Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD  On the other hand, inhibition of PP1 in vivo strongly increased CDK9 phosphorylation and profoundly inhibited Tat-activated HIV-1 transcription in vivo suggested that unlike the situation in vitro, CDK9 might be dephosphorylated by PP1 in the cells and that PP1 might have a regulatory role in HIV-1 transcription. Indeed, we previously showed that PP1 expression induced HIV-1 transcription in cultured cells in vitro, i.e Thr29 and C8 residues. One plausible candidate for PP1 dephosphorylation is CDK9 T-loop that includes Ser175 and Thr186 residues. Recently, CDK9 activation in stress-induced cells was shown to be cooperatively induced by PP1α and protein phosphatase 2B (PP2B) in vitro and in cultured cells reduced RNAPII CTD phosphorylation and the activity of CDK9, which indicated that PP1 is an activator of CDK9. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop and also in vivo phosphorylated CDK9 with Ser175 mutation showed that Ser175 could be dephosphorylated by PP1 in vitro. Further analysis of CDK9 phosphorylation residues was carried by Hunter 2D peptide mapping and LC-MS, pointing to CDK9 Ser 175 as one of the two phosphorylation sites. Analysis of the activity of CDK9's S175A and S175D mutants showed that CDK9 S175A mutant was active and S175D - inactive. Expression of cdNIPP1 prevented the interaction of CDK9 S175D with HEXIM1. Analysis of the effect of CDK9 point mutations alone or in combination with PP1 on HIV-1 transcription which is dependent on CDK9, indicate that dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to Ser175 as PP1-regulatory site in CDK9 which dephosphorylation upregulates CDK9 activity and may contribute to the regulation of HIV-1 transcription.CDK9/cyclin T1 is a protein kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) in vitro transcription reaction and its phosphorylation was visualized by immunoblotting with phospho-epitope specific monoclonal antibodies that recognized phospho Ser-2 residues in the heptapeptide repeats of the CTD  into the RNAPII in cultured cells. 293T cells were transfected with vectors expressing an inhibitory fragment of NIPP1 (cdNIPP1 wt) or the corresponding PP1 binding mutant  32P) orthophosphate for 3 hrs and also treated with 0.1 µM okadaic acid to inhibit PP1. RNAPII was immunoprecipitated from the cell lysates and its phosphorylation was analyzed. Expression of cdNIPP1 reduced the incorporation of (32P) into RNAPII  orthophosphate and also by the addition of 0.1 µM okadaic acid to inhibit cellular PP1 32P)-phosphorylated FLAG-CDK9 was immunoprecipitated and subjected to dephosphorylation by PP1, PP2A and, as a control, Cdc25, a dual-specificity phosphatase that dephosphorylates Thr14 and Tyr15 residues of CDK2 in vitro may not be specific toward their substrates, we compared PP1 and PP2A with a commercially available substrate, KR-pT-IRR peptide (Millipore) or a specially designed PP1 substrate, phospho-Rb peptide (HIPR-pS-PYKFPS-pS-PLR). While PP1 and PP2A both dephosphorylated the KR-pT-IRR peptide with PP2A being somewhat more efficient -labeled CDK9 by PP1 (32P)-labeled CDK9 is more efficiently dephosphorylated by PP1 than the Thr186-phosphorylated CDK9  or the T186A mutant were expressed in 293T cells. Subsequently, the cells were treated with okadaic acid, CDK9 was immunoprecipitated with anti-FLAG antibodies from the cell lysates and the immunoprecipitates were subjected to dephosphorylation by PP1 or PP2A. In contrast to the almost complete dephosphorylation of  and comprised both Ser175 and Thr 186 residues. We also used alanine mutants of Ser175 (S175A: 172RAFALAKNSQPNRYTNRVV190), Thr186 (T186A: 172RAFSLAKNSQPNRYANRVV190) or both residues (S175A/T186A: 172RAFALAKNSQPNRYANRVV190). The T-loop-derived WT CDK9 and CDK9 T186A peptides were efficiently phosphorylated by whole cell extracts whereas the S175A peptide and the double mutated S175A/T186A peptide were not phosphorylated suggesting that Ser175 but not Thr186 kinase activity was present abundantly in the cell extract (data not shown). Previously, CDK9/cyclin T1 was shown to autophosphorylate its T-loop 150KSFGSPNRIYTHQVV165; T160A: 150KSFGSPNRIYAHQVV165)  , lane 3.QVV165) , lane 5.QVV165) , lane 7 QVV165)   phosphorylated peptides 32P) in the presence of 0.1 µM okadaic acid and then immunoprecipitated with anti-FLAG antibodies  orthophosphate in the presence of okadaic acid and subjected to immunoprecipitation and dephosphorylation by PP1. Phosphorylated CDK9 was efficiently dephosphorylated by PP1  and also co-transfected with cyclin T1-expressing vector. CDK9 was immunoprecipitated with monoclonal anti-FLAG antibodies and its activity was analyzed using recombinant GST-CTD as substrate. Both WT CDK9 and CDK9 S175A were active in the phosphorylation of GST-CTD and were also autophosphorylated , lane 3.LacZ and Tat –expression vectors showed induction of HIV-1 transcription by CDK9 S175A but not any other mutants tested  in cultured cells with non-labeled CDK9 showed that residues in addition to Thr186 are likely to be dephosphorylated by PP1. Analysis of dephosphorylation of phosphopeptides and also CDK9 S175A mutant suggested that CDK9's Ser175 could be the additional dephosphorylation site. We analyzed CDK9 phosphorylation sites by combining Hunter peptide thin layer electrophoresis and LC-MS chromatography. In the cells treated with okadaic acid CDK9 was phosphorylated on two sites, which both were identified as Ser 175 containing phosphopeptides. Both of these sites were minimally phosphorylated in CDK9 S175A mutant. The third additional peptide which phosphorylation was not changed in the CDK9 S175 mutant was identified as C-terminal 346GSQITQQSTNQSR358 peptide. Recent study by Johnson and colleagues showed that Ser353 or Thr354 are autophosphorylated in vitro, in addition to the Thr186 residue which was also autophosphorylated in vitro was dephosphorylated by PP2A, but not PP1 in vitro with PP2A but not PP1 186NR peptide, but not the fully trypsinized YpT186NR peptide 175LAK peptide in the two spots resolved on Hunter thin layer electrophoresis, but were not able to perform the MS/MS analysis of the peptide. It is likely that the T-loop peptides are not well ionized and thus may not be easily detected. Previously, Morgan and colleagues detected CDK2-derived peptides by Hunter 2D peptide mapping system but noted difficulties in detection of T-loop derived peptides due to the strong binding of the peptides to the PVDF membrane in vitroIn a recent study, dephosphorylation of Thr 186 by PP1α was shown to disrupt the interaction between CDK9/cyclin T1 and 7SK RNA/HEXIM1 when PP2B acted in cooperation with PP1α Taken together, our study uncovered CDK9's Ser 175 as new target for PP1 thus providing further details on the regulation of CDK9 activity by PP1 and suggesting PP1 for further evaluation as a new therapeutic target.172RAFSLAKNSQPNRYTNRVV190, S175A: 172RAFALAKNSQPNRYTNRVV190, T186A: 172RAFSLAKNSQPNRYANRVV190, and S175A/T186A: 172RAFALAKNSQPNRYANRVV190) and peptides that span the T-loop of CDK7  were purchased from New England Peptides . Ser/Thr phosphatase assay kit that included KR-pT-IRR peptide was purchased from Upstate . Expression vectors for WT Flag-CDK9, CDK9 T175A and CDK9 T186A mutants and CDK9 with alanine substitutions of eight C-terminal serine and threonine (C8) were a gift from Dr. Qiang Zhou . Flag-CDK9 T29A, CDK9 T29E, CDK9 S175D and CDK9 T186E were created by mutagenesis using Quick-Change site-directed mutagenesis protocol of Stratagene , using the appropriate primers and templates. The sequences of the DNA constructs were verified by sequencing using a commercial service from Macrogen . The expression vectors for EGFP-fused central domain of NIPP1, NIPP1-(143–224) or NIPP1-(143–224) V201AF203A (RATA mutant) were described 293T and HeLa cells were purchased from ATCC . 293T-cdNIPP1 cells stably expressing NIPP1 (143–224) were previously described Anti-CDK9 phospho-Thr186 antibodies and anti-HEXIM1 antibodies were a gift from Dr. Qiang Zhou . Rabbit polyclonal antibodies to CDK9, rabbit polyclonal antibodies to cyclin T1 and rabbit polyclonal antibodies to EGFP were purchased from Santa Cruz Biotechnology . Anti-Flag monoclonal antibodies were purchased from Sigma . Monoclonal antibodies specific to the unphosphorylated form of CTD (8WG16) or CTD phosphorylated on Serine 2 (H5) were purchased from BabCo .32P)-orthophosphate and cells were further incubated for 3 hours at 37°C. To increase CDK9 phosphorylation, 0.1 µM okadaic acid (Sigma) was added to block cellular PPP-phosphatases. Cells were washed with PBS and lysed in a whole cell lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS and protease inhibitors cocktail (Sigma). After 10 min on ice, cellular material was scraped, incubated at 4°C for 30 min on a shaker and then centrifuged at 14,000 rpm, 4°C for 30 min. The supernatant was recovered and protein concentration was determined using Lowry protein assay (Bio-Rad). CDK9 was precipitated with monoclonal anti-Flag antibodies (Sigma) coupled to protein G-agarose for 2 h at 4°C in a TNN Buffer containing 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1% NP-40. The immunoprecipitated CDK9 was washed twice with TNN buffer and then with phosphatase reaction buffer  and incubated in the phosphatase reaction buffer with 0.01 or 0.1 Units of PP1, PP2A, cdc25A or non-treated (control sample) for 30 min at 30°C. Then the reactions were stopped with SDS-loading buffer, resolved on 10% SDS-PAGE  and subjected to autoradiography and quantification with PhosphorImager Storm 860 (Molecular Dynamics). Analysis of CDK9 phosphorylation and dephosphorylation on Thr186 was carried out in the same way except that CDK9 phosphorylation was detected by immunoblotting with phospho-specific Thr186 antibodies.293T cells were transfected with FLAG-CDK9-expression vector using Lipofectamine and Plus reagents (Life Technologies). After 48 hours incubation, the cells were placed in a phosphate-free DMEM media (Life Technologies) containing no serum for 2 hours. The media was changed to phosphate-free DMEM media supplemented with 0.5 mCi/ml of (2 (New England Biolabs) that also contained 150 µM KR-pT-IRR or phospho-Rb peptide. At indicated time points, 25 µl aliquots were removed and mixed with 100 µl of Malachite Green solution. Absorbance of malachite green was determined at 620 nm and the phosphate concentration was recalculated using a calibration curve of phosphate standards prepared using 1 mM KH2PO4 solution.The assays were carried out with the Ser/Thr phosphatase assay kit (Upstate). PP1 or PP2A was added in PP1 reaction buffer  supplemented with 1 mM MnCl2, 0.5 mM EDTA, 2 mM DTT and 10% glycerol). Transcription was carried out for 30 minutes at 30°C. The reaction was terminated by addition of 7 mM EDTA.RNAPII was phosphorylated in transcription reactions (20 µl) containing 50 µg of HeLa nuclear extract, 0.5 mM ATP, CTP, UTP and GTP, 0.2 µg of JK2 32P)-orthophosphate as described above and RNAPII was immunoprecipitated with anti-CTD 8WG16 monoclonal antibodies coupled to protein G agarose. The immunoprecipitated RNAPII was recovered by heating for 2 min at 100°C in SDS-loading buffer, resolved on 6% SDS-PAGE and the dried gel was exposed to Phosphor Imager screen . In a control, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane and analyzed with anti-RNAPII CTD 8WG16 antibodies.293T cells were transiently transfected with vectors expressing EGFP-fused NIPP1 (143–224) or NIPP1 (143–224) V201AF203A (RATA mutant). At 24 hours posttransfection, the cells were pulse-labeled with ATP for 30 minutes at 30°C. The reactions were stopped with Laemmli sample buffer, resolved on 10% Tris-Glycine SDS-PAGE  and subjected to autoradiography and quantification with Phosphor Imager .293T cells were seeded in 6-well plate and transfected with a combination of plasmids expressing CyclinT1 and FLAG-CDK9  using Lipofectamine-LTX (Invitrogen). At 48 hours posttransfection the cells were washed with PBS and lysed in a whole cell lysis buffer for 30 minutes. To precipitate chromatin the samples were centrifuged at 14,000 rpm at 4°C for 90 minutes. The lysates were equalized for protein concentration and diluted to 150 mM NaCl. CDK9 was immunoprecipitated with monoclonal anti-Flag antibodies bound to protein G agarose beads for 2 hrs at 4°C in TNN Buffer . The beads with precipitated CDK9 were washed with PBS and used for the kinase activity assay using GST-CTD as substrate in 50 mM HEPES-KOH buffer (pH 7.5), 10 mM MgCl2, 6 mM EGTA, 2.5 mM DTT, 100 µM cold ATP and 5 µCi γ-(32P)ATP for 30 minutes at 30°C. The reactions were stopped with Laemmli sample buffer, resolved on 15% Tris-Tricine SDS-PAGE and subjected to autoradiography and quantification with Phosphor Imager .About 2 µg of peptides that span T-loop of CDK9 or CDK7 as indicated were phosphorylated in 50 mM Hepes-KOH buffer (pH 7.5), 10 mM MgCl32P) orthophosphate as described above. (32P)-labeled FLAG-CDK9 WT and S175A mutant were resolved on 10% SDS-PAGE. Radioactive bands were visualized using Phosphor imager and excised from the gel. The gel pieces were reduced in 10 mM DTT, alkylated with 30 mM iodoacetamide and then digested overnight with 10 ng/µl Trypsin Gold (Promega) in 50 mM ammonium bicarbonate. The eluted peptides were applied to thin layer cellulose plates  and separated in first dimension by electrophoresis at pH 1.9  conducted at 1000 V, 12 mA for 1 hrs and followed by ascending chromatography in Phospho-Chromatography buffer  for 15 hrs. Cellulose plates were dried and exposed to Phosphor imager screen. To determine the content of the tryptic phosphopeptide, cellulose containing the phosphopeptides was scraped and the peptides were eluted in 70 acetonytril with 0.1% TFA. The eluted peptides were lyophilized, resuspended in 0.1% formic acid and subjected to mass spectrometry analysis.Tryptic phosphopeptide mapping was conducted using Hunter thin layer peptide mapping electrophoresis system . FLAG-CDK9 was expressed in 293T cells and metabolically labeled with  search with precursor and fragments tolerance 20 ppm. These high resolution data and search criteria reduce amount of false positives and dramatically decrease the search time.


CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser1270 compared to wt-ACE . The JNK inhibitor , in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor.Western blotting analysis showed that SS (18 h, 15 dyn/cm1270, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser1270.ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser Angiotensin-converting enzyme (ACE) is a key component of the renin-angiotensin system, which regulates blood pressure, electrolyte balance and fluid homeostasis 1270 (p-Ser1270) by ACE inhibitors (ACEi), which then triggers intracellular signaling cascade that leads to increase ACE expression 1270 mediated by casein kinase 2 activating MKK7 and JNK. Then, phosphorylated c-jun activates AP-1 transcription factor and increase ACE expression Recently, it has been demonstrated that the cytoplasmic tail of ACE is phosphorylated on Serin vitro using a cell system and in vivo in the rat aorta We have described that SS suppresses ACE gene expression and activity 1270 appear to be required for sensing and eliciting the SS-induced ACE downregulation.Considering that ACE is a transmembrane protein mainly expressed in EC, we now investigated whether the extra- and intracellular domains of ACE and the phosphorylation of a cytoplasmic residue are involved in the mechanotransduction of ACE regulation by SS. The results obtained suggest that phosphorylation of ACE and downstream JNK inactivation participate in this process in primary EC. Using the CHO cell model system, we provided additional evidence that both the extra- and the intracellular ACE domains and Ser2) in human SVEC is associated with a decrease in ACE protein expression  indicating that SS was accompanied by reduction in the percentage of p-ACE , p-JNK significantly decreased by 37%   or the cytoplasmic (Cyt-del-ACE) domains and carrying a point mutation at the cytoplasmic domain residue 1270 (S70A-ACE) . Each ce70A-ACE) .−1.mg−1; Cyt-del-ACE: 2815±345 uF.min−1.mg−1; S1270A-ACE: 3782±18 uF.min−1.mg−1; Extra-del-ACE: 192±18 uF.min−1.mg−1). Using the p-Ser1270 ACE antibody, it was demonstrated that wild-type CHO cells and S1270A-ACE fail to express phosphorylated ACE in the membrane fraction while the signal is clearly observed in the membrane fraction of the wt-ACE  was observed   . In cont2) . Notewor1270, Cyt-del-ACE, Extra-del-ACE, and S1270A-ACE cells, respectively. Interestingly, the SS-mediated response on ACE promoter activity was abrogated in all cell lines . ACE promoter activity remained unchanged by the SS-conditioned medium  , indicat1270 and ACE expression in vivo during ACE inhibition. Interestingly, the presence of both stimuli rendered the ratio of pACE/ACE as well as the ACE expression unchanged suggesting that the stimuli had opposing effects of similar magnitude on these responses  by ACE inhibitors (ACEi), which then triggers intracellular signaling cascade that leads to increase ACE expression 1270 mediated by casein kinase 2 activating MKK7 and JNK. Then, phosphorylated c-jun activates AP-1 transcription factor and increase ACE expression Recently, it has been demonstrated that the cytoplasmic tail of ACE is phosphorylated on Serin vitro and in vivo approaches 1270, downstream JNK inhibition and diminished ACE expression. Thus, SS modulates components of the same pathway described for ACEi, although in opposite direction as mentioned before.We previously demonstrated that SS decreases ACE expression and activity using a combination of The JNKs are traditionally considered stress-activated protein kinases involved in several cellular processes including cell proliferation, apoptosis, migration, cytoskeleton rearrangements, inflammation, metabolic disease, neurodegenerative disease, oncogenesis, and cancer progression The broad usage of ACEi in cardiovascular diseases is generally attributed to the conversion of Ang I and the accumulation of bradykinin, although the role of the latter is a matter of debate We also investigated whether the SS-induced ACE signaling activation is directly a consequence of the mechanical force or secondary to SS-induced released factor(s). The sustained exposure of ECs to laminar shear stress is accompanied by the release of several antiatherogenic factors, including prostacyclin (PGI2), transforming growth factor-β (TGF- β) and nitric oxide (NO) Thus, the decrease of ACE signaling by shear stress might also contribute to reduce Ang II formation and Bradykinin accumulation and all its consequences on diminishing the oxidative stress and Ang II-induced inflammatory responses The main claims of the present study were obtained using primary endothelial cells, although key aspects of the underlying molecular mechanisms were clarified using a versatile cell model system, which recapitulate the data observed with the endogenous system. An alternative to the use of CHO cell system, which lacks endogenous expression of ACE, is the use of endothelial cells lacking ACE from the ACE knockout mouse, however, these cells require the use of transient transfections since primary endothelial cells are not amenable to multiple passages required for the selection procedure. The transient transfection system is less robust than the permanently transfected cells expressing different ACE mutants. Thus, we believe the proposed strategy, despite its own limitations, was instrumental to give additional support to the main claims of the study.1270.Taken together, we provide the first evidence for an enzyme as a mechanosensor to shear stress. The data show that ACE influences its own regulation in response to SS and suggest that ACE extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on SerPrimary culture of endothelial cells was obtained as previously described Chinese hamster ovary (CHO) cells were obtained from Sigma-Aldrich. Collection. CHO cells were grown in 100-mm culture dishes in Dulbeccos's modified Eagle Medium (DMEM) High Glucose culture medium supplemented with L-glutamine, antibiotic (penicillin and streptomycin), Hepes buffer, and 10% fetal bovine serum. Cells were routinely sub cultured using trypsin-EDTA to mobilize them. For transfection, CHO cells were plated in 60-mm dish, 1 day prior to transfection.g for 5 min (4°C). The resulting supernatant was centrifuged again at 100.000 g for 30 min (4°C) to obtain the pellet (nucleus and cell membrane) and the cytoplasmic extract (supernatant). The pellet was incubated with the same lysis buffer containing 1% TritonX100 and DNAse (1 mg ml−1) for 30 min (4°C) and then centrifuged at 100.000 g for 30 min (4°C) to obtain the cell membrane (supernatant).Immediately following the experimental protocol, cell were lysed in ice-cold lysis buffer ). The homogenate was centrifuged at 100 1270A-ACE were kindly provided by Dr Pierre Corvol  cDNAs of human wild-type ACE, Cyt-del-ACE, Extra-del-ACE and STotal RNA was isolated with Trizol Reagent according to the manufacturer's instructions and cDNA synthesis was performed with radom hexamers (High Capacity cDNA Archive kit-PE Applied Biosystem). The reaction was carried out using Taq polymerase under the following conditions: initial denaturation for 5 min at 95°C followed by 40 cycles of denaturation for 15 s at 95°C, annealing for 1 min at 60°C, extension for 1 min at 72°C, and final extension for 10 min at 72°C. The PCR products were analysed by electrophoresis on agarose gel.5′-ACCTCGGAGCCGAACTGGGG-3′, sense 5′-GGCTGCTGCTCTTCCTGGGC-3′ amplify the fragment between 3801 and 3924 in the ACE cytoplasmic domain; Primer 2 antisense 5′-TCCGGGATGTGGCCATCACA-3′, sense 5′-CCTGCCCAGGAGCTGGAGGA-3′ amplify the fragment between 2219 and 2357 in the ACE extracellular domain.The primers used were: Primer 1 antisense CHO and CHO expressing wild-type ACE or ACE mutants were transfected with luciferase reporter plasmid  harboring the 1,274 bp of rat ACE promoter gene On 2 was produced by a cone plate viscometer as described before Primary cultures of ECs and CHO cell lineages were submitted to controlled shear stress as previously described 2, 5 mM KCl, 25 mM HEPES, 1 mM PMSF, 2 mM DTT, 0,1%Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich). After 10 min on ice, samples were centrifuged at 10,000 g for 10 min to remove cellular debris. Cell lysates (5 to 40 µg) supernatant were heated in SDS-PAGE sample buffer, fractioned by SDS-PAGE and transferred to Hybond membranes . Transfer efficiency was monitored by 0.5% Ponceau S staining. The blotted membranes were first blocked with 5% non-fat milk for 2 h at room temperature and then proteins were detected using their respective antibodies. The p-JNK and JNK antibodies were obtained from CellSignaling (1∶1000), the p-Ser1270 ACE antibody (1∶1000) was provided by Dr Ingrid Fleming from the Goethe University, Frankfurt, Germany; the Caveolin-1 , the ERK1/2 , and the ACE antibody by Dr Sergei M Danilov from the University of Illinois at Chicago, USA. Horseradish peroxidase-conjugated antibody was used as a secondary antibody, and signals were detected using the ECL detection kit .Cells were lysed in lysis buffer (1 mM EDTA, 1 mM EGTA, 2 mM MgClt-test or 1-way ANOVA and appropriate posthoc Tukey comparison. Statistical significance was accepted if p<0.05.All data are representative of at least 3 independent experiments. Numerical data era presented as mean±SEM. Comparisons among the groups were performed with student


ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates. The zinc finger gene family, one of the largest gene families in mammals, is defined by a conserved cysteine and histidine rich domain essential for the binding of zinc ions . This geIn accordance with their diverse structures, zinc finger proteins have been assigned multiple functions, including DNA recognition, transcriptional activation, RNA packaging, regulation of apoptosis, ubiquitination and many others , encodes a C3HC4-type zinc finger protein motif (ring finger motif), and, consistent with a role in premeiotic or postmeiotic sperm development, one of its transcripts has been identified in abundance in the testicular tissue of fertile men, but neither in fetus nor in azoospermic patients. This suggested that ZNF230 may be involved in spermatogenesis, and loss of its expression may lead to azoospermia  according to the manufacturer's instructions. SuperScript TM Reverse Transcriptase (Invitrogen) was used for reverse transcription.ZNF230 in zebrafish. This orthologue sequence was acquired by PSI-BLAST alignment. 5'-RACE experiments were performed using SMART RACE cDNA Amplification Kit (Clontech) (Primers for 5'-RACE (366L 5'-ACGAGACGC CTCTACCATTCCATCC-3') and the other three pairs of primers  were designed to obtain the complete coding sequence, based on the potential orthologue of rnf141 gene, total RNA was extracted from embryos of various developmental stages .Primer sequences used for amplifying 470 bp ß-actin were 5'-TGTGGCCCTTGACTTTGAGCAG-3' (forward) and 5'-TAGAAGCATTTGCGGTGGACGA-3'(reverse), according to rnf141 morpholino antisense oligonucleotide  was used to target rnf141 mRNA, and 5'mispaired control morpholino (5'-CCAcAAAcCTcCTGcCCCATgT TGG-3') served as a control. Both were designed by using Gene-tools . The coding region of rnf141 was ligated into vector pcDNA3 (Invitrogen) and linearized by appropriate restriction enzymes for mRNA was synthesis by using mMESSAGE mMACHINE® Kit (Ambion). rnf141-MO and control-MO were injected into 1-2 cell zebrafish embryos by using a Model PLI-90 Pico-Injector.rnf141 sense and antisense RNA probes were labeled with digoxigenin-11-UTP and synthesized by using DIG RNA Labeling Kit (SP6/T7) (Roche). The template for the probe was the entire 668 bp long cDNA. Whole-mount in situ hybridizations was performed as described by rnf141 based on the amino acid sequence of human ZNF230 and mouse znf230 by PSI-BLAST alignment and RT-PCR including RACE. As a result, four fragments were obtained that formed a816 bp cDNA contig. This was submitted to GenBank (accession number: AY621088). Previous studies have shown that human ZNF230 has two transcripts , suggesting functional similarity and conservation.The predicted open reading frame from 88 to 756 is 668 bp in length and encodes a polypeptide of 222 amino acid residues with a C3HC4 zinc finger domain from 146 to 187 amino acid residues . Proteinrnf141 during early embryonic development, whole-mount in situ hybridizations were performed on two-cell stage to five-day-old embryos using an antisense probe. As a result, rnf141 transcripts were already detected at the two-cell stage , a characteristic pattern was displayed with marked staining in the notochord  of the embryos to show phenotypes that are characteristic of embryonic ventralization at 24 hpf . In contrast, the ventral markers bmp2b and vent expanded dorsally during gastrulation . The ratios of embryos with altered marker gene expression are summarized in To further study the potential function of t 24 hpf . The exprnf141, a morpholino antisense oligonucleotide (rnf141-MO) was injected into one-cell embryos. As a result, 81% (n = 88) of the embryos injected with 12 ng rnf141-MO exhibited dorsalized phenotypes at 24 hpf: complete loss of the yolk sac extension and partial loss of the caudal ventral fin  tended to be opposite to those of rnf141 overexpression. In contrast, injection with 15 ng of control morpholino, which differs from rnf141-MO in five mismatched nucleotides, did not cause developmental defects  and lost-a-fin (laf) mutant embryos, which were first described by rnf141 mRNA may caused embryos to show ventralized phenotypes. We also noted that rnf141-MO-induced dorsalization could be neutralized by coinjection of a smaller amount of rnf141 mRNA, suggesting that rnf141-MO specifically targets rnf141 mRNA. The expression of ventral markers (bmp2b and vent) and of a dorsal marker (goosecoid) were impacted by altered expression of rnf141, thus suggesting that zebrafish rnf141 may participate in normal dorsoventral embryonic patterning. Further research is needed to better understand the respective biological pathway(s) and improve the knowledge on the function of rnf141.In conclusion, knockdown of


Activating mutations of Fms-like tyrosine kinase 3 (FLT3) constitute a major driver in the pathogenesis of acute myeloid leukaemia (AML). Hence, pharmacological inhibitors of FLT3 are of therapeutic interest for AML.in vitro and in vivo assays.The effects of inhibition of FLT3 activity by a novel potent FLT3 inhibitor, BPR1J-097, were investigated using 50) of BPR1J-097 required to inhibit FLT3 kinase activity ranged from 1 to 10 nM, and the 50% growth inhibition concentrations (GC50s) were 21±7 and 46±14 nM for MOLM-13 and MV4-11 cells, respectively. BPR1J-097 inhibited FLT3/signal transducer and activator of transcription 5 phosphorylation and triggered apoptosis in FLT3-driven AML cells. BPR1J-097 also showed favourable pharmacokinetic property and pronounced dose-dependent tumour growth inhibition and regression in FLT3-driven AML murine xenograft models.The 50% inhibitory concentration (ICin vivo anti-tumour activities and suggest that BPR1J-097 may be further developed in preclinical and clinical studies as therapeutics in AML treatments.These results indicate that BPR1J-097 is a novel small molecule FLT-3 inhibitor with promising  Acute myeloid leukaemia (AML) is the most common type of adult leukaemia. It is an aggressive disease that involves rapid growth of abnormal leukaemic cells in the bone marrow, resulting in failure of production of normal blood cells , 2002b. N1-(3-3-[(phenylsulphonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4-methylpiperazino) benzamide) , sunitinib (SU11248), midostaurin (PKC412), lestaurtinib (CEP-701), tandutinib (MLN518), ABT-869, KW-2449, and AC220 . Howevernzamide) , that waThese results demonstrate the potential of BPR1J-097 as a therapeutic candidate for treatment of AML patients. Further preclinical and clinical studies in AML patients are warranted.β-actin  antibodies were purchased for western blotting analysis. The N-terminal GST-tagged human FLT3 (residues Y567-S993), VEGFR1 (residues R781-I1338), and VEGFR2 (residues V789-V1356), expressed in Sf9 insect cells, were purified for kinase assay . The recombinant human FLT3 ligand (T27-P185) was purchased from R&D Systems . The anti-phospho and total  , anti-cleaved poly (ADP-ribose) polymerase , and anti-caspase 3 , anti-MOLM-13 cells were purchased from the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH . RS4;11, MV4-11, U937, and K562 cell lines were obtained from the American Type Culture Collection . All cell lines were grown in RPMI 1640  with 10% fetal bovine serum (FBS) . HEK293T- and FLT3-transfected HEK293T cells were cultured in DMEM (Invitrogen) medium with 10% FBS.μl, including 25 mM Tris pH 7.4, 10 mM MgCl2, 4 mM MnCl2, 1 mM DTT, 0.02% Triton X-100, 0.01% BSA, 1 μM ATP, 20 μM peptide (GGMEDIYFEFMGGKKK), 75 ng recombinant FLT3 proteins, and test compound at the indicated concentration. The VEGFR1 or VEGFR2 kinase assay was carried out in 96-well plates with tested compound in a final volume of 50 μl reaction at 30°C for 2 h with the following components: 25 mM HEPES pH 7.4, 10 mM MgCl2, 4 mM MnCl2, 0.5 mM Na3VO4, 2 mM DTT, 0.02% Triton X-100, 0.01% BSA, 1 μM ATP, 2 μM polyGlu4:Tyr peptide, 100 ng recombinant VEGFR1 or VEGFR2 protein. Aurora kinase A and Aurora kinase B assays were performed as reported by us in an earlier study  was added and incubated at 25°C for 20 min. A 70 μl aliquot of each reaction mixture was transferred to a black microtiter plate and the luminescence was measured on a Wallac Vector 1420 multilabel counter . Each IC50 value was determined by three different experiments. Kinase inhibition profiling and FLT3-D835Y-inhibitory activity were determined by Invitrogen SelectScreen kinase profiling service.The FLT3 Kinase-Glo kinase assays were carried out in 96-well plates at 30°C for 4 h in a final volume of 50 50 value was defined as the amount of compound that caused 50% reduction in cell viability in comparison with DMSO-treated (vehicle) control and was calculated using Prism version 4 software .Proliferation assays were performed by seeding 10 000 cells per well in a 96-well culture plate. After 16 h, cells were then treated with vehicle or test compounds at various concentrations of the tested compound in medium for 72 h. Cell viability was quantitated using the MTS method (Promega) according to the manufacturer's recommended protocol. The results were determined by measuring absorbance at 490 nm using a plate reader . The GCM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mM DTT). Protein lysates were resolved in SDS–PAGE and then transferred onto a polyvinylidene difluoride membrane . Membranes were immunoblotted with appropriate antibodies and reacted with the SuperSignal reagent , followed by exposure to X-ray film.Cells were lysed in lysis buffer  solution, was separately administered to groups of 3 rats each intravenously (i.v.) by a bolus injection through the jugular-vein cannula. Each animal received 1 ml of the dosing solution per kg of body weight i.v. At 0 (before dosing), 2, 5, 15, and 30 min and at 1, 2, 4, 6, 8, and 24 h after dosing, a blood sample (0.15 ml) was collected from each animal through the jugular-vein cannula and stored in ice (0–4°C). Immediately after collecting the blood sample, 150 μl of physiological saline (containing 30 Units of heparin per ml) was injected into the rat through the jugular-vein cannula. Plasma was separated from the blood by centrifugation (14 000 g for 15 min at 4°C in a Beckman Model AllegraTM 6R centrifuge) and stored in a freezer (−20°C). All samples were analysed for the parent drug by LC-MS/MS. Data were acquired through selected reaction ion monitoring. Plasma concentration data were analysed with non-compartmental method.Male Sprague–Dawley rats weighing 300–400 g each (8–12 weeks old) were obtained from BioLASCO . Animals were surgically prepared with a jugular-vein cannula one day before dosing and fasted overnight (for approximately 18–20 h) before dosing. Water was available n=5–7 per group) were inoculated subcutaneously with MOLM-13 (1 × 106 per flank) or MV4-11 cells (5 × 106 per flank). All human cancer cells were detected as free of Mycoplasma spp before they were injected into animals. When the tumour size reached 100–200 mm3, animals were grouped and treated with BPR1J-97 at various doses in a 2-week treatment period as indicated. Animals were treated with BPR1J-097  or vehicle as control at once daily for 5 days per week for 2 weeks. Tumour volumes were measured and calculated with the formula length × width2/2 after initiation of treatments. Tumour size and animal body weight were measured twice a week after tumour cell inoculation. At the end of the study, animals were killed by carbon dioxide inhalation followed by cervical dislocation. The significant difference between drug treatment and vehicle control were analysed using one-way ANOVA and Student–Newman–Keuls test. The level of a statistical significance was set at P<0.05. The uses and experimental procedures in animals were approved by the IACUC  of the National Health Research Institutes and met the standards required by the UKCCCR (United Kingdom Coordinating Committee on Cancer Research) guidelines  of 8 weeks of age were purchased from BioLASCO . Nude mice  was added for 5 min to prepare cell lysates for western analysis. As shown in M. The constitutive activation of STAT5 has a pivotal role in FLT3-ITD leukaemia cell expansion and survival . 293T cells were treated with BPR1J-097 at various concentrations for 2 h and then the FLT3 ligand  was observed in MOLM-13 cells treated with BPR1J-097 at 10 nM . Howeverat cells . It seem−1 i.v. dose administered to rats, achieved a maximum plasma level maximum concentration of 7.1 μM (3670 ng ml−1) at 2 min of dosing and, at 24 h after dosing, the estimated BPR1J-097 plasma concentration remained at a concentration of 1 ng ml−1 (1.9 nM). The total body clearance was 102.4±9.8 ml min−1 per kg and the volume of distribution at the steady state (Vss) was 15.5±4.8 l kg−1 for BPR1J-097 in rats. The apparent plasma half-life was ∼4.5 h.To assess the pharmacokinetic properties of BPR1J-097, the plasma concentration of BPR1J-097 over a 24-h period was measured after a single i.v. administration . BPR1J-0in vivo, MOLM-13 or MV4-11 cells were subcutaneously implanted into nude mice. In the MOLM-13 model, tumours were allowed to grow to a size of approximately 100–200 mm3. After i.v. administration of mice with BPR1J-097 at two cycles of 10 or 25 mg kg−1, a clear dose-dependent anti-tumour effect was observed  showed a significant tumour shrinkage effect on the subcutaneously growing MOLM-13 tumours in a size of >2000 mm3 (−1) also produced a dose-dependent growth reduction and shrinkage of another model using MV4-11 cells. It is noted that a prolonged disappearance of MV4-11 tumours was observed in mice treated with BPR1J-097 at 25 mg kg−1 (in vivo studies. It is interesting to note that although BPR1J-097 was able to trigger more apoptosis in MOLM-13 cells than in MV4-11 cells (To examine whether BPR1J-097 exhibited anti-tumour activity observed . Tumours2000 mm3 . Tumours mg kg−1 . There w11 cells , BPR1J-011 cells . FurtherAberrant regulation of tyrosine kinase has been implicated as a causal factor in tumourigenesis, cancer progression, and drug resistance in many tumours. Pharmacological inhibition of dysregulated kinases is an attractive therapeutic approach for treatment of cancers. Imatinib, a Bcr-Abl kinase inhibitor, and gefitinib, an EGFR tyrosine kinase inhibitor, showed remarkable efficacy in chronic myelogenous leukaemia and in a subset of non-small cell lung cancer, respectively . Fms-likSeveral existing drugs or experimental compounds with inhibitory activity against FLT3 kinase are being evaluated for treatment of AML patients . Thus, cin vitro and in vivo. The inhibitory activity of BPR1J-097 was characterised using various assays including in vitro kinase activity, cell-based phosphorylation of FLT3 and a major downstream signalling modulator, STAT5, and proliferation of FLT3-driven leukaemic cells under in vitro and in vivo conditions. We found that BPR1J-097 potently inhibits FLT3 activity in the in vitro kinase assay compared with other FLT3 inhibitors such as ABT-869, sorafenib, and PKC412 (50 of approximately 1–10 nM. Transfected cells with the FLT3-D835Y mutant was also inhibited by BPR1J-097 with similar IC50 values in 293T-FLT3-ITD cells. Proliferation and aberrant FLT3 signalling were both inhibited by BPR1J-097, with an IC50 of 10 nM. Treatment of FLT3-driven cell lines with BPR1J-097 led to induction of apoptosis.Through rational design, we discovered a potent FLT3 inhibitor, BPR1J-097, which effectively inhibits FLT3 activity d PKC412 . In addid PKC412 . To asse−1 administration to rats is >645-fold above the IC50 for FLT3-ITD inhibition in the biochemical and cellular assays. Even at 24 h after single dosing, plasma levels of BPR1J-097 were high enough for complete inhibition of FLT3-ITD. In addition, the high Vss indicated that the distribution of BPR1J-097 into a deep, including tumour, tissue compartment is expected. These good pharmacokinetic properties indicated that BPR1J-097 dosing once a day is sufficient for continuous inhibition of FLT3 activity in rats or mice.The maximum achievable plasma concentration of BPR1J-097 after a single dose of 3.4 mg kgin vivo, we used MOLM-13 and MV4-11 murine xenograft models. Results showed that BPR1J-097 administration resulted in significant tumour regression in those two models. In a previous report, ABT-869 was not able to produce regressions of tumours in the MOLM-13 model (3 in the MOLM-13 xenograft tumour model (To evaluate the anti-tumour efficacy of BPR1J-097 ur model . In the ur model . The prein vitro and in vivo assays, excellent selectivity among the kinases examined, and favourable pharmacokinetic properties. Further studies of the clinical features of BPR1J-097 will be required to evaluate whether BPR1J-097 may have therapeutic benefit for AML patients.In conclusion, these data demonstrate that BPR1J-097 exhibits potent FLT3-inhibitory activity in both


This study aimed to assess the prognostic value of a proliferation assay by using Ki-67 immunohistochemistry as compared with mitotic count scores.Although the prognostic significance of proliferation in early invasive breast cancer has been recognized for a long time, recent gene-expression profiling studies have reemphasized its biologic and prognostic value and the potential application of its assessment in routine practice, particularly to define prognostic subgroups of luminal/hormone receptor-positive  and well-characterized series of clinically annotated primary operable invasive breast cancer with long-term follow-up. Tumors were phenotyped based on their IHC profiles into luminal/HR+, HER2+, and triple-negative (TN) classes. We used a split-sample development and validation approach to determine the optimal Ki-67LI cut-offs.Proliferation was assessed by using Ki-67 labeling index (Ki-67LI) and mitotic scores in a large (+ or the TN classes.The optimal cut-points of Ki-67LI were 10% and 50% for the luminal class. Both Ki7LI and MS were able to split luminal tumors into subgroups with significantly variable outcomes, independent of other variables. Neither mitotic count scores nor Ki-67LI was associated with outcome in the HER2Assessment of proliferation by using Ki-67LI and MS can distinguish subgroups of patients within luminal/hormone receptor-positive breast cancer significantly different in clinical outcomes. Overall, both Ki-67 LI and mitotic-count scores showed comparable results. The method described could provide a cost-effective method for prognostic subclassification of luminal/hormone receptor-positive breast cancer in routine clinical practice. In tublished . Moreoveublished , was ava+: ER+ and/or PR+]), HER2+ (HER2+ regardless of the expression of other markers), basal-like [BLBC] , and triple-negative nonbasal BC  [Molecular classes were defined as luminal (HRegative) ,27.To assess the optimal Ki-67 LI cut-off in different molecular classes and to avoid data overfitting, each molecular class was randomly split into two subsets by using SPSS random sampling; one third of cases were used as a training set, and the remaining two thirds used as a validation (test) set. No differences between training and validation sets were identified in any of the molecular classes Table .2 test, Fisher Exact test, and Mann-Whitney U test. Survival curves for BCSS and DMFS were drawn by using the Kaplan-Meier estimates, and significance was assessed by using log-rank tests. Multivariate analyses of BCSS and DMFS were conducted by using Cox proportional hazard regression models. A two-tailed P value < 0.05 was considered significant.Statistical analysis was performed by using SPSS 15.0 statistical package . X-tile software   was usedThis study was approved by the Nottingham Research Ethics Committee 2 under the title "Development of a molecular genetic classification of breast cancer." All patients who participated in this study gave their written informed consent at the time of their donation.P < 0.0001; Table In the current study, informative results for Ki-67LI, MS, and follow-up data were available for 1,457 cases (94% of the whole series). To assess the optimal number of FFSs sufficient to report Ki-67LI reliably, four FFSs were cut from four different paraffin blocks representative of 25 invasive BC cases. These showed high levels of concordance between sections when Ki-67LI was analyzed as continuous variable , and between Ki-67LI and MS (P < 0.0001). High Ki-67LI and MS were associated with premenopausal status, larger tumor size, definite vascular invasion, and lymph node involvement (P < 0.0001). Significant differences appeared between BC molecular classes regarding their Ki-67LI   and HER2+ (intermediate Ki-67LI) and between HER2+ and triple-negative (highest Ki-67LI) classes. However, the difference between BLBC and TNnon-B was not significant (P = 0.263). Similar results were found when MS was used to define proliferation. When the prognostic results of Ki-67LI were expressed as a continuous variable in the class of luminal ER+/HER2- tumors, this showed association with outcome . Moreover, Ki-67LI cut-offs at 10% increments within the same class showed statistically significant differences between the resulting patients' subsets . Table In the training sets, the prognostic significance of previously defined Ki-67LI cut-off points including 10%, 13%, 17% (median), 20%, 25%, 30%, 35% ,27,29,30analysis . In the P < 0.001, HR = 2.3; 95% CI = 1.9 to 2.8), and DMFS . Low, moderate, and high proliferative subgroups of luminal class validation set displayed significant differences in BCSS  and DMFS  . However, the HR for Ki-67LI was slightly higher than that of MS, although the CIs of the high-proliferation subgroups overlapped. As Ki-67LI and MS are highly correlated, multivariate analysis including MS, tumor grade, nodal stage, and tumor size was repeated after the addition of Ki-67LI. This showed that Ki-67LI has a higher HR than that of MS for both BCSS and DMFS; Table Univariate survival analysis of the validation set revealed significant association between MS  and patients' outcomes, including BCSS  Figure . MultivaInterestingly, the HRs for both BCSS and DMFS were highest for Ki-67LI, followed by histologic grade, HER2 status, and then by MS, whereas the lowest HR was achieved by PR status; Table + cancers, luminal tumors were further classified after inclusion of HR+/HER2+ tumors [+ and HER2- , and the later being HR+ and HER2+. In luminal 1 subclass, both Ki-67LI and MS were associated with BCSS and DMFS, independent of other variables. However, no associations were found between either MS or Ki-67LI with BCSS or DMFS in luminal 2 subclass by using either 10% and 50% or 75% Ki-67LI cut-offs. When HER2 status and proliferation were considered within HR+ tumors, distinct subclasses with prognostic significance were identified  and moderate proliferative activity (moderately poor outcome), whereas the high-proliferative group was associated with the worst outcome that was not different from HER2+ tumors  and DMFS , and between MS and BCSS  and DMFS .In addition, analysis of 70 luminal tumors defined by global gene-expression profiling  showed sP = 0.611; HR, 1.1; 95% CI, 0.7 to 1.5) or DMFS  in HER2+ tumors. Combining MS1 and 2 and repeated testing against MS3 yielded the same results. Although 75% Ki-67LI cut-off split the HER2+ training set into low/moderate  and high-proliferative  subclasses with difference in outcomes, survival analysis within the HER2+ validation set showed no significant difference between these two subgroups for either BCSS (P = 0.445) or DMFS (P = 0.784).Kaplan-Meier plots showed no association between MS and either BCSS (P = 0.317) or DMFS (P = 0.590) in TN cancers. Similarly, no difference was found when MS1 and 2 were combined and tested against MS3. In addition, no association was found between Ki-67LI and BCSS or DMFS by using the X-tile generated cut-point in the TN BC training set . When TN tumors were subclassified into BLBC and TNnon-B BC, Kaplan-Meier plots showed no significant association between MS and Ki-67I and either BCSS or DMFS (P > 0.05).Univariate survival analysis revealed no association between MS and either BCSS (+ tumors) into prognostically distinct subclasses. Therefore, in the current study, we used a well-characterized series of operable invasive BC to assess the prognostic significance of Ki-67LI with regard to BC molecular classes compared with that provided by the routinely assessed MS and addressed some technical issues for use in routine practice in assessing tumor proliferation.Although the prognostic significance of proliferation in BC has been documented and validated in several independent studies, recent global gene-expression profiling studies have reemphasised its biologic and prognostic importance. Several studies have identified the proliferation signature as a key element in the molecular classification of BC and in the composition of different prognostic and predictive gene signatures . Therefo+ and TN tumors showed high proliferative activity in terms of high Ki-67LI and high MS, whereas up to half of luminal BCs were of low MS and showed low Ki-67LI.The distributions of Ki-67LI values in this series (mean and median) were consistent with those found in previous studies ,33, and + tumors trastuzumab (Herceptin)-naïve patients) that may be an appropriate subclass likely to benefit from chemotherapy. These findings confirm the prognostic relevance of routinely assessed MS in the luminal class, as previously reported in BC [In luminal BC, proliferation assays using MS and Ki-67LI identified subdivisions with statistically different patient outcomes. In this group, both MS and Ki-67LI retained their significant and independent association with outcome and identified three subclasses; one (low-proliferative group) associated with excellent prognosis where adjuvant chemotherapy is unlikely to provide benefit and could potentially be withheld, and one class (high-proliferative group) with a worse prognosis akin to HER2ed in BC ,7,8. It ed in BC . Suboptied in BC -36. Crited in BC . HoweverDifferent authorities have highlighted the prognostic significance of Ki-67LI in luminal BC, with emphasis on its ability to stratify patients into low-risk and high-risk populations. Although, in these studies, luminal BCs were subdivided into distinct subgroups, the optimal cut-off point remains nonstandardized, with most studies using a single, usually the median, cut-off point; dividing these tumors into two subgroups . In this+ and TN tumors are known to be poorly differentiated and highly proliferative [+ or TN classes. None of the previously reported Ki-67LI cut-off points was able to stratify HER2+ or TN tumors into clinically relevant subclasses. Although the high Ki-67LI cut-off generated in HER2+ and TN training sets appeared to stratify these tumors into proliferative subgroups based on outcome, no association with survival was identified in the validation sets. These findings could be explained by the small number of cases in these molecular classes after splitting them into training and validation sets or by their high proliferation rate, which limits the ability of a proliferation marker to identify clinically distinct subclasses.The majority of HER2The number of sections from each case required to assess Ki-67LI reliably in a clinical laboratory setting also was addressed. The results obtained from each of four sections assessed per tumor showed a high level of concordance, indicating that using a single FFS/case appears appropriate and representative.It is important, however, to mention that in the current study, outcome was assessed in a context in which the treatment given may not be homogeneous, and indeed, in some cases, the parameter being addressed (mitotic score as a grading component) may have affected the original systemic treatment decision. This may limit the ability of the prognostic analyses in making assertions that either mitotic counts or Ki-67LI can identify a group in which chemotherapy could be withheld.+ BC with significantly different clinical outcomes. Overall, both showed comparable results, with Ki-67LI having a marginal advantage in terms of patient-cohort separation. Neither MS nor Ki-67LI has additional prognostic value in HER2+ and TNBC. This study emphasises the importance of determination of appropriate clinically relevant cut points for Ki-67LI and demonstrates that sufficient IHC assessment of Ki-67LI can be achieved by using a single FFS. This method could provide a cost-effective method for prognostic subclassification of luminal/HR+ BC in routine clinical practice.In conclusion, proliferation assessment by using Ki-67LI and MS can distinguish subgroups of patients with luminal/HRin situ hybridization; ER: estrogen receptor; FFPE: formalin-fixed paraffin-embedded; HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry; Ki-67LI: Ki-67 labeling index; MSs: mitotic frequency scores; PR: progesterone receptor; TN: triple negative; TNnon-B: triple-negative non-basal.CISH: chromomeric The authors declare that they have no competing interests.MA performed the practical work, performed the statistical analysis, and drafted the manuscript. EAR conceived and designed the project, participated in the statistics and in writing the manuscript. AAB, FB, and ARG participated in the practical work and contributed to the manuscript. IOE and JSR-F contributed to the project design and to the manuscript. All authors read and approved the final manuscript.


Caveolin-2 is one of the major protein components of cholesterol- and glycosphingolipid-rich flask-shaped invaginations of plasma membrane caveolae. A new body of evidence suggests that caveolin-2 plays an important, and often more direct, role than caveolin-1 in regulating signaling and function in a cell- and tissue type-specific manner. The purpose of this paper is to primarily focus on discussing how these recent discoveries may help better understand the specific contribution of caveolin-2 to lipid raft- and caveolae-regulated cell/tissue-specific signaling and functions. Caveolins are critical membrane proteins of cholesterol lipid-rich lipid rafts and caveolae. Three members are known within the caveolin protein family: caveolin-1 (Cav-1), Cav-2, and Cav-3 , 5. In tUnlike the indispensable role for Cav-1 in caveolae assembly in nonmuscle cells and the respective role of Cav-3 in muscle cells, the role of Cav-2 is less clear. Caveolae have been identified in ultrathin sections from lung capillary endothelium and in perigonadal adipose tissue of Cav-2 knockout (KO), suggesting that Cav-2 is not necessary for caveolae formation, at least, in the aforementioned tissues . HoweverBecause in addition to plasma membrane caveolae and lipid rafts, Cav-2 can exist in other subcellular compartments such as ER, Golgi, cytosolic vesicles, lipid droplets, or nucleus, we have decided to examine if serine phosphorylation of Cav-2 depends on subcellular location. Using a combination of reconstituted epithelial cell-based systems such as LNCaP and FRT cells as well as endogenously expressing caveolins primary human endothelial cells, we have examined if phosphorylation of Cav-2 at serines 23 and 36 can be regulated by Cav-1 and subcellular targeting to noncaveolar intracellular compartments versus plasma membrane lipid raft/caveolae in the aforementioned epithelial and endothelial cells. Specifically, detergent insolubility and sucrose flotation gradients experiments revealed that Cav-2 was phosphorylated at serine 23 primarily in detergent resistant microdomains (DRMs). In contrast, serine 36 phosphorylation occurred in non-DRMs. Furthermore, immunofluorescence microscopy studies determined that in the presence of Cav-1 serine 23-phosphorylated Cav-2 localized mostly to plasma membrane, while serine 36-phosphorylated Cav-2 primarily resided in intracellular perinuclear regions. To directly address the role of Cav-1 in regulating phosphorylation of endogenous Cav-2, we have used siRNA approach. The specific knockdown of Cav-1 in human endothelial cells decreased Cav-2 phosphorylation at serine 23 but not serine 36. Thus, upregulation of serine 23 phosphorylation of Cav-2 depends on Cav-1-driven targeting to plasma membrane lipid rafts and caveolae .Cav-2 has been shown to undergo Src-induced phosphorylation on tyrosine 19, using phosphotyrosine 19 (pY 19)-Cav-2-specific antibody in NIH-3T3 cells stably overexpressing c-Src. Stimulation of adipocytes with insulin resulted in increase in pY 19-Cav-2. Unlike total Cav-2, pY 19-Cav-2 primarily localized near focal adhesion sites. Using Gst-fused Cav-2, several Src homology 2 (SH2) domain containing proteins were identified, such as c-Src, Nck, and Ras-GAP to potentially interact with Cav-2 in a phosphorylation-dependent manner. These data suggest that tyrosine 19-phosphorylated Cav-2 could possibly be a docking site for SH2 domain containing proteins . Interes In addition to tyrosine 19, Cav-2 can also be phosphorylated at tyrosine 27. Remarkably, pY 27-Cav-2 appeared to be more critical than pY 19-Cav-2 for binding c-Src, Nck, and Ras-GAP. EGF stimulated Cav-2 phosphorylation at both tyrosines 19 and 27 in A431 cells; however, the temporal response to EGF was different; that is, Cav-2 was phosphorylated at tyrosine 19 in a rapid and transient fashion, whereas phosphorylation at tyrosine 27 was sustained. Interestingly, just as pY 19-, pY 27-Cav-2 also associated with lipid rafts/caveolae but did not form high molecular heterooligomeric complexes with Cav-1 .The possibility for the involvement of Cav-2 in regulating endothelial cell proliferation and differentiation was suggested by the observation that Cav-2 KO mice develop a hyperproliferative phenotype in the lungs involving vascular endothelial growth factor receptor 2 (VEGFR-2 = Flk-1 in mouse) positive cells . BecauseWe are now investigating the mechanistic nature of this negative regulation by Cav-2. Since Cav-2 almost entirely targets to lipid rafts/caveolar microdomains in mouse lung endothelial cells used in our experiments , our datHow could the inhibitory signal be transduced from caveolar Cav-2 to downstream target proteins regulating endothelial cell proliferation? It may be either through the negative effect on some of many growth factor receptors that are present in caveolae or even via a direct effect on intracellular proteins involved in cell proliferation. For example, the N and to a lesser degree C-terminal ends of caveolae-localized Cav-2 face the cytosol and could potentially interact with signaling proteins located in cytosol, membrane vesicles, and other compartments which can come in a close proximity with caveolae.β) in mouse lung endothelial cells. Although treatment with TGF-β resulted only in a modest inhibitory effect on WT lung endothelial cells, it profoundly inhibited proliferation of Cav-2 KO lung endothelial cells. To confirm the specificity of the observed difference between WT and Cav-2 KO endothelial cells in response to TGF-β, we have stably re-expressed human Cav-2 in Cav-2 KO endothelial cells using a retroviral approach. Similar to WT endothelial cells, the antiproliferative effect of TGF-β was dramatically reduced in the Cav-2 re-expressing endothelial cells. This reduced antiproliferative effect of TGF- β in Cav-2 positive cells was demonstrated by three independent proliferation assays and correlated with a loss of TGF-β-mediated upregulation of p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the Rb protein in Cav-2 re-expressing endothelial cells. Mechanistically, Cav-2 inhibits antiproliferative action of TGF-β by suppressing Alk5/Smad2/3 pathway manifested by reduced magnitude and length of TGF-β-induced Smad2/3 phosphorylation as well as activation of Alk5/Smad2/3 target genes, plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive endothelial cells. To examine possible changes in targeting of TGF-β receptors (TβRs) or other components of TGF-β signaling pathway to lipid raft and caveolar domains, we performed detergent-free sucrose fractionation gradient. Our preliminary data suggest that expression of Cav-2 does not significantly change targeting of TGF-β receptors type I, Alk5 and Alk1 as well as Smad2/3 to caveolar and lipid raft microdomains. However, additional studies with control versus TGF-β-treated endothelial cells using various subcellular fractionation and immunofluorescence microscopy localization techniques will be needed for a more comprehensive analysis. One could possibly argue that perhaps the fact that Cav-1 expression levels are reduced at least by c.a. 50% in Cav-2 KO, relative to WT endothelial cells, could contribute to the enhanced antiproliferative effect of TGF-β in Cav-2 KO endothelial cells. This is, however, not the case, because the levels of re-expressed Cav-2 in Cav-2 KO endothelial cells were insufficient to significantly upregulate the expression levels of Cav-1 or change its targeting to lipid raft/caveolar microdomains. In addition, just as endogenous Cav-2 in WT endothelial cells, the re-expressed Cav-2 displayed normal targeting to plasma membrane and lipid raft/caveolae and did not affect subcellular targeting of endogenous Cav-1. Moreover, these low levels of re-expressed Cav-2 were adequate to reduce TGF-β-mediated responses in Cav-2 re-expressing cells to the levels that were comparable with WT endothelial cells [β signaling and function by Cav-2 is independent of both the expression levels and targeting of Cav-1 to lipid raft/caveolar microdomains. Further studies examining the detailed mechanisms responsible for inhibitory regulation of TGF-β-induced signaling and function in endothelial cells by Cav-2 will be necessary. For example, it will be important to examine for possible interactions of Cav-2 and Cav-1 with Alk5 or other components of TGF-β pathway in the absence and presence of TGF- β. In addition, it will be interesting to examine possible regulation of subcellular localization of Alk5, TβRII, or accessory receptors such as endoglin and β-glycan by Cav-2. Also, it will be important to examine the specific role of previously identified serine and tyrosine phosphorylation of Cav-2 [in vivo significance of our results, for example, how our findings may translate into a possible role of Cav-2 in regulating angiogenesis using various models of angiogenesis in which TGF-β pathway plays an important role, is clearly warranted.Our most recent findings suggest that the role of Cav-2 in regulating lung microvascular EC proliferation is more complex and appears to be context specific . Specifial cells . Thus, tof Cav-2 –18. Finaβ-mediated antiproliferative effect in endothelial cells [β, the role of Cav-2 could switch from anti- to proproliferative effect through the negative regulation of the growth inhibitory action of TGF-β/Alk5/Smad2/3 pathway [It is important to reconcile the role for Cav-2 in inhibiting TGF-al cells  with preal cells . Althougal cells . Under t pathway . Thus, iβ are not altered [Although in Cav-2 KO mice, adipose tissue remains normal and the protein levels of insulin receptor- altered , the fun altered . Further altered . In subs altered . How could Cav-2 play an opposite role in regulating endothelial and fibroblast cell proliferation? The most likely reason is subcellular targeting. Although lipid raft/caveolar localization was not examined in HircB fibroblasts, unlike endothelial cells, HircB cells do not seem to express Cav-1 . Thus, iThe Cav-2 gene was colocalized with the Cav-1 gene to the locus D7S522 of human chromosome 7q31.1, a known fragile site (FRA7G) that is frequently deleted in human cancers, including squamous cell carcinomas of the head and neck, prostate cancers, renal cell carcinomas, ovarian adenocarcinomas, colon carcinomas, and breast cancers . AlthougPseudomonas aeruginosa and that lipid raft targeting and tyrosine phosphorylation of Cav-2 as well as interaction with Csk and c-Src was important [P. aeruginosa to invade MLE-12 cells. In addition, the siRNA knockdown of Cav-1 also resulted in reduction of P. aeruginosa invasion. However, unlike Cav-2 siRNA which did not change Cav-1 expression level, Cav-1 siRNA reduced Cav-2 expression levels, suggesting that Cav-2 rather than Cav-1 could be directly responsible [P. aeruginosa invasion and Cav-2 phosphorylation was determined on the basis of various pharmacological inhibitors, including cholesterol affecting agents such as methyl-β-cyclodextrin or filipin [P. aeruginosa using sucrose floatation gradients and immunofluorescence microscopy. Cav-2 has been shown to facilitate infection of murine lung epithelial cell line MLE-12 with mportant , 37. Spemportant , while oponsible . Lipid r filipin , 37. HowRickettsia conorii in HeLa cells [In another study, using siRNA approach, it was demonstrated that Cav-2, actin, E3 ubiquitin ligase, c-Cbl, and clathrin but not Cav-1 is involved in invasion of La cells . Conside The role of noncaveolar Cav-2 was also suggested in chlamydial infection of various epithelial cell lines including HeLa and FRT cells, where Cav-2 associated with the chlamydial inclusion independently of Cav-1 . However2-terminal (residues 70–86) and COOH-terminal (residues 120–150) hydrophilic domains are all necessary for the targeting to lipid droplets. The NH2- and COOH-terminal domains appeared to be important to membrane binding and exit from endoplasmic reticulum, respectively, indicating that Cav-2 is synthesized and transported to lipid droplets as a membrane protein. In conjunction with findings that lipid droplets contain unesterified cholesterol and raft proteins, the result suggests that the lipid droplet surface may function as a membrane domain. It also suggests that lipid droplets could be related to trafficking of lipid molecules mediated by caveolins [Lipid droplets are organelles that play important role in lipid metabolism and beyond . It was aveolins . Althoug As expected on the basis of lipid droplet targeting of Cav-2, later studies also implicated Cav-2 in regulating lipid metabolism due to changes in its expression levels associated with obesity and type 2 diabetes mellitus or adenoviral overexpression studies , 44; howAlthough originally only Cav-1 but not Cav-3 have been shown to interact and form high molecular weight heterooligomeric complexes with Cav-2 , later sCav-2 has also been shown to regulate endocytosis and trafficking of the M1 muscarinic acetylcholine receptor (mAChR) in MDCK cells . The mecCaenorhabditis elegans [Cav-2 has also been shown to be required for apical lipid trafficking in the intestine of  elegans .Coimmunoprecipitation and fluorescence microscopic colocalization studies have revealed that Cav-2 interacts and colocalizes with Connexin 43 in rat epidermal keratinocytes, suggesting that both caveolins could regulate gap junctional intercellular communication .Generation of and studies involving Cav-2 KO mice started revealing more information as to the pathophysiological significance of Cav-2. Initially, it was shown that Cav-2 KO mice develop hyperplasia in the lung associated with an increased number of Flk-1-positive, most likely endothelial cells , suggestStudies of Woodman et al. , revealeSkeletal muscle abnormalities including tubular aggregate formation, mitochondrial aggregation, and increased numbers of M-Cadherin positive satellite cells that are skeletal muscle-specific stem/precursor cells were also reported in Cav-2 KO mice . BecauseIn another study, an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-2 KO brains could be observed, suggesting that Cav-2 may play a negative role in regulating proliferation of adult neural stem cells . SimilarThe most recent studies have reported increased sensitivity of Cav-2 KO but not Cav-1 KO mice to endotoxemia upon intraperitoneal injection with lipopolysaccharide (LPS) . This auFurther studies will be required to precisely determine pathophysiological and mechanistic aspects of the reported phenotypes involving Cav-2 KO mice.β-induced signaling and function? What new phenotypes are waiting to be discovered in properly challenged Cav-2 KO mice and how they may help to better understand the contribution of Cav-2 to cell- and tissue-specific role of caveolae and lipid rafts?Multiple new findings from functional and mechanistic studies involving both in vivo and cell-based models have been recently reported, suggesting that one of the major protein components of plasma membrane caveolae, Cav-2, is not just an accessory protein, but it is also an important contributor to lipid raft- and caveolae-mediated signaling and function. The evidence continues to accumulate suggesting that in most cases in which loss or reduction of Cav-2 or Cav-1 results in a similar phenotype, Cav-2 plays a more direct role. The simple reason is that unlike Cav-1 which targets lipid rafts and caveolae on its own, interaction with Cav-1 is required for Cav-2 targeting to caveolae. Thus, whenever Cav-1 is lost or significantly reduced, Cav-2 loses its lipid raft/caveolar location and function, for which proper targeting of Cav-2 to lipid raft/caveolae is essential. In contrast, when Cav-2 is lost or reduced, Cav-1 still targets to lipid rafts/caveolae and retains all functions which do not depend on Cav-2. Despite considerable progress in the field, there are many questions to be answered. For example, what is the specific contribution of Cav-2 to lipid raft- and caveolae-mediated regulation of cell signaling and function? Exactly, how Cav-2 performs its functions in caveolae? Which specific domains of Cav-2 are mostly involved and what is the contribution of each phosphorylation site identified on Cav-2? Are there any signaling proteins of which targeting to caveolae depends more directly on Cav-2 than Cav-1 that could explain previously reported functions of Cav-2? What is the functional significance of serine 23 phosphorylation, which strictly depends on targeting of Cav-2 to caveolae in epithelial and endothelial cells? Are there additional posttranslational modifications of Cav-2 in addition to serine 23 which occur when Cav-2 reaches plasma membrane lipid rafts and caveolae? How important is tyrosine 19 and 27 phosphorylation or perhaps dephosphorylation of Cav-2 for the cell-specific role of lipid rafts/caveolae? How is it possible for tyrosine phosphorylated-Cav-2 to associate with caveolae/lipid rafts without being able to form high molecular heterooligomeric complexes with Cav-1? What are the interacting partners for Cav-2 necessary for inhibition of lung endothelial cell proliferation and TGF-


Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening “gap4” region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity. The Wnt/β-catenin signaling pathway is essential for embryonic development and adult tissue homeostasis, consequently mutation of many of the components result in human birth defects, cancer and other diseases LRP5 and LRP6 are the two LRP type of Wnt receptors in the human and mouse genome, and are both widely/ubiquitiously expressed Xenopus embryos Xenopus embryos and mammalian cells Other studies have peripherally compared the relative activities of LRP5 and LRP6. Expression of LRP6 alone, but not LRP5 alone, is able to induce a secondary axis in Given the importance of LRP5 and LRP6 in development and human diseases, we chose to dissect the molecular basis that distinguishes and regulates LRP5 and LRP6 functions by generating a series of LRP5/6 chimeric proteins. Our results suggest that despite highly conserved PPPSPxS motifs shared by both receptors, LRP6 cytoplasmic domain (LRP6C) harbors stronger signaling activity than that of LRP5 (LRP5C), likely owing to the fact that PPPSPxS motifs are more readily phosphorylated in LRP6 than in LRP5. We further identified between the two most carboxyl PPPSPxS motifs an intervening region that appears to account for most of the difference in phosphorylation and signaling capacity between LRP5C and LRP6C. Finally we show that LRP5/LRP6-binding to Axin is likely direct without GSK3 acting as an intermediary physical bridge.To compare the activity of the endogenous Lrp5 and Lrp6 we derived mouse embryonic fibroblasts (MEFs) from the wild-type (WT) +/+, Lrp5−/−, and Lrp6−/− embryonic day 14 (E14) embryos. We analyzed the expression of the endogenous receptors and confirmed the absence of protein in our knockout cells . FurtherWe set out to generate chimeric receptors between LRP5 and LRP6 to dissect their functional differences . In the We first generated *LRP5/6C and LRP6/5C by swapping the cytoplasmic domain between *LRP5 and LRP6 . ComparePstI site located after PPPSPxS motif B in both LRP5 and LRP6 to divide the cytoplasmic domain into two parts, which contain motifs A plus B, and motifs C plus D plus E, respectively >LRP6/5CDE (which contained the A and B motifs of LRP6)>LRP6/5C , mirroring the little effect of insertion of these gaps each in LRP5 . SimilarXenopus (85%) Lrp6 is greater than human LRP5 to mouse (94%), chicken (88%) and Xenopus (80%) Lrp5 , indicating that LRP6 is more conserved during evolution whereas changes in LRP5 may be more tolerable to the organism. These observations may also in part explain the small number of identified human LRP6 mutations in the literature compared to those of LRP5 The LRP5 and LRP6 genes likely arose from duplication of a single ancestral gene approximately 500 million years ago during the emergence of the chordate phylum. Since then LRP5 and LRP6 have retained a relatively high degree structural similarity and paralogous conservation, resulting in partially redundant functions as Wnt receptors in vertebrates. However since this early gene duplication, it is likely that LRP5 and LRP6 have independently evolved to the receptors they are today in the human genome. Genetic studies suggest that LRP6 has a more dominant role in Wnt signal transduction By generating a series of reciprocal chimeric receptors between LRP5 and LRP6, we found that difference between the cytoplasmic domain of LRP5 and LRP6 underlies, to a significant degree, the different signaling activities of these two receptors . Our preIt has been established that Axin binds to both LRP5 Drosophila embryos The gap4 region lies between motifs D and E, but it has a strong effect on phosphorylation of not only motif E (and possibly D), but also of motifs C and A that are some distance away . We beliThe gap4 sequence SYF is conserved among vertebrate Lrp5 proteins, and is distinct from SYSH in gap4 that is invariable among vertebrate Lrp6 orthologs . Such a s interaction in response to parathyroid hormone (PTH) binding to LRP6 We note that while other gap regions, including gap2, do not appear to influence LRP5/6 activities in Wnt/β-catenin signaling, a recent study has shown that the RMTSV region of LRP6 gap2 serves as a potential phosphorylation site (T1558) for Protein Kinase A (PKA) and mediates LRP6 and GαOur study further helps to resolve a controversial issue regarding LRP5/6-Axin interaction. Earlier findings based on yeast two-hybrid assays suggested that LRP5/6-Axin association is likely direct In summary we have determined that the cytoplasmic domain of LRP5 and LRP6 plays a major role in the different signaling activity of these two Wnt receptors, and identified between the last two carboxyl PPPSPxS motifs an intervening gap4 region that has a key modulatory function in LRP5/LRP6 phosphorylation and signaling output. We have also provided evidence that argues for direct LRP5/LRP6-Axin interaction. Collectively our data provide significant new insights into the molecular mechanism of LRP5/LRP6 in Wnt signal transduction in development and human diseases.ClaI site before the transmembrane domain. LNCX-Wnt1, LNCX-Wnt3a or an empty LNCX vector, was used for co-expression assays. GST-LRP5C (amino acids 1409–1615) was cloned into pGEX4 using similar boundaries based on GST-LRP6C (amino acids 1396–1613). GST-LRP6CΔS/T contains a 14 amino acid deletion , 50 ng of LNCX/Wnt1 and 100 ng of LRP5/6 constructs or pCS2 vector control]. Dual luciferase reporter assays were performed as previously described Mammalian cell transfections were done in HEK 293T cells (ATCC# CRL-11268) using FuGENE 6 and performed in triplicate. Cells were plated at 1×10Recombinant GSK3  and CKI  were used for in vitro phosphorylation and binding assays as previously described Lrp5 null Lrp6 null Polyclonal anti-VSVG  was used to detect the expressed LRP5/6 chimeric receptors by western blot analysis as previously described Figure S1Location of VSVG epitope tag and alignment of the LRP5 and LRP6 cytoplasmic domains. (A) The predicted signal peptide cleavage site for LRP5 and LRP6. Insertion of a VSVG epitope tag and BspEI site is a few amino acid residues after the cleavage site. Underlined residues represent the beginning of the first YWTD β-propeller structure. (B) ClustalW alignment of LRP5 and LRP6 proteins from human, chicken, frog, and zebrafish. Amino acid residues highlighted in black, grey, and light grey represent identical, conservative, and similar amino acids, respectively. Green boxes indicate PPPSPxS motifs A, B, C, D and E. The conserved PstI site located after motif B in LRP5 and LRP6 cDNAs was used to create the LRPAB/CDE swap constructs. The extra amino acid residues in LRP6 between the PPPSPxS motifs compared to LRP5 in gaps 1–4 are highlighted in yellow. Deleted residues in GST-LRP6CΔS/T, Δ1466–1479, are shown with asterisks. Proteins used for alignment: LRP5, Human , Gallus gallus , Xenopus laevis , and Danio rerio ; LRP6: Human , Gallus gallus , Xenopus laevis , and Danio rerio .(TIF)Click here for additional data file.Figure S2Comparison of the LRP5 and LRP6 cytoplasmic domains using the endogenous LRP5 signal peptide. TOPFLASH reporter activity of LRP5/6 and LRP6/5 chimeric receptors co-transfected with Wnt1 (A) or Wnt3a (B). The dotted line represents the activity of Wnt alone (through the endogenous receptor).(TIF)Click here for additional data file.Figure S3Secondary structure prediction for LRP5 and LRP6 cytoplamic domain containing the most carboxyl terminal PPPSPxS motifs D and E (boxed) and the gap4 region in between (underlined). Garnier-Robson secondary structure prediction (TIF)Click here for additional data file.


Trichoderma reesei (anamorph to Hypocrea jecorina) contains two cellobiohydolases (CBHs), cellobiohydrolase I (TrCel7A) and cellobiohydrolase II (TrCeI6A), that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs) of variable lengths with respect to their binding and inhibition of both TrCel7A and an enzyme variant without the cellulose-binding domain (CBM).The well-studied cellulase mixture secreted by TrCel7A by isothermal titration calorimetry. We found that XOSs bind to TrCel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of TrCel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number.We studied the binding of XOSs to TrCel7A with phosphoric swollen cellulose as a substrate.On the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of  Biomass is the most abundant raw material in the biosphere, and enzymatic biomass conversion of sugars and subsequent fermentation to transportation fuels has attracted enormous scientific and industrial interest . In typiSeveral phenomenological studies have documented a negative influence of xylans and xylan oligosaccharides (XOSs) on the performance of industrial cellulase mixtures -7. HowevTrichoderma reesei (anamorph to Hypocrea jecorina) contain several endoglucanases and two cellobiohydrolases (CBHs) which process exo-acting, cellobiose-releasing enzymes. The CBHs exhibit quite low reaction rates and non-Michaelis-Menten kinetics, which include an initial burst followed by a biphasic slowdown . The protein sequence of the catalytic domain used was the same sequence as that of TrCel7A but truncated by the carboxy-terminal CBM and the linker sequence. The mature TrCel7A core contained the first 436 amino acids after the signal peptides (17 amino acids). TrCel7A [UniProtKB:P62694] and TrCel7A core were both produced heterologously in Aspergillus oryzae by Novozymes A/S  according to patent WO/2000/039322 and were purified as described by Praestgaard et al. [TrCel7A and TrCel7A core was measured at 30°C in an iTC200 power-compensated isothermal titration calorimeter  with a cell volume of 200 μl and by using an injector equipped with a 40-μl syringe. All binding experiments were carried out at 30°C with 1,000 rpm stirring. In a typical binding experiment, the cell was filled with 40 μM cellobiohydrolase, and pure XOS (1.25 mM) or pools of mixed oligosaccharides (low XOS 5 g/L about 8.4 mM or high XOS 5 g/L about 4.4 mM) were injected according to the following protocol: initial equilibration for 16 minutes, then nine injections of 1 μl after 150 seconds, followed by fifteen injections of 2 μl. Data were analysed using Origin 7.0 software  with the add-on provided by MicroCal/GE Healthcare.The binding affinity of TrCel7A and TrCel7A core amounts were adjusted to release 100 μM or 127 μM pNP from pNP-lac when incubated at 50°C for one hour. After one hour of incubation, PASC was removed by short centrifugation for 20 seconds in a tabletop centrifuge. An aliquot of the supernatant (0.5 ml) was removed and incubated at 99°C for 20 minutes at 300 rpm in an Eppendorf heating block to inactivate the cellobiohydrolase. We analysed 10 μl of the supernatant by HPAEC-PAD. XOSs (200 μl) were added to a final concentration of 1 g/L, which is approximately equivalent to 1.7 mM low XOS or 0.9 mM high XOS. The amount of free cellobiohydrolase was quantified as pNP release from pNP-lac as described by Jalak and Väljamäe [Cellobiohydrolase activity was measured as cellobiose release from 1 g/L PASC at 50°C or 30°C with 300 rpm shaking in an Eppendorf heating block. Väljamäe .TrCel7A activity is hindered by several parameters. TrCel7A is product-inhibited by cellobiose. In the hydrolysis of bacterial cellulose, the product inhibition by cellobiose was measured by Gruno et al. [Ki approximately 1.5 mM [pNP-lac activity are most likely underestimates, since the Ki for cellobiose is 20 μM for the hydrolysis of pNP-lac [A simple quantification of o et al.  as Ki apy 1.5 mM ,21. Thuskcat: catalytic rate constant; Ki: inhibition constant; K: binding constantMJB and KB are employed by Novozymes A/S , which is a major enzyme producer. PW declares no competing interests.MJB designed, planned and conducted the study, and drafted the manuscript. KB provided the enzymes and reviewed the manuscript. PW participated in the critical discussion of the results and finalized the manuscript. All authors read and approved the final manuscript.


The natural history of cow’s milk allergy is usually benign. Among non-IgE mediated forms, proctocolitis resolves in practically all cases close to the first year of life; FPIES is dealt with more conservatively, nevertheless, it also resolves in most cases before the age of 5 years. All studies focusing on IgE mediated CMA have demonstrated a time-dependent decrease of prevalence due to the development of tolerance in increasing proportions of the relevant populations. Epidemiological studies have also been ubiquitous in confirming that, in contrast to childhood, cases of IgE-CMA in adults are very rare. Although it is not clear whether such cases have a childhood onset, there is no doubt that only a handful of pediatric patients will continue to suffer post adolescence. Nevertheless, it has been put forward that CMA may currently last longer than previously suggested, with proportions developing tolerance at school age dropping from ~90% to ~40% in the two most cited studies. In order to inform such discussion, factors that may affect the natural history should be taken into account. These include, but are not confined to, severity of the disease, pattern of IgE sensitization and events relevant to antigen exposure. The infant diet may be crucial, however, few studies have addressed the effects of different variations of it on natural history. A considerable proportion of CMA children may tolerate partially hydrolyzed and/or heated milk, however the effects of these on the natural history are not clear. Finally, several successful attempts to introduce CM in the context of SOTI protocols, seems to be able to permit milk consumption in these children, while inducing tolerance in some.