deletion. These observations thus suggest that PP2A, like Sgo1, may protect centromeric but not arm cohesion of sister chromatids. Consistent with this notion, fission yeast mutants with deletion of Par1B' or Sgo1 are impaired in retaining centromeric Rec8 following meiosis I whereas deficiency in PP2A activity does not have an effect on expression as well as centromeric localization of Rec8 before the onset of anaphase I [Using affinity protein purification followed by tandem mass spectrometry, the Nasmyth group found that protein phosphatase 2A (PP2A) subunits are the prevalent components that are co-purified in meiotic I cells of both fission and budding yeasts . PP2A isdeletion , a defecaphase I .The mechanism by which PP2A protects centromeric cohesin is fully conserved in mammalian cells. Through a combination of co-immunoprecipitation and mass spectrometry, the Watanabe group identified PP2A as a major component in Sgo1, but not in control, immunoprecipitates , suggestSimilar results regarding PP2A's physical interaction with Sgo1 in human cells are also reported by the Yu group . Co-immuDrosophila, Polo phosphorylates MEI-S332, which is essential for its removal from centromeres at anaphase [In eukaryotes, a hallmark for mitotic entry is the activation of mitotic kinases , resulting in phosphorylation of numerous mitotic targets essential for mediating mitotic progression. Early studies established that Bub1 is upstream of Sgo1 in the regulatory hierarchy ,19. Receanaphase ; (iv) Planaphase  althoughGiven the close involvement of Plk1/Polo in regulating anaphase entry and chromosome segregation, it is relevant to examine how Plk1 regulates PP2A activity, which in turn modulates centromeric cohesion. Interestingly, the Yu group showed that co-depletion of Plk1 and a key PP2A subunit through transfection of specific siRNAs causes retention of Sgo1 at centromeres in human cells, which is correlated with significant reduction of chromosomal missegregation; in contrast, co-depletion of Plk1 and Sgo1 fails to prevent missegregation of chromosomes ,15. ThusSgo1 deletion during meiosis I, implying that the localization of PP2A to the centromeric region depends on Sgo1 [The identification of PP2A as a new component in protecting centromeric cohesion of sister chromatids prompts additional studies determining its position in the regulatory hierarchy. As Sgo1 is primarily localized at centromeres during meiosis or early mitosis whereas PP2A signals are detected throughout the cell, it is possible that Sgo1 may help to recruit a specific form of PP2A to centromeres. This notion is consistent with the fact that most PP2A is not associated with Sgo1 . Support on Sgo1 . On the  on Sgo1 . Consist on Sgo1 .Although it remains not entirely clear how Sgo1 regulates PP2A or vice versa with regard to protection of centrometic coehsin/cohesion during meiosis and mitosis, a simple model is proposed to illustrate the regulatory hierarchy in animal cells (Figure