Modulation of RGD sequence motifs regulates disintegrin recognition of αIIbβ3 and α5β1 integrin complexes

Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the αIIbβ3 and α5β1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards αIIbβ3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the αIIbβ3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10–13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the α5β1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg. AM but inhibited poorly by Eleg. PM and Kistrin. In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the α5β1 complex. In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an α5β1-dependent manner. Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the α5β1 complex by the PRGD molecules. These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.

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Enhanced Adhesion, Migration and Pyk2 Expression in K562 Cells Following Imatinib Treatment

Blood ◽

10.1182/blood.v120.21.4426.4426 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4426-4426

Author(s):

Adelina Ovcharenko ◽

Galit Granot ◽

Jeniffer Park ◽

Ofer Shpilberg ◽

Pia Raanani

Keyword(s):

Cell Adhesion ◽

Targeted Therapy ◽

Mrna Expression ◽

Cell Line ◽

Focal Adhesion ◽

K562 Cell ◽

K562 Cells ◽

Fold Increase ◽

And Migration

Abstract Abstract 4426 Background/Aims: Despite improved prognosis of CML patients with the use of imatinib (IM), its administration is associated with extramedullary disease (EMD) occurrence. We postulate that, like in the metastatic processes, changes in migration and adherence potential may enable leukemic cells to inhabit extramedullary sites. Focal adhesion complexes linking between extracellular matrix and the cell cytoskeleton are likely to play an important role in these processes. Pyk2 is a tyrosine kinase highly expressed in hematopoietic cells, localized to focal adhesion complexes, and known to participate in adhesion and migration processes. We have previously shown that Pyk2 participates in NB4 (an acute promyelocytic leukemia [APL] cell line) cells' adhesion and migration following exposure to the APL targeted therapy ATRA. We postulate that similar to the effect of ATRA on NB4 cells, IM being also a targeted therapy, might also be associated with enhanced adhesion and migration abilities of the CML cell line K562. Our objectives were to identify the effect of IM administration on pyk2 expression and on K562 cell adhesion and migration ability and to establish the role of these changes in treatment-associated EMD. Results: We found a 2.6-fold increase in pyk2 mRNA expression in K562 cells following exposure to IM. We also found that 30% of IM-treated K562 cells adhered to fibronectin (FN) compared to untreated cells having no adhesion ability. In addition, a 3-fold induction in migration was seen in K562 cells following treatment. Furthermore, K562 cells treated with IM demonstrated a 2-fold increase in invasion potential as compared to untreated cells. In order to assess whether Pyk2 is essential for IM-dependent adhesion and migration of K562 cells, these cells were infected with pyk2 specific shRNAs. While 30% of the non-infected NB4 cells adhered to FN following IM treatment, only 12% of the pyk2-shRNA–infected K562 cells exhibited adhesion potential (Pvalue<0.002). In addition, we witnessed over a 3-fold reduction in the ability of pyk2-shRNA–infected K562 cells to migrate following exposure to IM when compared to parental K562 cells. These data support the role of Pyk2 in IM-mediated adhesion and migration. Finally, we found that IM treatment induced an in-vivo increase in pyk2 mRNA expression level in leukocytes derived from 3 out of 5 CML patients studied. Conclusions: IM induces K562 cell adhesion, migration and invasion accompanied by increased pyk2 expression. Pyk2 is one of the key proteins regulating IM-induced cell migration and adhesion. Collectively our data suggest a critical role of Pyk2 in adhesion and migration initiated by the targeted therapy IM and a possible role in EMD development. These data support a common mechanism for the development of EMD in hematological malignancies treated by targeted therapies via pyk2 expression. Disclosures: No relevant conflicts of interest to declare.

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Investigation of the Roles of Non-neuronal Acetylcholine in Chronic Myeloid Leukemic Cells and their Erythroid or Megakaryocytic Differentiated Lines

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180406123154 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1440-1447 ◽

Cited By ~ 2

Author(s):

Banu Aydın ◽

Hülya Cabadak ◽

M. Zafer Gören

Keyword(s):

Cell Lines ◽

K562 Cell ◽

Ache Activity ◽

K562 Cells ◽

Myelogenous Leukemia ◽

Choline Uptake ◽

Dependent Manner ◽

Uptake Inhibition ◽

Cell Counts

Background: Many studies suggested that Acetylcholine (ACh) might serve as an autocrine/ paracrine growth factor in several types of tumors or tumor cell lines. High levels of Acetylcholinesterase (AChE) activity have been reported in primary brain tumors, ovarian, colon and lung tumors. Objectives: The role of cholinergic signaling needs to be clarified in in leukemia. Method: K562 cells were derived from a chronic myelogenous leukemia patient during blast crisis serving as pluripotent hematopoietic stem cells. K562 cells were incubated with various cholinergic agonists or antagonists to investigate the role of ACh in different differentiated cell lines. Results: Our experiments showed that AChE activity was increased in response to ACh in undifferentiated K562 cells, but in the erythroid differentiated K562 cells a high concentration of ACh (1 mM) decreased the AChE activity. ACh failed to elevate the AChE activity in the megakaryocytic differentiated K562 cells. An AChE inhibitor, eserine, also suppressed the AChE activity in a concentration-dependent manner. Choline uptake inhibition by hemicholinium did increase the AChE activity but not in the erythroid differentiated K562 cell line. Likewise, megakaryocytic differentiated K562 cells also displayed a similar pattern. Vesamicole, a vesicular choline uptake inhibitor, produced similar results. Curare, a nicotinic antagonist, elevated the cell counts of the megakaryocytic differentiated cells. Conclusion: Our findings may suggest excess extracellular ACh will decrease the cell growth in undifferentiated and megakaryocytic differentiated K562 cell lines through nicotinic type cholinoceptors.

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CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Blood ◽

10.1182/blood-2019-129873 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1734-1734

Author(s):

Orit Uziel ◽

Zinab Sarsur- Amer ◽

Einat Beery ◽

Pia Raanani ◽

Uri Rozovski

Keyword(s):

B Cells ◽

Time Dependent ◽

Dependent Manner ◽

Environmental Resources ◽

Type B ◽

Wild Type ◽

Cell Competition ◽

Western Immunoblotting ◽

Competition Theory

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

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Abstract 110: Thrombospondin Type-1 Domain-Containing Protein 1 (THSD1), an Intracranial Aneurysm Susceptibility Gene, Mediates Cell Adhesion in Human Umbilical Vein Endothelial Cells

Stroke ◽

10.1161/str.46.suppl_1.110 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Xiaoqian Fang ◽

Dong H Kim ◽

Teresa Santiago-Sim

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Focal Adhesion ◽

Umbilical Vein ◽

Adhesion Formation ◽

Wild Type ◽

Focal Adhesion Formation ◽

Umbilical Vein Endothelial Cells

Introduction: An intracranial aneurysm (IA) is a weak spot in cerebral blood vessel wall that can lead to its abnormal bulging. Previously, we reported that mutations in THSD1 , encoding thrombospondin type-1 domain-containing protein 1, are associated with IA in a subset of patients. THSD1 is a transmembrane molecule with a thrombospondin type-1 repeat (TSR). Proteins with TSR domain have been implicated in a variety of processes including regulation of matrix organization, cell adhesion and migration. We have shown that in mouse brain Thsd1 is expressed in endothelial cells. Hypothesis: THSD1 plays an important role in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane. Methods: Human umbilical vein endothelial cells are used to investigate the role of THSD1 in vitro . THSD1 expression was knocked-down by RNA interference. Cell adhesion assay was done on collagen I-coated plates and focal adhesion formation was visualized using immunofluorescence by paxillin and phosphorylated focal adhesion kinase (pFAK) staining. THSD1 re-expression is accomplished by transfection with a pCR3.1-THSD1-encoding plasmid. Results: Knockdown of THSD1 caused striking change in cell morphology and size. Compared to control siRNA-treated cells that exhibited typical cobblestone morphology, THSD1 knockdown cells were narrow and elongated, and were significantly smaller ( p <0.01). Cell adherence to collagen I-coated plates was also attenuated in THSD1 knockdown cells ( p <0.01). Consistent with this finding is the observation that the number and size of focal adhesions, based on paxillin and pFAK staining, were significantly reduced after THSD1 knockdown ( p <0.01). These defects in cell adhesion and focal adhesion formation were rescued by re-expression of wild type THSD1 ( p <0.05). In contrast, initial studies indicate that expression of mutated versions of THSD1 as seen in human patients (L5F, R450*, E466G, P639L) could not restore cell adhesion and focal adhesion formation to wild type levels. Conclusions: Our studies provide evidence for a role of THSD1 and THSD1 mutations in endothelial cell adhesion and suggest a possible mechanism underlying THSD1 -mediated aneurysm disease.

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Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

Circulation Research ◽

10.1161/res.119.suppl_1.361 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Jie Liu ◽

Yanmei Qi ◽

Shu-Chan Hsu ◽

Siavash Saadat ◽

Saum Rahimi ◽

...

Keyword(s):

Cell Adhesion ◽

Es Cells ◽

Embryonic Stem ◽

Intercellular Junctions ◽

Amino Acid Residues ◽

Loss Of Function ◽

Wild Type ◽

Adhesion Sites ◽

Cell Cell

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

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ROLE OF PLATELET GLYCOPROTEINS lb AND llb/llla IN TUMOR CELL INDUCED PLATELET AGGREGATION AND TUMOR CELL ADHESION TO EXTRACELLULAR MATRIX. I. Grossi

10.1055/s-0038-1644670 ◽

1987 ◽

Author(s):

L Grossi ◽

K V Honn ◽

B F Sloane ◽

J Thomopson ◽

D Ohannesian ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Adhesion ◽

Platelet Aggregation ◽

Tumor Cell ◽

Platelet Adhesion ◽

Eicosatetraenoic Acid ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Tumor Cell Adhesion ◽

Dose Dependent

Platelet glycoproteins are known to play a role in platelet platelet interactions, platelet activation, and platelet adhesion to extracellular matrix (ECM). Monoclonal antibody to human platelet glycoprotein lb (mAblb) and polyclonal antibodies to the llb/llla complex (pAbllb/llla) were used to evaluate the involvement of these glycoproteins in tumor cellinduced platelet aggregation (TCIPA and tumor cell adhesion to the ECM. We have demonstrated that human cervical carcinoma (MS5I7), human colon carcinoma (Clone A), and rat Walker 256 carcinosarcoma (W256) cells induce aggregation of homologous platelets via thrombin generation. MAblb and pAbllb/llla were shown to inhibit TCIPA by MS517, Clone A, and W256 in a dose dependent manner. MAblb was also shown to inhibit platelet thromboxane B2 production in response to tumor cells in a dose dependent manner. Neither mAblb nor pAbllb/llla had any effect on ADP stimulated platelet aggregation. Concentrations of mAblb and pAbllb/llla which produced half maximal inhibition alone were combined resulting in complete inhibition of TCIPA. Preincubation of MS5I7 and W256 with mAblb also resulted in inhibition of TCIPA, while preincubation of Clone A with mAblb did not, suggesting the presence of this glycoprotein on the cell membranes of MS5I7 and W256, but not on Clone A. Immunofluorescence studies confirmed the presence of this glycoprotein on the cell plasma membrane of the MS5I7 and W256, but not on Clone A. Preincubation of MS5I7 and W256 with both mAblb and pAbllb/llla alone or in combination, also resulted in decreased (12S)-12 -hydroxy -5, 8,10, 14 -eicosatetraenoic acid (12-HETE) production, while platelets preincubated with these antibodies had no effect on the concentration of 12-HETE produced. Isolation of platelet membranes and released platelet contentswere tested separately and in combination on platelet adhesion to ECM. Platelet release factors were ineffective, while isolated platelet membrane ghosts enhanced adhesion. Disruption of the platelet cytoskeleton andinhibition of the formation of the llb/llla complex decreased platelet enhanced tumor cell adhesion. These findings suggest a role for these platelet glycoproteins in TCIPA, platelet enhanced tumor cell adhesion to ECM and subsequent tumor metastasis.

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Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽

10.1182/blood.v96.6.2172 ◽

2000 ◽

Vol 96 (6) ◽

pp. 2172-2180 ◽

Cited By ~ 92

Author(s):

Kotaro Suzuki ◽

Hiroshi Nakajima ◽

Norihiko Watanabe ◽

Shin-ichiro Kagami ◽

Akira Suto ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Cytokine Receptor ◽

Type I ◽

Dependent Manner ◽

Type Ii ◽

Wild Type ◽

Peritoneal Mast Cells ◽

The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

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The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation

The Journal of Cell Biology ◽

10.1083/jcb.145.5.961 ◽

1999 ◽

Vol 145 (5) ◽

pp. 961-972 ◽

Cited By ~ 36

Author(s):

Alessio Merlin ◽

Wolfgang Voos ◽

Ammy C. Maarse ◽

Michiel Meijer ◽

Nikolaus Pfanner ◽

...

Keyword(s):

Protein Import ◽

Protein Translocation ◽

Adaptor Protein ◽

Dependent Manner ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

J Proteins

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Δ18 in addition to the endogenous wild-type Tim44. Tim44Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

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The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v112.11.112.112 ◽

2008 ◽

Vol 112 (11) ◽

pp. 112-112

Author(s):

Aleksandra Stojanovic ◽

Matvey Gorovoy ◽

Tatyana Voyno-Yasenetskaya ◽

Xiaoping Du

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Wild Type ◽

Glycoprotein Ib ◽

Lim Kinase ◽

And Function

Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.

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Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽

10.1182/blood.v112.11.1870.1870 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1870-1870

Author(s):

Sirisha Kodeboyina ◽

Sima Zein ◽

Moosueng Lee ◽

Parimaladevi Balamurugan ◽

Xiao Yao ◽

...

Keyword(s):

Electrophoretic Mobility ◽

Globin Gene ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Luciferase Reporter ◽

Complete Analysis ◽

Dependent Manner ◽

Mobility Shift ◽

Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

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