Novel Lyn-Mediated Regulation of CLEC-2 Signaling By Gq-Coupled Receptors in Platelets

Abstract The CLEC-2 agonist, rhodocytin, elicits powerful platelet activation signals in conjunction with Src family kinases, Syk, and PLCg2. Previous reports have shown that rhodocytin-induced aggregation is dependent on secondary mediators. However, the role of secondary mediators in CLEC-2 signaling is not defined. In this study we report that CLEC-2-induced Syk activation is aspirin-sensitive and that TxA2 plays an important role in the most proximal events such as CLEC-2 phosphorylation and Syk activation. We also show that the activation of other GPCRs, such as the ADP receptors and PARs, can also potentiate CLEC-2 signaling. By using the Gq-inhibitor, UBO-QIC, or P2Y1/P2Y12 antagonists, we show that the Gq-signaling, but not G12/13 or Gi, is essential for GPCR-induced Syk phosphorylation downstream of CLEC-2. We further elucidated the signaling involved in Gq-mediated Syk phosphorylation and identified an important role for PKCs downstream of PLCb regulating SFK activation (Figure 1A). Using Lyn-knock out murine platelets we identified a potential role for Lyn downstream of the Gq-pathway in potentiating CLEC-2 signaling by using. We suggest that, at low concentration of CLEC-2 agonist, the unclustered CLEC-2 receptors are phosphorylated by the Gq-activated Lyn resulting in the potentiation of the initial CLEC-2 signal (Figure 1B). However, Gq receptors by themselves cannot phosphorylate CLEC-2 receptors and require minimal levels of ITAM-containing receptor stimulation in order to initiate unclustered CLEC-2 receptor phosphorylation. Together, these results provide evidence for a novel Lyn-mediated regulation of CLEC-2 signaling by Gq-coupled receptors thereby leading to potentiation of CLEC-2-induced signaling (Figure 1C). Figure 1 A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling. Figure 1. A) Western blot showingeffect Gq-pathway inhibitors on CLEC-2 signaling. B) Effect of low concentration of CLEC-2 agonist in Lyn-knock out murine platelets. C) Model showing role of Gq-activated Lyn in CLEC-2 signaling. Disclosures No relevant conflicts of interest to declare.

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148 EXPRESSION OF OVIDUCT-SPECIFIC GLYCOPROTEIN IN THE CANINE OVIDUCT DURING THE PERIOVULATORY PERIOD

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab148 ◽

2014 ◽

Vol 26 (1) ◽

pp. 187 ◽

Cited By ~ 1

Author(s):

C. Marnier ◽

M. Saint-Dizier ◽

M. Z. Tahir ◽

S. Chastant-Maillard ◽

K. Reynaud

Keyword(s):

Protein Expression ◽

Western Blot ◽

Potential Role ◽

Germinal Vesicle ◽

Blastocyst Stage ◽

Santa Cruz ◽

Immuno Histochemistry ◽

Imagej Software ◽

Mouse Antibody

In the canine species, the oocyte is ovulated at the immature germinal vesicle (GV) stage and will reach metaphase II stage after 3 to 4 days spent in the oviduct. Fertilization and embryonic development to the blastocyst stage also take place in the oviduct. In a previous study (Tahir et al. 2012 Reprod. Domest. Anim. 47, 487), we reported the expression of oviductin (oviduct-specific glycoprotein) mRNA in the oviduct. The present study aimed to describe the oviductin protein expression (immunolocalization and Western blot quantification) and the effect of the oviducal region and the ovarian cycle. Beagle bitches were ovariectomized at 6 stages (6 bitches/stage): anestrus, after the onset of proestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov), 1 day (Day 1), 4 days (Day 4), and 7 days (Day 7) after ovulation. Three oviducal regions were collected [i.e. ampulla, isthmus, and ampulla-isthmus junction (AIJ)]. Ampulla and isthmus were fixed in paraformaldehyde, embedded in paraffin, and 7-μm sections were used for immuno-histochemistry using a goat polyclonal anti-human oviductin (N20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ImmPress kit (Vector Laboratories, Burlingame, CA, USA). Total protein from the AIJ was extracted and used for Western Blot using a mouse monoclonal anti-mouse antibody (H8; Santa Cruz Biotechnology). The expression of oviductin in AIJ was quantified in duplicate on blots using ImageJ software and normalized with actin levels. Relative amounts of oviductin were compared between stages by ANOVA followed by a Tukey test. Immuno-histochemistry revealed that oviductin was specifically expressed in the nonciliated cells of the oviducal epithelium from Pre-LH to Day 7, with a stronger staining in the isthmus than in the ampulla at all stages. Furthermore, the expression of oviduct-specific glycoprotein, detected by Western Blot, varied significantly with the stage (P < 0.0001). The oviductin protein expression was at its lowest level at anestrus, then increased significantly at Pre-LH and Pre-ov (35- and 41-fold higher levels than anestrus, respectively), reached a maximal level at Day 1 (66-fold higher than anestrus), then decreased at Days 4 and 7 (47- and 20-fold higher than anestrus, respectively). In conclusion, this is the first report of oviductin protein expression in the canine oviduct. The region-specific higher expression of oviductin at Day 1 post-ovulation suggests a potential role of this glycoprotein in gamete maturation and fertilization in the bitch.

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HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis

Blood ◽

10.1182/blood.v112.11.2743.2743 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2743-2743

Author(s):

Simona Colla ◽

Nicola Giuliani ◽

Paola Storti ◽

Mirca Lazzaretti ◽

Katia Todoerti ◽

...

Keyword(s):

Multiple Myeloma ◽

Western Blot ◽

Cell Line ◽

Real Time ◽

Real Time Pcr ◽

Potential Role ◽

Angiogenic Switch ◽

Bone Biopsies

Abstract Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls. In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

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Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽

10.1182/blood.v114.22.3470.3470 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3470-3470

Author(s):

Daniela Cilloni ◽

Monica Pradotto ◽

Francesca Messa ◽

Francesca Arruga ◽

Enrico Bracco ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Western Blot ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Small Gtpases ◽

Loss Of Function ◽

Imatinib Resistance ◽

Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

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Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽

10.1182/blood.v116.21.2474.2474 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2474-2474

Author(s):

Jesus Duque-Afonso ◽

Aitomi Essig ◽

Leticia M Solari ◽

Tobias Berg ◽

Heike L. Pahl ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Functional Role ◽

Target Gene ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Leukemia Cells ◽

Myeloid Differentiation ◽

Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

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Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,

Blood ◽

10.1182/blood.v118.21.3206.3206 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3206-3206

Author(s):

Carlos E Vazquez ◽

Gregory N Prado ◽

Enrique R Maldonado ◽

Gabriela Saca ◽

Iren M Ortiz ◽

...

Keyword(s):

Western Blot ◽

Respiratory Burst ◽

Mineralocorticoid Receptor ◽

Conflicts Of Interest ◽

Critical Role ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Dead Cell ◽

Cardiovascular Morbidity And Mortality

Abstract Abstract 3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

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The Role of the IAP Livin in Megakaryocytes Differentiation and Thrombopoiesis

Blood ◽

10.1182/blood.v120.21.3297.3297 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3297-3297

Author(s):

Ihab Abd-Elrahman ◽

Varda R Deutsch ◽

Marjorie Pick ◽

Sigi Kay ◽

Tzahi Neuman ◽

...

Keyword(s):

Progenitor Cells ◽

Potential Role ◽

Conflicts Of Interest ◽

Cell Activity ◽

Human Cell Line ◽

Over Expression ◽

Immunohistochemistry Staining ◽

Apoptotic Activity ◽

Apoptosis Proteins

Abstract Abstract 3297 In this study we observe that Livin plays a role in thrombopoiesis. Livin is a member of the Inhibitor of Apoptosis Proteins (IAP) family of intracellular anti-apoptotic proteins that acts by binding and inhibiting caspases. Previously we found that Livin is unique among the IAP members as upon strong apoptotic stimuli, it is specifically cleaved by caspases to produce a truncated protein (tLivin) that has a paradoxical pro-apoptotic activity. Here we have shown that Livin is expressed in normal mature bone marrow MK and in platelets. Objective: The objective of this study was to evaluate the potential role of Livin in thrombopoiesis. Methods: We studied Livin expression in normal BM using immunohistochemistry staining. We studied the ability of primary MK generated from CD34+progenitor cells over-expressing Livin to produce platelets. While, the human cell line, LAMA-84, was induced by a phorbol myristic acid (PMA) to differentiate to megakaryocytes (MK) to evaluate the potential role of Livin in thrombopoiesis. Results and conclusions: Livin over-expression in CD34+ progenitor cells induced differentiation of these cells into MK and increased the ability of these primary MK to produce platelets. LAMA-84, upon differentiation into MK, produces platelets that are functional and capable of aggregating. This thrombopoiesis was accompanied by increased Livin expression and downregulation of other IAPs: XIAP and Survivin. Our results show that Livin plays a dual role in thrombopoiesis, demonstrating both an anti and pro-apoptotic role in cell activity. Disclosures: No relevant conflicts of interest to declare.

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Homoharringtonine Contributes to Imatinib Sensitivity by Blocking EphB4/RhoA Pathway in Ph+ Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.4420.4420 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4420-4420

Author(s):

Liu Xiaoli ◽

Bintao Huang ◽

Qingfeng Du ◽

Jinfang Zhang ◽

Na Xu ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Annexin V ◽

Apoptosis Rate ◽

Xenograft Models ◽

Stimulation Of ◽

In Cells

Abstract Abstract 4420 Objective: The purpose was to investigate the role of the EphB4 in imatinib (IM) resistance and the mechanism why the homoharringtonine (HHT)+IM regimen gained more treatment profits than simple HHT or IM treating myeloid leukemia. Method: The stable under-expressing EphB4 cells (K562-R-EphB4-sh) were obtained. The cell viability and IC50 under the incubation of IM or HHT+IM was tested by MTT. PE Annexin V apoptosis detected the apoptosis rate of K562-R cells. Subcutaneous K562 xenograft models were established. The activated signal proteins in cells and tissues such as RhoA, MEK and ERK were tested by Western blot. Result: K562-R-EphB4-sh cell and xenograft were sensitivity to IM. Activated RhoA was not involved in K562-R-EphB4-sh cell and xenograft tissue. But phosphorylation of MEK/ERK was overexpression in K562-R-EphB4-sh cell and tissue. The apoptosis rate reached 58.71 ± 2.39% with K562-R cell incubated with HHT+IM, which was higher to K562-R cell incubated with IM (P=0.002). IC50 of K562-R cell incubated by IM was 5.45 mg/L. But under the stimulation of HHT+IM, IC50 of K562-R decreased from 5.45 to 1.17 mg/L (P<0.001). K562-R xenograft volumes significantly decreased with IM+HHT treatment comparing with before treatment (1692.82 ± 317.14 mm3 versus 975.56 ± 132.42 mm3, P<0.001). HHT blocked the expressions of EphB4/RhoA in K562-R cell and xenograft, but HHT cannot down-regulate the expression of P- MEK/ERK. Conclusions: A new marker of IM resistance mediated by the activation of EphB4/RhoA pathway. HHT+IM regimen gained more treatment profits than simple HHT or IM treating myeloid leukemia by blocking EphB4/RhoA pathway in Ph+ myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

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A HoxD11 Homeobox Gene Mutation Associated With Congenital Absent Radius But Without The Thrombocytopenia Of The TAR Syndrome Or a HoxA11 Mutation

Blood ◽

10.1182/blood.v122.21.4837.4837 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4837-4837

Author(s):

Roger A. Fleischman

Keyword(s):

Potential Role ◽

Conflicts Of Interest ◽

Homeobox Genes ◽

Homeobox Gene ◽

Incomplete Penetrance ◽

Potential Candidate ◽

Negative Finding ◽

Tar Syndrome ◽

Absent Radius

HoxA11 and HoxD11 are homeobox genes critical for normal development of the forearm and thus are potential candidate genes for involvement in the pathogenesis of the thrombocytopenia/absent radius (TAR) syndrome. However, we previously reported an absence of coding sequence mutations in either HoxA11 or HoxD11 in a series of 10 unrelated TAR syndrome patients (Fleischman RA et al., Br J Haematol., 116:367-75, 2002). Despite this negative finding, interest in the potential role of homeobox genes in the TAR syndrome has been supported by a report of a HoxA11 mutation occurring in two kindreds with amegakaryocytic thrombocytopenia and radio-ulnar synostosis, a less pronounced more proximal pattern of radial malformation (Thompson AA and Nguyen LT. Nat Genet., 26:397-8, 2000). Unlike HoxA11, however, no mutations in the human HoxD11 gene have been described thus far that would help elucidate the potential role of this paralogous gene in megakaryopoiesis or the TAR syndrome. We now describe a novel mutation in human HoxD11 that results in a polyalanine sequence expansion, (GCG)6→ (GCG)8, and report that this mutation is associated with a unilateral absent radius in the affected propositus. A familial syndrome is suggested in this kindred, moreover, by the prior observation of a bilateral absent radius in a deceased maternal aunt. This mutation was not present in more than 100 unrelated normal subjects or 8 other unrelated individuals with sporadic absence of the radius. Two other living maternal relatives also carried the mutation but did not exhibit any radial defects, a finding consistent with autosomal dominance with incomplete penetrance, an inheritance pattern reported for short polyalanine expansion mutations in the related homeobox gene HoxD13 which cause synpolydactyly. In contrast to the reported HoxA11 mutation, however, neither the propositus nor the mutation carriers of this HoxD11 mutation exhibited thrombocytopenia or any other cytopenias or congenital defect. The results suggest that at least one class of mutation in human HoxD11 may be sufficient to cause an absent radius syndrome but unlike the reported HoxA11 mutation, does not adversely affect megakaryopoiesis. The findings further suggest that additional studies of the TAR syndrome may be necessary to exclude as yet undetected non-coding mutations in promoter or enhancer sequences that alter the expression of HoxA11, HoxD11 or other homeobox genes critical for radial development and/or megakaryopoiesis. This work was supported by a VA Merit Award. Disclosures: No relevant conflicts of interest to declare.

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Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation

PLoS ONE ◽

10.1371/journal.pone.0051451 ◽

2012 ◽

Vol 7 (12) ◽

pp. e51451 ◽

Cited By ~ 18

Author(s):

Carmen Aravena ◽

Ana R. Beltrán ◽

Marcelo Cornejo ◽

Viviana Torres ◽

Emilce S. Díaz ◽

...

Keyword(s):

Cell Proliferation ◽

Arsenic Trioxide ◽

Intracellular Ph ◽

Potential Role ◽

Low Concentration

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C-Cbl-PI3K Interaction Is Important for Platelet Outside-In Signaling

Blood ◽

10.1182/blood.v116.21.2014.2014 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2014-2014

Author(s):

Claudia Lorena Buitrago ◽

Satya P. Kunapuli ◽

Archana Sanjay

Keyword(s):

Actin Cytoskeleton ◽

Protein Interactions ◽

Conflicts Of Interest ◽

Physiological Role ◽

Human Platelets ◽

Scaffolding Protein ◽

Protein Protein Interactions ◽

Clot Retraction ◽

Knock Out

Abstract Abstract 2014 Platelet activation by outside-in signaling is initiated by the binding of fibrinogen to alphaIIbbeta3, an integrin only expressed in platelets and megakaryocytes. Signals transduced by alphaIIbbeta3 regulate actin cytoskeleton resulting in filopodia and lamellipodia formation, cell spreading and retraction. c-Cbl protein is abundantly expressed in platelets and functions as E3 ubiquitin ligase and scaffolding protein to mediate protein-protein interactions. Importantly, c-Cbl tyrosine 731 has been shown to interact with p85 subunit of phosphotidylinositol 3-kinase (PI3K) modulating the actin cytoskeleton. Although previous reports showed c-Cbl activation downstream of alphaIIbbeta3, the mechanisms and implications of this activation or the downstream targets remain to be elucidated. We have studied the role of c-Cbl in platelet outside-in signaling: Using human platelets we have demonstrated that c-Cbl Y700, Y731 and Y774 residues undergoes tyrosine phosphorylation upon platelet adhesion to immobilized fibrinogen. These phosphorylation events are completely inhibited in the presence of the pan Src Family Kinases (SFKs) inhibitor (PP2) suggesting that c-Cbl is phosphorylated downstream of SFKs. Spleen tyrosine kinase (Syk) is also involved in this signaling pathway since its inhibition significantly reduce c-Cbl phosphorylation at residues Y774 and Y700; interestingly, tyrosine 731 phosphorylation, which allows the interaction with the p85-subunit of PI3K, is not affected by Syk inhibition. The physiological role of c-Cbl in platelet outside-in signaling was studied using c-Cbl knock-out mice. We found that in contrast to WT platelets, c-Cbl KO platelets had a significantly reduced spreading over a fibrinogen-coated surface. Furthermore, clot retraction analysis demonstrated that c-Cbl KO platelets retraction time was delayed when compared to WT platelets, suggesting a retraction defect. To further elucidate the physiological role of c-Cbl-PI3K interaction we used a knock-in mouse in which the c-Cbl residue Y 731 was substituted with phenylalanine (Y731F) thereby abolishing the PI3K binding site on c-Cbl. Importantly, platelets from Y731F mice showed spreading and clot retraction defect that were comparable with the c-Cbl KO. These result indicates that in large part, the role of c-Cbl in platelets outside-in signaling is determined by its interaction with PI3K. In conclusion, we have demonstrated that c-Cbl plays an important role in platelet outside-in signaling, and its interaction with PI3K through tyrosine 731 is of pivotal importance in platelet spreading and retraction. Disclosures: No relevant conflicts of interest to declare.

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