Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes

During differentiation, megakaryocytes increase ploidy through a process called endomitosis, whose mechanisms remain unknown. As it corresponds to abortive mitosis at anaphase and is associated with a multipolar spindle, investigation of chromosome segregation may help to better understand this cell-cycle abnormality. To examine this variation, a new method was developed to combine primed in situ labeling to label centromeres of one chromosome category and immunostaining of tubulin. Human megakaryocytes were obtained from normal bone marrow culture. By confocal microscopy, this study demonstrates an asymmetrical distribution of chromosomes (1 or 7) either between the spindle poles at anaphase stage of endomitosis and between the different lobes of interphase megakaryocyte nuclei. The metaphase/anaphase checkpoint appears normal on the evidence that under nocodazole treatment megakaryocytes progressively accumulate in pseudo-metaphase, without spontaneous escape from this blockage. Immunostaining of p55CDC/hCDC20 with similar kinetochore localization and dynamics as during normal mitosis confirms this result. HCdh1 was also expressed in megakaryocytes, and its main target, cyclin B1, was normally degraded at anaphase, suggesting that the hCdh1-anaphase-promoting complex checkpoint was also functional. This study found the explanation for these unexpected results of an asymmetrical segregation coupled to normal checkpoints by careful analysis of multipolar endomitotic spindles: whereas each aster is connected to more than one other aster, one chromosome may segregate symmetrically between 2 spindle poles and still show asymmetrical segregation when the entire complex spindle is considered.     

The activity of extracellular signal-regulated kinase is required during G2/M phase before metaphase–anaphase transition in synchronized leukemia cell lines

The pharmacological inhibitors of extracellular signal-regulated kinase (ERK) have been suggested as a novel molecular target-based therapy for acute myeloid leukemia. Several studies have established the role of ERK in cell cycle progression from G₁ to S phase in response to mitogen, but the role of ERK after the restriction point is less clarified. In this study, we used models of aphidicolin and nocodazole-synchronized HL-60 and NB4 leukemia cell lines to determine the kinetics of ERK activity during the progression of the cell cycle and to test the effects of commercially available inhibitors on G₂/M progression of synchronized leukemia cells. In aphidicolin-synchronized cells, the activity of ERK was low during early S phase and increased at late S and G₂/M phase of the cell cycle. The presence of MEK inhibitors PD 98059 and U0126 caused a delay in G₂/M phase. In nocodazole-synchronized cells, the activity of ERK was low during M/G₁ transition and MEK inhibitors had no effects on return of the cells to G₁ phase. These results demonstrate that the activity of ERK is required during G₂/M phase of leukemia cell cycle before the cells reach metaphase–anaphase transition.     

From the Cover: Regulation of ciliary polarity by the APC/C

Planar cell polarity signaling controls a variety of polarized cell behaviors. In multiciliated Xenopus epidermal cells, recruitment of Dishevelled (Dvl) to the basal body and its localization to the center of the ciliary rootlet are required to correctly position the motile cilia. We now report that the anaphase-promoting complex (APC/C) recognizes a D-box motif of Dvl and ubiquitylates Dvl on a highly conserved lysine residue. Inhibition of APC/C function by knockdown of the ANAPC2 subunit disrupts the polarity of motile cilia and alters the directionality of the fluid movement along the epidermis of the Xenopus embryo. Our results suggest that the APC/C activity enables cilia to correctly polarize in Xenopus epidermal cells.     

Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome

Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis.     

Regulation of APC development, immune response, and autoimmunity by Bach1/HO-1 pathway in mice

APCs are essential for innate and adaptive immunity as well as self-immune tolerance. Here, we show that the Cap'n'collar member Bach1 regulates the generation of APCs, specifically macrophages and dendritic cells, in mice. The impaired APC development in Bach1(-/-) mice was accompanied by defects in downstream T-cell responses and partial protection from experimental autoimmune encephalomyelitis. Genomewide analyses identified a panel of Bach1 target genes and ablation of the direct Bach1 target gene HO-1 exacerbated the impaired APC development observed in Bach1(-/-) mice. This was attributed to the impaired ability of HO-1(-/-)Bach1(-/-) double mutants to produce upstream APC progenitor cells, including common myeloid progenitor (CMP)-Flk2(+). By contrast, we observed an increase in hematopoietic stem-progenitor cells (HSPCs) in these mice, suggesting a developmental block in the progression of HSPCs to CMP-Flk2(+) and subsequently APCs.     

Lkb1/Stk11 regulation of mTOR signaling controls the transition of chondrocyte fates and suppresses skeletal tumor formation

Liver kinase b1 (Lkb1) protein kinase activity regulates cell growth and cell polarity. Here, we show Lkb1 is essential for maintaining a balance between mitotic and postmitotic cell fates in development of the mammalian skeleton. In this process, Lkb1 activity controls the progression of mitotic chondrocytes to a mature, postmitotic hypertrophic fate. Loss of this Lkb1-dependent switch leads to a dramatic expansion of immature chondrocytes and formation of enchondroma-like tumors. Pathway analysis points to a mammalian target of rapamycin complex 1-dependent mechanism that can be partially suppressed by rapamycin treatment. These findings highlight a critical requirement for integration of mammalian target of rapamycin activity into developmental decision-making during mammalian skeletogenesis.Growth of the endochondral skeleton is dependent on a cartilaginous growth plate. In the growth plate, mitotic chondrocytes transition to a postmitotic, terminal hypertrophic chondrocyte fate (Fig. S1A). Reciprocal signaling between prehypertrophic chondrocyte-derived Indian hedgehog (Ihh) and epiphyseal secreted parathyroid hormone-related peptide (Pthrp; also known as Pthlh) controls the spatial positioning of the hypertrophic transition and the normal growth properties of the skeleton (1–3). The present study demonstrates an unexpected role for liver kinase b1 (Lkb1; also known as Stk11) in growth plate regulation.Lkb1 is a multifunctional serine/threonine kinase inhibitor of mTOR signaling whose activity regulates cell cycle progression, cellular energy homeostasis, and cell polarity (4, 5). Mouse embryos lacking Lkb1 die at midgestation with vascular and neural tube defects (6), and germ-line inactivating mutations of Lkb1 in the human population underlie Peutz–Jeghers syndrome, characterized by development of benign polyps in the gastrointestinal tract, and an increased risk of various types of epithelial cancers (7, 8). Conditional ablation of Lkb1 in pancreatic, vascular, neural and cardiac tissue links Lkb1 to tissue specific actions in a variety of organ systems (9). Here, we provide evidence that Lkb1 regulation of mammalian target of rapamycin complex 1 (mTORC1) action is a critical step in the transition of mitotic chondrocytes to postmitotic hypertrophic fates suppressing cartilaginous tumor-like growths in the postnatal mammalian skeleton.     

Complement components C1q,C1r/C1s,and C1inh in rheumatoid arthritis

Objective. To analyze the synovial site and the cell types expressing C1q, C1r/C1s, and C1–esterase inhibitor (C1INH) and to characterize newly synthesized C1q in patients with rheumatoid arthritis (RA). Methods. Tissue and primary cell cultures of synovium from RA patients were analyzed for C1q, C1r/C1s, and C1INH by Northern blotting, in situ hybridization, and pulse-chase experiments for C1q. Results. The de novo synthesis of C1q, C1r/C1s, and C1INH in synovium and primary cell cultures was proven by Northern blot and by antigenic and functional analysis. In in situ hybridization experiments, the synovial lining cell layer was identified as the site of C1q, C1r, and C1INH expression. In contrast, immunohistologic analysis showed that C1q, C1s, and C1INH proteins were present in a thin film covering the synovial lining cells. In situ hybridization performed on primary cell cultures provided evidence that only macrophages were able to express C1q, whereas fibroblasts and stellate cells synthesized C1r. Conclusion. The synovium is important for the synthesis and secretion of C1q and C1r/C1s, as well as the control protein C1INH, which supports the idea of a locally occurring inflammatory process in RA patients.     

C7a, a biphosphinic cyclopalladated compound, efficiently controls the development of a patient-derived xenograft model of adult T cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a highly aggressive disease that occurs in individuals infected with the human T lymphotropic virus type 1 (HTLV-1). Patients with aggressive ATLL have a poor prognosis because the leukemic cells are resistant to conventional chemotherapy. We have investigated the therapeutic efficacy of a biphosphinic cyclopalladated complex {Pd(2) [S(-)C(2), N-dmpa](2) (μ-dppe)Cl(2)}, termed C7a, in a patient-derived xenograft model of ATLL, and investigated the mechanism of C7a action in HTLV-1-positive and negative transformed T cell lines in vitro. In vivo survival studies in immunocompromised mice inoculated with human RV-ATL cells and intraperitoneally treated with C7a led to significantly increased survival of the treated mice. We investigated the mechanism of C7a activity in vitro and found that it induced mitochondrial release of cytochrome c, caspase activation, nuclear condensation and DNA degradation. These results suggest that C7a triggers apoptotic cell death in both HTLV-1 infected and uninfected human transformed T-cell lines. Significantly, C7a was not cytotoxic to peripheral blood mononuclear cells (PBMC) from healthy donors and HTLV-1-infected individuals. C7a inhibited more than 60% of the ex vivo spontaneous proliferation of PBMC from HTLV-1-infected individuals. These results support a potential therapeutic role for C7a in both ATLL and HTLV-1-negative T-cell lymphomas.     

An intermolecular electrostatic interaction controls the prepore-to-pore transition in a cholesterol-dependent cytolysin

β-Barrel pore-forming toxins (βPFTs) form an obligatory oligomeric prepore intermediate before the formation of the β-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for βPFTs. Using the archetype βPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant β-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a βPFT.The cholesterol-dependent cytolysins (CDCs) make up the largest class of bacterial β-barrel pore-forming toxins (βPFTs) and are present in nearly 50 Gram-positive opportunistic pathogens. A central paradigm of βPFTs is the formation of an obligatory intermediate termed the prepore (1–6). The prepore is a membrane-bound oligomerized ring-shaped complex in which the membrane-spanning β-barrel pore has not formed. Pore conversion, characterized by β-barrel insertion, only occurs after prepore completion. For all βPFTs, the molecular mechanism or mechanisms that control the transition from the prepore to the pore remains unknown.The archetype CDC, perfringolysin O (PFO), coordinates the assembly of about 37 monomers into a prepore and then assembles and inserts into the membrane a β-barrel pore composed of 74 amphipathic hairpins (7–12). On prepore completion, a signal is generated within the oligomeric complex that triggers this transition. The nexus of the prepore-to-pore transition is the disruption of the interface that domain 3 (D3) forms with domains 1 and 2 (D1,2) (10) (Fig. 1). This interface is formed between D1,2 and one of the two D3 α-helical bundles. The D3 α-helical structures ultimately unfurl and refold into the two transmembrane β-hairpins (TMH1 and TMH2), which contribute to the formation of the giant β-barrel pore (Fig. 1) (11–13). Pore formation also requires that the prepore undergo a 40-Å vertical collapse that allows the TMHs to span the bilayer (7, 8, 14).Open in a separate windowFig. 1.PFO structure and pore complex assembly. (A) The PFO ribbon structure (19) with magnified D3 (Right) with pertinent residues and structures identified. The D3 core β-sheet (β1–β4) is shown in cyan, and the β5α1 loop is shown in magenta. The α-helical bundles that refold into TMH1 and TMH2 are shown in yellow and purple, respectively (11, 12). The waters surrounding N197 are shown as red spheres. The membrane-binding interface (34–37) is circled. Structures were created and rendered using UCSF Chimera (38). (B) Schematic overview of the PFO prepore assembly and its conversion to the pore complex (39). The D3 counterparts (dashed circle) are colored the same as in A. Bound monomers oligomerize into the prepore structure, and then the D3 interface with domains 1 and 2 (D1,2) is disrupted and the α-helical bundles unfurl and refold into the TMHs to form the β-barrel pore.Here we show that the prepore-to-pore transition is controlled by an intermolecular electrostatic interaction between E183 and K336 in PFO. Loss of either charged residue traps PFO in a stable prepore state, whereas pore formation can be restored by weakening the D3–D1,2 interface by a single mutation or by increasing the temperature. Molecular measurements with cross-linkers and molecular dynamics simulations suggest these two residues are positioned to form a strong electrostatic interaction or salt bridge. Our studies suggest the interactions that stabilize the D3–D1,2 interface are the primary barrier to the prepore-to-pore transition and further show that the E–K interaction provides the necessary free energy to overcome this transition state barrier.     

Analysis of phenotype resistance to activated protein C (APC resistance)

The laboratory diagnosis of resistance to activated C protein (APC-resistance) involves examination of the phenotype and genotype of this thrombophilia. For examination of the phenotype coagulation and chromogenic tests are used. Their essence is examination in the presence and absence of exogenous APC. While the result of the original coagulation examination of APC-resistance which uses the APTT principle is influenced by a number of factors, the sensitivity and specificity of the modification of this examination (dilution of the examined plasma sample by FV deficient plasma before making the test) in relation to detection of FV Leiden is almost 100% and eliminates the majority of limitations of the original examination. The chromogenic assessment of APC-resistance has similar advantages, however, it cannot differentiate between the heterozygous and homozygous form of FV Leiden. During examination of the genotype of subjects with APC-resistance the mutation of FV Leiden is detected in as many as 90%. The group of subjects with the phenotype of APC-resistance comprises in particular subjects with acquired APC-resistance caused by conditions which lead to a disbalance between procoagulation and anticoagulation proteins of haemostasis which influence the reactions of the applied laboratory examinations. The acquired phenotype of APC-resistance can be also associated with an increased risk of thrombosis and the clinical manifestations of this thrombophilia resemble the classical, FV Leiden conditioned APC resistance. Rarely also congenital causes of the phenotype of APC-resistance are encountered caused by another mutation than the Leiden mutation of gene FV. The concurrent examination of the patient's plasma with the original and modified coagulation test makes it possible to assess the inborn cause of APC-resistance (positive finding also in modified examination). The presence of FV Leiden is then confirmed by examination of the genotype by the polymerase chain reaction.     

Structural analysis of human Cdc20 supports multisite degron recognition by APC/C

The anaphase-promoting complex/cyclosome (APC/C) promotes anaphase onset and mitotic exit through ubiquitinating securin and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 (Cdc20) protein, directly interacts with APC/C degrons––the destruction (D) and KEN boxes. APC/C^Cdc20 is the target of the spindle checkpoint. Checkpoint inhibition of APC/C^Cdc20 requires the binding of a BubR1 KEN box to Cdc20. How APC/C recognizes substrates is not understood. We report the crystal structures of human Cdc20 alone or bound to a BubR1 KEN box. Cdc20 has a disordered N-terminal region and a C-terminal WD40 β propeller with a preformed KEN-box-binding site at its top face. We identify a second conserved surface at the side of the Cdc20 β propeller as a D-box-binding site. The D box of securin, but not its KEN box, is critical for securin ubiquitination by APC/C^Cdc20. Although both motifs contribute to securin ubiquitination by APC/C^Cdh1, securin mutants lacking either motif are efficiently ubiquitinated. Furthermore, D-box peptides diminish the ubiquitination of KEN-box substrates by APC/C^Cdh1, suggesting possible competition between the two motifs. Our results indicate the lack of strong positive cooperativity between the two degrons of securin. We propose that low-cooperativity, multisite target recognition enables APC/C to robustly ubiquitinate diverse substrates and helps to drive cell cycle oscillations.     

Blockade of gC1qR/p33, a receptor for C1q, inhibits adherence of Staphylococcus aureus to the microvascular endothelium

Endovascular infections with Staphylococcus aureus (S. aureus) are associated with high mortality. gC1qR/p33 (gC1qR), a receptor for the complement component C1q expressed on endothelial cells, interacts with protein A of S. aureus and gC1qR blockade reduces S. aureus colonization during infective endocarditis. The aim of this study was to analyze in vivo whether this observation is due to a decreased interaction of S. aureus with the microvascular endothelium. A dorsal skinfold chamber was prepared in Syrian golden hamsters, which were treated with the monoclonal antibody (MAb) 74.5.2 directed against gC1qR or vehicle. The interaction of fluorescein isothiocyanate (FITC)-labeled staphylococci and leukocytes with the endothelium was analyzed under physiological conditions as well as after TNF-α-induced inflammation using intravital fluorescence microscopy. Administration of MAb 74.5.2 significantly reduced adherence of S. aureus to the endothelium in untreated and TNF-α-exposed tissue. In addition, we could demonstrate in vitro that S. aureus adherence to human endothelial cells was inhibited by MAb 74.5.2. Blockade of gC1qR did not affect leukocyte-endothelial cell interaction. In conclusion, our findings indicate that immunological inhibition of gC1qR may be therapeutically used to decrease the interaction of S. aureus with the microvascular endothelium.     

APC I1307K increases risk of transition from polyp to colorectal carcinoma in Ashkenazi Jews

BACKGROUND & AIMS: The I1307K allele of the APC gene has been shown to confer a modestly elevated risk of colorectal cancer in the Ashkenazi Jewish population (relative risk, 1.5-1.7). However, it is unclear whether the alteration predisposes to adenomas and whether the genetic information can be used in clinical practice. To further address the pathogenic significance of I1307K, we offered both a genetic test and a screening program to individuals considered to be at increased risk for colorectal cancer. We compared the prevalence of polyps and their characteristics between carriers and noncarriers. METHODS: Invitations to participate in a DNA and colonoscopy screening program were mailed, together with a family questionnaire, to 3540 households forming the Jewish Community in Ottawa. The I1307K variant was analyzed in 242 eligible respondents who were selected because they had a personal or family history of colon cancer. Nearly 80% of these respondents (n = 189; age range, 32-83 years) consented to undergo a single colonoscopic examination. RESULTS: The overall carrier frequency of I1307K in the study group was 10.3%. A higher proportion of heterozygous gene carriers was found in the subgroup of colon cancer survivors (27%) than among asymptomatic individuals (8%, P < 0.02). A total of 59 polyps were identified in 44 subjects. Histologically confirmed adenomatous polyps were diagnosed in 11.8% of carriers and 12.8% of noncarriers (P > 0.5). No significant differences in polyp size, multiplicity, location, degree of villosity, or age-dependent prevalence were found between the 2 groups of participants. CONCLUSIONS: The high frequency of I1307K colorectal cancer patients found in the Ashkenazi Jewish community of Ottawa and the equivalent proportion of carriers and noncarriers who developed adenomatous polyps suggest that in this community, I1307K is associated with a significant predisposition to carcinoma but not adenoma.     

Silencing of a metaphase I-specific gene results in a phenotype similar to that of the Pairing homeologous 1 (Ph1) gene mutations

Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I.The Pairing homeologous 1 (Ph1) gene was discovered in 1958 based on the observation that plants lacking wheat chromosome 5B exhibit homeologous pairing (1, 2). Lack of the gene results in multivalents during metaphase I (MI) of meiosis, resulting in partial sterility. Conversely, six doses of the gene in the triisosomic line of chromosome 5BL resulted in interlocking of the bivalents and reduced chiasmata frequency even among homologs, along with rare multivalents (3). Several other genes promoting or suppressing homeologous chromosome pairing (HECP) have also been reported (4, 5), although their effect is difficult to measure in the presence of the Ph1 gene (6). Ph1-like genes were also reported in other sexually propagating polyploids, including Avena sativa, Festuca arundinacea, Brassica napus, Gossypium hirsutum, and Gossypium barbadense, as well as in some diploids, including Lolium perenne, Lolium multiflorum, and Lolium rigidum (7–11).Ph1 gene mutants in tetraploid (ph1c) (12, 13) and hexaploid (ph1b) (14) wheat were shown to be interstitial deletions involving an ∼0.84-μm region and an ∼1.05-μm region around the gene, respectively (15, 16) (SI Appendix, Fig. S1). Physical mapping localized the gene to an ∼2.5-Mb chromosomal region referred to as “Ph1 gene region,” bracketed by the distal breakpoint of ph1c deletion on the distal end and the breakpoint of deletion line 5BL-1 on the proximal end (16) (SI Appendix, Fig. S1). Various marker enrichment efforts identified nine markers for the region (17). Detailed microsynteny analyses and comparative mapping identified a 450-kb region of rice chromosome 9 (17). The corresponding rice region contained 91 genes. The major objective of the present study is to identify the gene(s) responsible for the Ph1 gene-like function using the available mapping information.     

Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-β-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.     

FrzA/sFRP-1, a secreted antagonist of the Wnt-Frizzled pathway, controls vascular cell proliferation in vitro and in vivo

OBJECTIVE: FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt/frizzled signaling pathway. We have recently demonstrated its role in vascular cell growth control in vitro. In this study, we aimed to examine the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and its potential effect in vivo. METHODS AND RESULTS: On synchronized, growth-arrested endothelial cells (EC) and smooth muscle cells (SMC) treated with the recombinant purified FrzA protein, flow cytometry analysis showed that the recombinant FrzA protein delayed G1 phase and entry into S-phase. Western blot experiments demonstrated that the treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclins and cyclin-dependent kinases and an increase in cytosolic phospho-beta-catenin levels. The FrzA-induced cell cycle delay was resolved by 24 h. C57BL/6J mice underwent surgery to produce unilateral hindlimb ischemia and empty adenoviruses (AdE) or adenoviruses coding for FrzA (AdFrzA) were injected at the time of the surgery. In AdFrzA-treated mice in the 7 days following surgery, we showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle compared to that in the AdE-treated ischemic muscle. To gain insight into the pathway activated by FrzA overexpression, we showed an increase in the level of cytosolic phospho-beta-catenin, a marker of beta-catenin degradation, in AdFrzA-treated ischemic muscle compared to that in control AdE-treated ischemic muscle. CONCLUSION: We provided the first evidence that an impairment of the Wnt-Frizzled pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia.