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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2266.)
© 2007 American Heart Association, Inc.

Thrombosis

Silencing of a Targeted Protein in In Vivo Platelets Using a Lentiviral Vector Delivering Short Hairpin RNA Sequence

Tsukasa Ohmori; Yuji Kashiwakura; Akira Ishiwata; Seiji Madoiwa; Jun Mimuro; Yoichi Sakata

From the Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan.

Correspondence to Tsukasa Ohmori, MD, PhD or Yoichi Sakata, MD, PhD, Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, 3111-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail tohmori{at}jichi.ac.jp or yoisaka@jichi.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets.

Methods and Results— To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin IIb caused a significant reduction of integrin IIbβ3 (IIbβ3) expression in GFP-positive platelets. It also inhibited IIbβ3 activation assessed by the binding of JON/A, an antibody that recognizes activated IIbβ3. Talin-1 silencing by the same method resulted in normal IIbβ3 expression but deficient inside-out IIbβ3 signaling.

Conclusions— shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.

The expression of short hairpin RNA (shRNA) in hematopoietic stem cells by a lentiviral vector resulted in inhibition of targeted protein in platelets, suggesting that shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. Talin silencing by this method caused significant reduction of inside-out IIbβ3 signaling in platelets.

Key Words: shRNA • RNA interference • platelets • talin • integrin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Platelets are terminally-differentiated circulating anucleate cells whose adhesive and signaling functions are essential for normal hemostasis. Platelets are produced in the bone marrow from megakaryocytes as cytoplasmic fragments without genomic DNA.¹ Although platelets contain mRNA within their cytoplasm and can respond to physiological stimuli using biosynthetic processes regulated at the protein translation level,^2,3 application of direct genetic manipulation in platelets has not been reported. Alternatively, megakaryocyte lineage cells derived from embryonic or hematopoietic stem cells are amenable to genetic manipulation using gene transduction systems, enabling molecular studies of adhesion and signaling in megakaryocytes in a way not possible with platelets.^4,5

Because platelets and their precursor megakaryocytes have a finite lifespan, hematopoietic stem cells are preferable targets for genetic transfer to establish long-term in vivo expression of the targeted protein in platelets.⁶ When a retroviral vector containing the integrin β3 (β3) gene driven by the integrin IIb (IIb) promoter was transduced into CD34⁺ cells from a Glanzmann thrombasthenia patient with defects in the β3 gene, integrin IIbβ3 (IIbβ3) was detected after in vitro megakaryocyte differentiation.⁷ We have previously shown that transduction of hematopoietic stem cells with lentiviral vector harboring the glycoprotein (GP) Ib promoter enables specific and efficient expression of the targeted protein in platelets in vivo.⁸ Further, the therapeutic expression of IIbβ3 in β3-deficient mice using a lentivirus vector containing β3 complimentary DNA (cDNA) under the control of the IIb promoter has been reported.⁹ These data indicate that gene expression driven by a platelet-specific promoter using the transduction of hematopoietic stem cells with lentiviral vector can be applied to investigations of the involvement of specific proteins in platelet signaling pathways. However, there has been no evaluation of whether the knock-down of targeted proteins in hematopoietic stem cells using a short hairpin RNA (shRNA) sequence results in sufficient protein reduction in platelets. In this study, we examined whether shRNA expression driven by the RNA polymerase III promoter is sustained during megakaryopoiesis, and whether gene silencing with shRNAs is applicable to analyzing the functions of IIbβ3 in in vivo platelets.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials, cDNA cloning, transfection, construction of lentiviral vector, stem cell transplantation, flow cytometry, immunoblotting, RT–polymerase chain reaction (PCR), platelet adhesion assay, and immunohistochemistry are described in detail in the supplemental materials, available online at http://atvb.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Efficient GFP Expression in Platelets Using LentiLox Vector Harboring GPIb Promoter In Vivo
We first substituted the CMV promoter of pLL3.7, a lentiviral gene transfer vector which simultaneously expresses shRNA and GFP (LentiLox-CMV),¹⁰ with the platelet-specific GPIb promoter (LentiLox-GPIb) (supplemental Figure I). To compare strengths and the specificities of the CMV and GPIb promoters and to assess GFP transduction using these lentiviral vectors in vivo, bone marrow cells transduced with LentiLox-CMV or LentiLox-GPIb were transplanted into the recipient mice. When bone marrow cells transduced with LentiLox-CMV were transplanted, GFP expression was observed in 14% to 32% of CD45⁺ cells and in 0.7 to 2.4% of platelets in peripheral blood (supplemental Figure II). As described previously,⁸ transduction with LentiLox-GPIb resulted in efficient GFP gene marking in platelets (10% to 22%); however, only marginal GFP expression was observed in CD45⁺ and red blood cells (supplemental Figure II). These data suggested that the LentiLox-GPIb system enables specific GFP marking of platelets derived from transduced hematopoietic stem cells.

Efficiency of Lentiviral shRNA for Silencing of Targeted Protein Expression
We next validated the effects of shRNA sequence expression driven by the U6 promoter in LentiLox on expression of the targeted protein. We selected the shRNA sequences for integrin IIb (supplemental Figure IB), so that it was easy to validate the expression and function of this protein using flow cytometry. We also chose talin-1 sequences as a test case for intracellular protein (supplemental Figure IB); because talin is responsible for β integrin activation but its function in in vivo platelets has not been evaluated. Because a molecular defect affecting 1 of the 2 integrin-coding genes is sufficient to cause a concomitant deficit of both IIb and β3,¹¹ we prepared the 2 expression plasmids containing cDNA of IIb and β3 for the expression of IIbβ3 complexes. To determine the efficiency of lentiviral shRNA for IIb-A, the surface expression of IIbβ3 in HEK293 cells was determined after cotransfection of the shRNA constructs with integrin IIb and β3 expression plasmids. As shown in Figure 1A, IIbβ3 expression on the cell surface was significantly inhibited by cotransfection with the shRNA constructs for IIb. On the other hand, other shRNA sequences did not affect the surface expression of IIbβ3 (Figure 1A). As well, shRNA sequences for talin-1 specifically inhibited ectopically-expressed talin in HEK293 cells (Figure 1C). Sequences for IIb-A and talin did not influence the expressions of GPVI and vinculin (Figure 1B and 1D). The degradation of mRNA by shRNA expression was confirmed by real-time quantitative RT-PCR (supplemental Figure III). The construct expressing the shRNA sequence IIb-A and talin-A caused a more powerful inhibition of the expressions of IIb and talin, respectively (Figure 1A and 1C). To rule out the off-targeting effect caused by the high concentration of shRNA, we validated the specificity of these shRNA sequences by a lower concentration of plasmid vectors (0.2 µg; data not shown). Hence, we selected the IIbA and talin-A sequences for in vivo experiments.

View larger version (41K): [in this window] [in a new window]   Figure 1. Inhibition of ectopic expression of the target protein by a lentiviral vector construct delivering shRNA in HEK293 cells. HEK293 cells were cotransfected with expression plasmid containing cDNA of mouse integrin IIb and integrin β3 (A), GPVI (B), Talin (C), or vinculin (D) and the lentiviral siRNA vector plasmid containing no RNA sequence (Empty), scramble RNA sequence (Scramble), IIbA sequence (IIb-A), IIbB sequence (IIb-B), talinA sequence (Talin-A), or talinB sequence (Talin-B). After 48 hours, protein expressions were examined by flow cytometry or immunoblotting. The data are representative of 3 experiments. In lower panel, protein expression after transfection was quantified. Columns and error bars represent the mean±SD (n=3 per group).

Silencing of IIbβ3 Expression In Vivo Platelets by Transplantation of Transduced Bone Marrow Cells With shRNA Lentiviral Vector
We next examined the effects of shRNA driven by the U6 promoter during megakaryopoiesis in vivo. The bone marrow cells transduced with LentiLox-GPIb containing the scramble sequence (LentiLox-scramble-GPIb) or IIbA sequence (LentiLox-IIbA-GPIb) were transplanted into recipient mice. After transplantation, GFP expression was observed in 15% to 20% of platelets in both transplanted groups (data not shown). It is of note that IIbβ3 expression in GFP-positive platelets was significantly reduced in the recipient mice transplanted with cells transduced with LentiLox-IIbA-GPIb (Figure 2A and 2B). As well, JON/A binding after ADP stimulation, which recognizes activated IIbβ3, was reduced to a greater extent by the transduction with LentiLox-IIbA-GPIb (Figure 2B and 2D). The discrepancy in the results between IIbβ3 expression and JON/A binding is thought to be partly the result of the expression of an incompetent IIbβ3 complex that is recognized as antigen but does not act as functional receptor. Because IIb is the most abundant protein in platelets, it is possible that mRNA degradation of IIb by siRNA in platelets becomes incomplete. Under the same conditions, GPIb and GPVI expressions were not affected (data not shown). Additionally, P-selectin expression after platelet activation was hardly affected (Figure 2C and 2D). These data suggested that the expression of the shRNA sequence driven by the U6 promoter is maintained during megakaryopoiesis, and that this method can be applied to investigations of the involvement of specific proteins in platelet activation.

View larger version (33K): [in this window] [in a new window]   Figure 2. Silencing of endogenous integrin IIbβ3 in platelets. Bone marrow cells from donor mice were transduced with LentiLox-scramble-GPIb or LentiLox-IIbA-GPIb at an MOI of 30. Each irradiated recipient mouse received 2 000 000 transduced cells. A, Representative flow cytometry analyses of integrin IIbβ3 expression in platelets of peripheral blood are shown 30 days after transplantation. The plots represent the degree of GFP expression (horizontal) and specific antibody binding for integrin IIb (vertical). The specific antibody binding in GFP-positive platelets is shown (right panel; white:Lentilox-scramble-GPIb, gray: LentiLox-IIbA-GPIb). B, Columns and error bars represent the mean±SD of mean fluorescence intensity of antibody binding for IIb in GFP-positive platelets (n=9 per group). C, The activation of IIbβ3 assessed by JON/A binding and P-selectin expression in platelets stimulated with 150 ng/mL of convulxin. The plots represent the degree of P-selectin expression (horizontal) and JON/A binding (vertical) in GFP-positive platelets. D, Columns and error bars represent the mean±SD of mean fluorescence intensity (MFI) of antibody binding after stimulation with 6 µmol/L ADP or 150 ng/mL of convulxin to GFP-positive platelets (n=5 to 7 per group).

Silencing of Talin in Platelets Decreases IIbβ3 Activation
We next examined whether talin-1 knockdown affects IIbβ3 activation in in vivo platelets, using shRNA silencing. Although talin is believed to be involved in the final common step of integrin IIbβ3 activation,¹² functional analysis in in vivo platelets has not been performed. When bone marrow cells transduced with LentiLox-scramble-GPIb or LentiLox-talinA-GPIb at an MOI of 30 were transplanted, we confirmed the inhibition of talin expression in platelets derived from the cells transduced with LentiLox-talinA-GPIb by intracellular flowcytometry (Figure 3A). In addition, talin reduction was also verified after sorting of GFP-positive platelets by immunoblotting (Figure 3B). On the other hand, the expressions of IIbβ3, GPIb, and GPVI were not affected (data not shown). As shown in Figure 3C through 3E, in talin-deficient platelets identified as GFP-positive cells, IIbβ3 activation after ADP or convulxin stimulation was significantly decreased. Furthermore, talin-deficient platelet partly affected the expression of P-selectin after the platelet stimulation (Figure 3D and 3E). These data clarified that talin was involved not only in IIbβ3-dependent platelet activation but also in the release reaction in actual platelets.

View larger version (28K): [in this window] [in a new window]   Figure 3. Knockdown of talin-1 inhibits integrin IIbβ3 activation and P-selectin expression in platelets. Bone marrow cells transfected with LentiLox-scramble-GPIb or LentiLox-talinA-GPIb were transplanted into recipient mice. A, Representative flowcytometry analyses of intracellular talin expression in GFP-positive (white: LentiLox-scramble-GPIb; gray: Lentilox-talinA-GPIb). B, After the sorting of GFP-positive platelets, the platelet lysates were immunoblotted with anti-talin MoAb (upper panel), anti-vinculin polyclonal antibody (middle panel), or anti-paxillin antibody (lower panel). C, Activation of IIbβ3 was assessed by JON/A binding to platelets incubated with 150 ng/mL of convulxin. The plots represent the degree of GFP expression (horizontal) and specific antibody binding (vertical). D, Activation of IIbβ3 assessed by JON/A binding and P-selectin expression in GFP-positive platelets stimulated with 150 ng/mL of convulxin. The plots represent the degree of P-selectin expression Figure 3 (Continued). (horizontal) and JON/A binding (vertical). E, Columns and error bars represent the mean±SD of mean fluorescence intensity (MFI) of antibody binding after stimulation with 6 µmol/L ADP or 150 ng/mL of convulxin to GFP-positive platelets (n=7 to 11 per group).

Finally, using a platelet adhesion assay we attempted to determine whether talin-deficient platelets influenced the spreading onto fibrinogen. Platelet adhesion to immobilized fibrinogen by itself was not inhibited by the deficiency of talin (Figure 4). However, platelet spreading after stimulation with PMA was markedly suppressed (Figure 4), suggesting that talin is required for platelet spreading on fibrinogen.

View larger version (45K): [in this window] [in a new window]   Figure 4. Failure of talin-deficient platelets to spread onto immobilized fibirinogen after stimulation with PMA. Bone marrow cells transfected with LentiLox-scramble-GPIb (A) or LentiLox-talinA-GPIb (B) were transplanted into recipient mice. Washed platelets treated without (upper panel) or with 1 µmol/L PMA (lower panel) were placed on immobilized fibrinogen for 30 minutes. Cells were fixed and then stained with anti-GFP antibody (green; left panel) and rhodamine-conjugated phalloidin (red; middle panel), as described in Materials and Methods. The merged images show colocalization of GFP and actin staining (yellow; right panel). The data are representative of 3 independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
RNA interference using siRNAs to inhibit specific gene expression is a powerful and promising technology for both basic research and therapeutic intervention.^13,14 A number of vector systems have been reported to mediate stable transduction and expression of shRNAs in mammalian cells.¹³ Among them, lentiviral vectors have been demonstrated to have the ability to stably transduce nondividing cells such as stem cells through integration of the vector DNA into the genome.^10,15 RNA polymerase III promoters, most commonly the H1 and U6 promoters, have been incorporated into the lentiviral vectors for stable expression of shRNAs.¹³ The potential problem for functional genomics studies inherent in transfection with shRNA is variability in transfection efficiency. A solution to this problem is coexpression of reporter genes such as GFP, which facilitates the selection of transduced cells. To obtain efficient gene marking of platelets derived from transduced hematopoietic stem cells, we substituted the ubiquitous CMV promoter of LentiLox with the platelet-specific GPIb promoter and showed that transplantation of bone marrow cells transduced with the vector enabled specific marking of platelets derived from transduced hematopoietic stem cells expressing shRNA sequence.

Platelets are terminally-differentiated anucleate cells, and for this reason direct gene transfer and silencing using virus or plasmid vector have been thought to be impossible. Since the development of gene targeting technologies in embryonic stem cells,¹⁶ the "gold standard" for the analysis of gene function in platelets has been the creation of knockout mice. A large number of knockout studies have shown aberrant platelet phenotypes.^17–20 However, one of the major drawbacks of conventional knockouts is that if the gene product is essential in many tissues, then it is quite likely that the consequence of homozygosity for the mutated allele will be lethality. Several experimental strategies are used or have the potential to overcome the problem of lethality. First, improvements in the technological procedures have allowed for refined analyses of gene functions at specific developmental stages or in specific tissues, based on conditional knock-out strategies by means of Cre-lox–regulated recombination.²¹ Another solution, which has been applied in a few cases, is to use hematopoietic cells from fetal liver of knockout embryos to reconstitute the hematopoietic system of lethally-irradiated wild-type animals.^22,23 Despite these significant improvements, the creation of loss-of-function alleles in the mouse remains time-consuming and costly. Our demonstration that the expression of shRNAs driven by RNA polymerase III promoters can be used to functionally silence protein expression in platelets suggests that RNAi-based technologies might represent a convenient strategy for the study of platelet signal transduction. Our methods could in fact detect platelets derived from transduced stem cells as GFP-positive platelets, and so enable the examination of the involvement of target proteins in platelet signal transduction by flow cytometry and adhesion assay. However, GFP-positive platelets after the transplantation were limited by up to 20% in our protocols. Hence, platelet aggregation testing and the analysis of intracellular signaling pathways including tyrosine phosphorylation are not thought impossible. Higher transfection efficiencies would be required to demonstrate genuine effects and to be a valid alternative to making gene knockout mice.

Cellular control of integrin activation is essential for normal development because it controls cell adhesion, migration, and assembly of the extracellular matrix.^24,25 Platelets express members of the β1 subfamily (vβ1, 2β1, and 6β1) that support platelet adhesion to the extracellular matrix proteins including collagen and laminins, as well as expressing members of the β3 subfamily (vβ3 and IIbβ3).²⁴ Among them, IIbβ3, a receptor for fibrinogen, von Willebrand factor (VWF), fibronectin, and vitronectin is an essential requirement for platelet aggregation. Integrin activation can be controlled by signaling pathways that are thought to act by regulating specific interactions between cytoplasmic proteins and the integrin—or β-subunit—cytoplasmic tail.^25,26 Although many types of proteins interacting with integrin cytoplasmic tails have been reported to be involved in platelet aggregation,^12,27–31 functional analysis of in vivo platelets has not been reported; embryos lacking these proteins, including talin, vinculin, FAK, and Cas do not normally grow in the uterus.^32–35 Talin is a major cytoskeletal protein that colocalizes with activated integrins and binds to integrin β cytoplasmic domains; with the overexpression of the N-terminal region of talin results in activation of integrins.^14,28,36 Additionally, binding of talin to integrin β tails has been shown to be a common final step in integrin activation.¹² In this study, using a method involving RNA interference, we clearly demonstrated that talin is involved in IIbβ3-dependent platelet activation in in vivo platelets. Furthermore, talin might partly participate in the -granule release reaction. These results confirmed that this strategy could be useful as a convenient and powerful method to investigate the role of specific proteins in platelet activation in vivo.

	Acknowledgments

 
We thank N. Matsumoto and M. Ito for their excellent technical assistance.

Sources of Funding

This work was supported by Grants from the Mitsubishi Pharma Research Foundation; Grants-in-aid for Scientific Research from the Ministry of Education and Science; Health and Labor Science Research Grants for Research from Ministry of Health, Labor, and Welfare; and Grants for "High-Tech Center Research" Projects for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science, and Technology), 2002–2006.

Disclosures

None.

	Footnotes

 
Original received March 26, 2007; final version accepted July 19, 2007.

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 27/10/2266    most recent ATVBAHA.107.149872v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ohmori, T. Articles by Sakata, Y. Search for Related Content PubMed PubMed Citation Articles by Ohmori, T. Articles by Sakata, Y.