标题: Reduction of Shp-2 Expression by Small Interfering RNA Reduces Murine Embryonic [打印本页] 作者: 江边孤钓 时间: 2009-3-5 00:10 标题: Reduction of Shp-2 Expression by Small Interfering RNA Reduces Murine Embryonic
作者：Gang-Ming Zou, Rebecca J. Chan, W. Christopher Shelley, Mervin C. Yoder作者单位：Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA ! P6 `5 F" ]0 ?5 H0 Z
. Z9 H# U- b) p6 k3 A+ y+ @" Q& d' b
- @& s. y- G2 M/ }
# U' \9 }$ n$ }" D4 B
* J7 d& |- }, M* b- w$ x8 {7 e " y/ `2 u+ P" y0 [; `4 M
9 H& N3 F! E1 ?. J4 n 1 l- J' ?' E& Y3 x6 ^6 K 9 J. o9 q3 V! E* r+ U
1 r1 k. P/ D; V' F " `, H$ v$ ~5 a) u5 j
+ A# G0 P( ?* b# V( C* T% t
) U. t. R' R7 h3 S+ i5 J& r 【摘要】 ; q, _, k7 |% c) E9 ]/ X Shp-2 is a member of a small family of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases. Although Shp-2 has been shown to be necessary for hematopoiesis using a mouse model expressing a mutant residual protein (Shp-2/), we used small interfering RNA (siRNA) to reduce Shp-2 expression and examined the consequences on embryonic stem cell (ESC)-derived hemangioblast, primitive, and definitive hematopoietic development. We found that at a concentration of 50 nM, Shp-2 siRNA effectively diminished Shp-2 expression in differentiating embryoid bodies. Hemangioblast, primitive, and definitive hematopoietic progenitor formation was decreased significantly after transfection with Shp-2 siRNA but not with scrambled siRNA. Because Shp-2 is involved in signals emanating from the basic fibroblast growth factor (bFGF) receptor, we asked whether Shp-2 functions in bFGF-mediated hemangioblast development. Reduction of Shp-2 expression using siRNA, but not scrambled siRNA, blocked the bFGF-induced increase in hemangioblast development. Using siRNA as an independent method of reducing Shp-2 function, in contrast to the mutant mouse model (Shp-2/) previously used, we demonstrate that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development and that Shp-2 is integrally necessary for bFGF-mediated hemangioblast production. , E8 ^4 Z% s1 P y# C+ }
【关键词】 Shp- Embryonic stem cell RNA interference Small interfering RNA Hemangioblast9 m2 l, [ I& [: Y
INTRODUCTION 7 ]9 R0 p0 z$ y9 t$ A4 A; Z7 \3 z. m! S" Z e. `2 \ ?; a
Shp-2 is a cytoplasmic protein tyrosine phosphatase that contains two Src homology-2 (SH2) domains and a tyrosine phosphatase domain .% L4 q u" T( y3 z0 V+ M( o2 \
: N- Q p7 j: F: D0 G8 LTo examine the function of Shp-2, the murine Shp-2 locus has previously been targeted by homologous recombination to yield an in-frame mRNA encoding a mutant Shp-2 protein lacking amino acids 46 through 110 within the N-terminal SH2 domain (Shp-246¨C110). Shp-2/ mice die in utero by day 9.5 of gestation due to severe defects in gastrulation and mesodermal patterning ; however, the studies examining the role of Shp-2 in embryonic stem cell (ESC) and hematopoietic development, though extensive, are limited to the animal model bearing the residual mutant protein, Shp-246¨C110. 6 `% P5 b( C& ^5 y9 i( M& n! C* g) F1 t& k) X
Studies using Shp-2/ ESCs demonstrated that Shp-2 is required for normal ESC differentiation . Collectively, these findings strongly support a positive role for Shp-2 in ESC differentiation and hematopoietic development. However, interpretation of the data has been confounded by the persistent expression of the residual mutant protein. 6 X& m* S, I- H; G5 A) J " Y/ Q, q {0 U) j; |# w0 ^& GTo better understand this question, we used an RNA interference (RNAi) approach to knockdown Shp-2 expression in murine embryoid body (EB)-derived cells. Consistent with studies using the Shp-2/ ESC model, we found that reduction of Shp-2 expression by small interfering RNA (siRNA) causes diminished hemangioblast and primitive and definitive hematopoietic progenitor formation. Furthermore, consistent with reduced basic fibroblast growth factor (bFGF)-induced signaling in Shp-2/ murine embryonic fibroblasts , we found that reduction of Shp-2 expression using siRNA ablates bFGF induction of hemangioblast formation. Collectively, these data validate the previous findings generated using the Shp-2/ model and highlight the importance of Shp-2 in mediating proficient hematopoietic differentiation.! X L( I7 J l! d& M
. M( u$ X$ I) \$ nMATERIALS AND METHODS" x; E2 {8 e' p5 r" ?. C) y. \* S
. F g5 {- b- M/ C" `) y, R* B8 g
ESC Culture and In Vitro Differentiation ! Q8 a! F: [1 N6 { / G3 B0 Q6 }3 a' x+ P, s/ QThe mouse CCE ESCs (passage 10) were obtained from the Indiana University Transgenic and Knock-Out Mouse Facility (Indianapolis). R1 ESCs were a gift from Dr. Feng. Both CCE ESCs and R1 cells were maintained on gelatinized tissue culture dishes (100 mm; Costar, Cambridge, MA, http://www.corning.com) in standard ESC media consisting of Dulbecco¡¯s modified Eagle¡¯s medium supplemented with 15% fetal calf serum (FCS; Gibco, Grand Island, NY, http://www.invitrogen.com), 0.1 mM L-glutamine, 150 µM monothioglycerol (MTG, Sigma, St. Louis, http://www.sigmaaldrich.com), penicillin 100 U/ml, streptomycin 100 mg/ml, and LIF 1000 U/ml (Chemicon, Temecula, CA, http://www.chemicon.com). To initiate differentiation into EBs, dissociated ESCs were added to Iscove¡¯s modified Dulbecco¡¯s medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 15% differentiation serum (HCC6900; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), and 450 µM MTG at a cell concentration of 5000 to 10,000 cells per ml plated in 100-mm low-adhesion dishes, and cultured for up to 10 days to differentiate ESCs to EBs. " B. |" j* q& L& c$ A0 e; ]8 e& J! S5 ~. u8 e
Harvesting EBs V' D g# @) W! h. L4 j
7 _, _, Q+ T3 c; i
Day 3, 6, or 10 EBs were collected by centrifugation, washed twice with phosphate-buffered saline containing 1% albumin (PBSA), and resuspended in 0.25% (wt/vol) Trypsin mixed with 15% fetal bovine serum. The EBs were incubated at 37¡ãC for 5 minutes and dissociated into a single-cell suspension by passing through a 20-gauge needle. Remaining small aggregates were removed by filtration through a 40-µm mesh (Falcon; BD Biosciences Discovery Labware, Bedford, MA, http://www.bdbiosciences.com). The EB cells were spun and resuspended in 0.5% bovine serum albumin (BSA) (Path-O-Cyte 4; Bayer Corporation, Kankakee, IL, http://www.bayerus.com) in PBSA at 5 x 106 cells per ml.! l% }& v" `4 {0 t
9 P: T& I0 d- D" M7 ?* j
Transfection of EB Cells with siRNA * P+ O' X4 K' q& n5 E5 N; ?( W& W' ^. r# w
All Shp-2 siRNA and scrambled siRNA were commercially obtained from Dharmacon (Lafayette, CO, http://www.dharmacon.com). The 21-base sequence was subjected to a Basic Local Alignment Search Tool (BLAST) search (National Center for Biotechnology Information, Bethesda, MD, http://www.ncbi.nlm.nih.gov) database to ensure that only the Shp-2 message was targeted. Sense and antisense RNA oligonucleotides for Shp-2 corresponded to coding sequences from nucleotide 1624 to 1644 (5'-aag gac aug aau aua cca aua-3'; Shp-2 siRNA1), nucleotide 1899 to 1921 (5'aat gtc aag act aga cga gcg-3'; Shp-2 siRNA2), and the scrambled siRNA sequence 5'-gaa cag aug aau aua aca auc-3' were obtained as RNA duplexes. Transient transfection of siRNAs was carried out as previously described . Total EB cells were diluted with fresh medium without antibiotics and transferred to 12-well plates with 1 x 105 cells per well (500 µl per well). Transient transfection of siRNAs was carried out using Oligofectamine according to the manufacturer¡¯s instructions (Life Technologies, Carlsbad, CA, http://www.lifetech.com). The cells were exposed to scrambled Shp-2 siRNA (50 nM) or Shp-2 siRNA (25, 50, and 75 nM). Control treated cells were exposed to the transfection reagent only.9 ?. \- U- F; L( h& w
% y! x6 d/ ^/ R: B1 ^; u
Secondary Plating Assays for Hematopoietic Progenitors; e1 u) Y+ h7 k
0 y o4 f, k6 d
For the hemangioblast assay, siRNA-transfected day 3 EB cells were cultured in 0.9% methylcellulose, 20% FCS, 10% BSA, 100 ug/ml bovine transferrin, 10 ng/ml of bovine insulin, 100 µM MTG, 25% D4T-conditioned media (kindly provided by Gordon Keller), vascular endothelial growth factor (VEGF) 5 ng/ml, and SCF 100 ng/ml, as described . In some experiments, bFGF was added at a concentration of 10 or 20 ng/ml. For primitive erythroid progenitor (EryP) assays, siRNA-transfected day 6 EB cells were cultured in 0.9% methylcellulose, 15% plasma derived serum (Animal Technologies, Antech, TX, http://www.animaltechnologies.com), and erythropoietin 5 U/ml. For definitive erythroid progenitor (EryD) assays, siRNA-transfected day 10 EB cells were cultured in 0.9% methylcellulose, 15% FCS with 100 ng/ml SCF, 1 ng/ml IL-3, and 5 U/ml Epo. For granulocyte/macrophage progenitor assays, 10 ng/ml of GM-CSF was also included.5 c: u8 ~5 ^' |& e% @6 m$ C4 F6 A
z& x2 [$ u$ j; {) s7 ]2 m' lTotal RNA was extracted from undifferentiated ESCs and EB cell populations using TRIZOL reagent (Invitrogen). Reverse transcription (RT) was performed using Superscript II reverse transcriptase (Gibco-BRL, Carlsbad, CA, http://www.gibcobrl.com), and polymerase chain reaction (PCR) was performed using Pyrobest DNA polymerase. The primer sequences for Brachyury, Flk-1, and scl and ß-actin have been reported . * K7 a$ ^" Z4 F/ x% l/ M# a; |6 G7 u" E1 ?7 T
Western Blot Analysis 9 v0 c% K' h B/ m0 A5 N( f $ s! ~2 Q) ?, W YWhole-cell extracts were prepared using hot Laemmli buffer from both Shp-2 siRNA, scrambled siRNA transfected and untransfected cells. Proteins were separated by SDS-polyacrylamide electrophoresis using a 10% (wt/vol) polyacrylamide resolving gel and transferred electrophoretically to a nitrocellulose membrane. Immunoblotting was performed using anti-Shp-2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) at a 1:400 dilution at room temperature, anti-phospho-Akt (Ser473) at 1:500, anti-Akt (New England Biolab) at 1:800 dilution, or anti-Shp-1 antibody at 1:300 dilution (Santa Cruz Biotechnology Inc.). All immunoblots were visualized by enhanced chemiluminescence according to the manufacturer¡¯s instructions (Amersham Pharmacia Biotech, Piscataway, NJ, http://www.amersham.com). 2 D9 Y, |, X( B3 L& S1 y( O7 v" N N6 j L9 M2 ]7 F+ {
Apoptosis Assay/ p! `% P7 j( O- ]$ P1 z% g7 e
+ r4 N% r0 n y/ N" K n5 ^) P
Day 3 and 10 EB cells or CD34 from day 10 EB cells (collected by fluorescence-activated cell sorting after incubating with anti-CD34 conjugated to biotin and secondarily stained with streptavidin-allophycocyanin) were transfected with Shp-2 siRNA or control scrambled siRNA and plated in six-well plates for 24 hours. The cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) and analyzed by flow cytometry. ) G6 ^6 \" t$ C" Q; d( d5 F 4 N2 z* H9 o- s& ATransfection of EB Cells with FITC-Labeled siRNA7 f. R9 g9 |& A1 F( |* D
" x3 B9 f9 e7 v, U+ JAfter 1 day of plating, EB cells were transfected with varying doses of FITC-labeled siRNAs in 400 µl serum-free Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen). After 24 hours of transfection, the cells were aspirated from the plate, stained with phycoerythrin (PE)-labeled rat anti-mouse CD71 antibody (BD Pharmingen), washed, and analyzed by flow cytometry (FACS Calibur instrument; Becton, Dickinson and Company, San Jose, CA, http://www.bc.com). ) R0 `1 G f( @0 e- \* y2 x7 S: |/ ` @4 C4 X% w3 k! n
RESULTS + D" i% N8 f9 z% }# V- y- d; _ ! L8 s) a w0 HShp-2 Expression in Differentiating EBs and Hemangioblast Development2 b" ]& u. y$ e3 Q& e
* K8 {2 R( Z+ n; S7 ITo establish a baseline for experiments examining the effect of siRNA knockdown of Shp-2 expression on hematopoietic development, we first demonstrated that Shp-2 is highly expressed in undifferentiated CCE ESCs as well as in differentiating CCE EBs as determined by Western blot. Similar results were obtained from R1 ESCs and R1 EB cells (data not shown). Re-plating day 3 EB cells into culture with VEGF and SCF resulted in the formation of hemangioblast colonies (BL-CFC), as previously described . 9 g- _$ H! V$ S/ c* S9 H5 `8 n6 O G3 E+ {/ L
Transfection of Shp-2-Specific siRNA Efficiently Reduces Shp-2 Expression and Inhibits BL-CFC Formation ! Z5 Q+ ~' R" D ( g I2 M- ~/ t# @' N( v+ t( e( ]Having established a baseline of Shp-2 expression in undifferentiated ESCs as well as EBs and having demonstrated competent differentiation to hemangioblasts, we next determined whether knockdown of Shp-2 expression altered hemangioblast development. To examine this question, day 3 EBs were dissociated into a single-cell suspension and transfected with either scrambled siRNA or Shp-2 siRNA. Forty-eight hours after siRNA transfection, protein lysates were prepared to examine Shp2 expression using Western blot analysis. In cells transfected with control siRNA or with 25 nM Shp-2, Shp-2 expression was not diminished (Fig. 1A). However, when the Shp-2 siRNA concentration was raised to 50 and 100 nM, significant reduction of Shp-2 expression was revealed (Fig. 1A). In contradistinction, Shp-1 expression was unaffected (Fig. 1A). Transfection of Shp-2 siRNA into day 3 EB cells induced a significant reduction of BL-CFC formation compared with untransfected cells or cells transfected with scrambled Shp-2 siRNA (Fig. 1B). These findings are consistent with the findings using Shp-2/ ESCs . " Q' B( b1 Z9 o+ j# D+ y1 W9 e$ M/ X, N* s! N" X, X% H
Figure 1. Transfection of Shp-2 siRNA significantly reduces Shp-2 expression and hemangioblast development. (A): Western blot analysis demonstrating reduction of Shp-2 expression after transfection with 50 and 100 nM Shp-2 siRNA1 or 50 and 100 nM Shp-2 siRNA2 into cells derived from day 3 EBs. No effect on Shp-2 expression was observed using 25 nM Shp-2 siRNA or scrambled siRNA. (B): Hemangioblast progenitor assay after transfection of Shp-2 siRNA or scrambled siRNA into day 3 EB-derived cells. *, p 5 u! _6 M& `/ N
/ o) Q' g. ~4 o9 P! |' xReduction of Shp-2 Expression Ablates Fibroblast Growth Factor-Dependent Hemangioblast Development2 o; t& S5 I. C+ z. N. |1 f& F% v
* @2 `. t7 O# z! N1 p
bFGF signaling is critical for mesoderm induction and patterning events , suggesting that Shp-2 is needed as a positive signal for mesoderm development. We hypothesized that downregulation of Shp-2 expression would ablate the effect of bFGF on hemangioblast development. Consistent with previous reports, the addition of bFGF at 10 and 20 ng increased the number of hemangioblasts in a dose-dependent manner (Fig. 2). As predicted, siRNA-dependent reduction of Shp-2 expression ablated the bFGF-dependent increase in hemangioblast numbers. Together, these data support the role of Shp-2 as a positive mediator of mesoderm and hemangioblast development and provide a signaling pathway in which Shp-2 is critically necessary in early hematopoietic development. ' r' F% {2 D* s! ^; E# ]4 ^5 z 6 [( C, G& Z) ?0 L/ j: T( tFigure 2. Reduction of Shp-2 expression ablates bFGF-mediated hemangioblast development. (A): FGF induces Shp-2 phosphorylation in day 3 CCE EB cells. (B): Cells derived from day-3 EBs were either untreated, transfected with Shp-2 siRNA1, Shp2 siRNA2, or scrambled siRNA and plated into hemangioblast progenitor assays in the presence or absence of bFGF 10 or 20 ng/ml. Reduction of Shp-2 expression significantly reduced the bFGF-mediated increase in hemangioblast formation. *, p " ^6 y. F8 W& I. Y7 G8 |3 T; I, \ j% k
Reduction of Shp-2 Expression Diminishes Primitive and Definitive Hematopoietic Differentiation R3 X" t4 J/ m& N% X$ W) u, ^( y, T! Y: \
We next examined the effect of reduced Shp-2 expression on primitive and definitive hematopoiesis using hematopoietic progenitor colony assays. To test the role of Shp-2 in EryP development, day 6 EBs were dissociated and single EB cells were replated in methylcellulose supplemented with plasma-derived serum and erythropoietin. Knockdown of Shp-2 expression in day 6 EB cells blunted their potential to form EryP (Fig. 3A). To determine whether a reduction of Shp-2 expression affected definitive hematopoiesis, Shp-2 siRNA was introduced into dissociated day 10 EB cells followed by progenitor assays. Cells with reduced Shp-2 expression generated significantly fewer EryD colonies compared with untreated cells or cells transfected with scrambled siRNA (Fig. 3B). To examine myeloid differentiation, CFU-GMs were scored by replating control or Shp-2 siRNA-treated day 10 EB cells in the presence of Epo, SCF, GM-CSF, and IL-3. As shown in Figure 3C, the frequencies of CFU-GM colony were decreased in Shp-2 siRNA-transfected group compared with control scrambled siRNA-transfected group (p ) F' |( W7 Q5 r) J3 L ' J1 G" p, Y f" EFigure 3. Reduction of Shp-2 expression abrogates primitive and definitive hematopoiesis. (A): Cells derived from day 6 CCE EBs were trans-fected with Shp-2 siRNA1, Shp-2 siRNA2, or scrambled siRNA and plated into primitive progenitor assays. (B): Cells derived from day 10 EBs were transfected with 50 or 100 nM of Shp-2 siRNA1, Shp-2 siRNA2, or scrambled siRNA and plated into either definitive erythroid progenitor assays or (C) granulocyte-macrophage colony assays. At both 50 and 100 nM of Shp-2 siRNA1, Shp-2 siRNA2 in all assays conducted, significantly fewer progenitors developed. *, p 1 Z k/ v+ e4 o* I9 m3 U P
5 x6 l6 w; b$ o2 ~Findings from the primitive and definitive progenitor assays, along with the results of the hemangioblast assays, demonstrate that Shp-2 is required at multiple levels of hematopoietic progenitor differentiation. These findings support the results obtained using Shp-2/ ESCs. However, our results are also significant because previous studies examining the role of Shp-2 in hematopoietic development used a system lacking Shp-2 at the earliest point of differentiation within undifferentiated ESCs. As noted, there is a block in mesoderm formation (this of course would diminish all downstream lineages such as the hematopoietic elements). Results from the present study suggest that ablation of Shp-2 expression even at later points of hematopoietic development (when progenitor cells have emerged from the mesoderm precursors) also reduces the capacity for progenitor cells to contribute to hematopoietic differentiation. 3 K4 Z2 M1 [1 o* b/ X - S( s s1 T! q. N" P* LReduced Shp-2 Expression in EB Cells Does Not Trigger Increased Hematopoietic Progenitor Apoptosis9 J4 H8 _% B6 ^ |, D
' `. N( Z; N6 } mThe data presented thus far are consistent with the findings using Shp-2/ ESCs and additionally suggest that Shp-2 is required at multiple levels of hematopoietic development for proficient differentiation. However, one possibility is that Shp-2 siRNA-induced reduction of Shp-2 expression may be decreasing hematopoietic progenitor formation detected in colony assays via increasing apoptosis in hematopoietic precursors within the EBs. To examine this possibility, we assayed apoptosis levels in Shp-2 siRNA- and control siRNA-transfected cells using Annexin V and PI costaining. Treating day 3, day 10, or day 10-derived sorted CD34 EB cells with Shp-2 siRNA caused negligible levels of apoptosis and levels similar to that observed in untransfected control cells or scrambled siRNA-transfected cells (Fig. 4). In contrast, treatment of these cell populations with interferon- led to significant apoptosis, exceeding 80% of the cell populations examined. These findings indicate that reduction of Shp-2 expression using siRNA does not merely induce apoptosis of hematopoietic precursors within the EBs, supporting the notion that Shp-2 is necessary as a positive mediator of hematopoietic differentiation. 0 G; s+ `! ~) G$ S7 e/ ~( i 1 t! W$ K0 ^9 l r6 G6 nFigure 4. Transfection of Shp-2 siRNA does not induce increased apoptosis of hematopoietic progenitors. Cells derived from day 3 CCE EBs, day 10 CCE EBs, or CD34 cells from day 10 CCE EBs were transfected with Shp-2 siRNA or scrambled siRNA and were assessed for apoptosis after 24 hours in culture using annexin V-FITC and PI staining. Data represent the mean ¡À the SD of three independent experiments. IFN--treated cells were used as positive control in apoptosis assay. Similar results have been obtained in R1 EB cells (data not shown). Abbreviations: EB, embryoid body; FITC, fluorescein isothio-cyanate; IFN, interferon; PI, propidium iodide; siRNA, small interfering RNA.3 i0 L. C3 P1 x
* h @' i u S2 k% R" }) F
Knockdown of Shp-2 Gene Expression in EB Cells Affects bFGF-Induced Activation of ERK1/2 and PI3 Kinase in EB Cells, ` k4 l: c0 J3 D0 J, A+ V
, F, V/ Q, q c
Stimulation of EB cells with bFGF (20 ng/ml) stimulated both ERK1/2 and Akt phosphorylation at Ser473 (Fig. 5). Further studies revealed that transfection of control siRNA did not affect bFGF-induced ERK1/2 activation. However, knockdown of Shp-2 gene expression by transfection of specific Shp-2 siRNA diminished fibroblast growth factor-mediated ERK1/2 and Akt phosphorylation in EB cells (Fig. 5).) K" C2 H1 s& w4 l, v& j2 {
! Y+ C4 Q, M6 t: |Figure 5. Shp-2 is essential for bFGF-mediated activation of ERK and Akt. bFGF treatment increased activation of ERK1/2 in EB cells. Transfection of control siRNA did not change FGF-induced activation of ERK1/2. In contrast, cells transfected with Shp-2 siRNA displayed diminished activation of ERK1/2 upon FGF stimulation. bFGF treatment also increased activation of Akt in EB cells. Transfection of control siRNA did not affect FGF-induced activation of Akt. In contrast, EB cells transfected with Shp-2 siRNA displayed diminished activation of Akt. One representative result from three experiments is shown. Abbreviations: ERK, extracellular signal-regulated kinase; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; FGF, fibroblast growth factor; siRNA, small interfering RNA.* T( z$ m7 I3 \" b1 X$ A4 d, l
4 p _1 ]4 d2 X5 t* ]$ NEfficiency of siRNA Transfection in EB Cells 2 J( m. j% X. \2 @: J9 ]: y2 |( u3 C& I3 ?9 n
We evaluated the ability of siRNA transfection using FITC-labeled siRNA at doses ranging from 25 to 100 nM. PE-labeled CD71 expression was used to identify the EB cells (all ESCs and EB cells express CD71). The transfection efficiency of the CD71 expressing cells was 96% at 25 nM, 100% at 50 nM, and 100% at 100 nM (Fig. 6). However, the mean fluorescence intensities were significantly different among these cells: 33.24 at 25 nM siRNA, 233.28 at 50 nM siRNA, and 298.08 at 100 nM siRNA. These results suggest that failure of 25 nM Shp-2 siRNA to reduce Shp-2 expression is a threshold effect in individual cells and not the consequence of a poor rate of transfection within a portion of the cellular population. 8 d( Y, R- O; n* c$ H+ R3 l F # Y( [0 b# @& xFigure 6. Transfection efficiency of siRNA in EB cells at different siRNA concentrations. EB cells were transfected with varying doses of Shp-2 siRNA: (A) 25 nM, (B) 50 nM, and (C) 100 nM. The cells were collected after 24 hours of siRNA transfection and analyzed for siRNA-associated fluorescence (FITC label). Nearly all of the CD71 expressing EB cells coexpressed the siRNA: (A) 96% for 25 nM siRNA, (B) 100% for 50 nM siRNA, and (C) 100% for 100 nM siRNA. However, the MFI varied from 33.24 for 25 nM siRNA, 233.28 for 50 nM siRNA, and 298.08 for 100 nM siRNA. One representative from three independent experiments is shown. Abbreviations: EB, embryoid body; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; siRNA, small interfering RNA.( m' X( F# T; A3 {" N* _
7 ]; f H: {+ N: ZDISCUSSION 0 i: D1 I0 A0 B9 | & v& z0 Q0 p% g" i. f; ^Previous reports using ESCs derived from mice bearing homozygous germline mutations of the Shp-2 allele demonstrated that Shp-2 is involved in hematopoietic cell development , suggesting that an initial block in mesoderm formation contributes at least in part to the defect in mesoderm-derived structures. Therefore, reduced detection of hematopoietic progenitors derived from Shp-2/ ESCs could be secondary to reduced differentiation to mesoderm. ; k, ]- S% |$ |1 I # `0 P3 ], e6 Y3 U+ S5 qTo examine the effect of Shp-2 loss on ESC-derived hematopoiesis and to determine whether Shp-2 is required at multiple points of hematopoietic differentiation, not merely for derivation from mesoderm, we used an siRNA method to knock down Shp-2 expression at various points of ESC hematopoietic development. We were able to significantly reduce Shp-2 expression using transfection of siRNA specific for the Shp-2 message. In contrast, the level of Shp-1 expression was unchanged, demonstrating that we specifically targeted Shp-2 expression. No effects of a scrambled Shp-2 siRNA control were observed in the transfected EBs. Upon introduction of the Shp-2 siRNA into cells derived from differentiated EBs at various ages (days 3, 6, and 10), and secondary plating into hematopoietic progenitor assays, we observed a clear abrogation of hematopoietic colony formation at multiple levels of hematopoietic differentiation (hemangioblast, primitive, and definitive hematopoiesis). These studies validate the findings of the Shp-2/ ESC and mouse models and further suggest that Shp-2 is not only needed for the initial formation of mesoderm but is also required for proficient hematopoietic differentiation from hematopoietic progenitor cells. Importantly, we did not observe an increase in the level of apoptosis after transfection with the siRNA, suggesting that the hematopoietic progenitors were viable but still failed to differentiate and grow into a detectable colony. . b7 ]2 _" `1 D7 T- T( k3 a . I& E; F, w* \+ I* ~9 I5 Y* }The studies presented here also examined the role of Shp-2 in bFGF-mediated hemangioblast development. Initial studies using murine embryonic fibroblasts derived from Shp-2/ mice demonstrated that the signals emanating from the bFGF receptor were significantly reduced . bFGF has also been shown to play a role in hemangioblast development. Given that reduction of Shp-2 expression using siRNA ablated the effect of bFGF on hemangioblast growth, our studies demonstrate that Shp-2 is a critical mediator of bFGF-stimulated hemangioblast development. ! p u) [1 d1 P c # k6 n: D3 f9 A' d% w# nIn sum, we have used siRNA to effectively reduce Shp-2 expression in murine ESC-derived EBs and to examine consequent effects on hematopoietic differentiation. Our findings indicate that Shp-2 is necessary for proficient hematopoietic differentiation at multiple levels of the hematopoietic hierarchy and are in line with the results obtained using the Shp-2/ mouse model. These findings support the notion that the residual mutant protein (Shp-246¨C110) is biologically inert and re-emphasize the importance of Shp-2 function in controlling normal hematopoiesis./ \$ [& {9 Z k# T; {$ z
" J1 @. L4 W; V/ l9 ]The authors indicate no potential conflicts of interest. ) K! T- f" I4 m' f! f ' C N3 z* L9 P& lACKNOWLEDGMENTS5 I) T1 f3 g- z* d, m
( h% @, S7 @+ u5 F# x$ fWe thank Bill Carter for CCE cells, Gen-Sheng Feng (The Burnham Institute) for R1 ESCs, Yuan-Jun Li for technical assistance on the hemangioblast assay, and Arliene K. Britt and Janice L. Walls for assistance with manuscript preparation. This study was supported by R01HL63169 of the National Institutes of Health. 1 b y# }$ J( P3 Z1 d* N/ G& q( u 【参考文献】 $ N E ~3 Y+ b2 N% }' v d: t* b3 L' @+ r3 P # o7 ?, y. Q' `; \5 d9 nNeel BG. Structure and function of SH2-domain containing tyrosine phosphatases. Semin Cell Biol 1993;4:419¨C432. 3 ?' {# [4 p( ^7 E# O: V2 t . {* L; _9 V! ?0 l% jFeng GS. Shp-2 tyrosine phosphatase: Signaling one cell or many. Exp Cell Res 1999;253:47¨C54. ' Z* U2 `) o" q M7 g; o3 ~) w2 y; h# j0 f
Tauchi T, Feng GS, Marshall MS et al. The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells. J Biol Chem 1994;269:25206¨C25211. 7 K( a0 s8 Y6 n- a . {* u; e- ]+ mTauchi T, Feng GS, Shen R et al. Involvement of SH2-containing phosphotyrosine phosphatase Syp in erythropoietin receptor signal transduction pathways. J Biol Chem 1995;270:5631¨C5635., w# P# I- h* v- F3 C; P
7 v5 I' D# ]. e# rWheadon H, Paling NR, Welham MJ. Molecular interactions of SHP1 and SHP2 in IL-3-signalling. Cell Signal 2002;14:219¨C229.( B7 S. ]) M8 u) M
" D8 g! D' ]' f6 D5 @
Liu Y, Jenkins B, Shin JL et al. Scaffolding protein Gab2 mediates differentiation signaling downstream of Fms receptor tyrosine kinase. Mol Cell Biol 2001;21:3047¨C3056.% A: A: T5 }3 [" Q
/ \. V3 ^4 q: m9 q
Burdon T, Stracey C, Chambers I et al. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev Biol 1999;210:30¨C43.6 g, F$ X/ B! t- {
* N7 g6 H1 Y8 Z$ ?# H
Qu CK, Shi ZQ, Shen R et al. A deletion mutation in the SH2-N domain of Shp-2 severely suppresses hematopoietic cell development. Mol Cell Biol 1997;17:5499¨C5507. ( ^' N. a Y2 A$ x3 A! I" {0 Z% p* v6 ^" {
Yu DH, Qu CK, Henegariu O et al. Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion. J Biol Chem 1998;273:21125¨C21131. . c8 y3 D) W9 r& v: n8 n/ z% x+ Y! G
Yuan L, Yu WM, Qu CK. DNA damage-induced G2/M checkpoint in SV40 large T antigen-immortalized embryonic fibroblast cells requires SHP-2 tyrosine phosphatase. J Biol Chem 2003;278:42812¨C42820., W% Q3 X( a% ^, r# m& O( d
% |5 r- \0 q% [
Yuan L, Yu WM, Yuan Z et al. Role of SHP-2 tyrosine phosphatase in the DNA damage-induced cell death response. J Biol Chem 2003;278: 15208¨C15216.! `( c6 [! V7 r+ V2 M
! a) v: E* V! {( |/ t6 JZhang SQ, Tsiaras WG, Araki T et al. Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2. Mol Cell Biol 2002;22:4062¨C4072.+ }- J1 Q! _5 I
# V6 G# }7 z) n. u& n/ ^+ r1 ]. Q! p
Ivins Zito C, Kontaridis MI, Fornaro M et al. SHP-2 regulates the phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis. J Cell Physiol 2004;199:227¨C236. " d( k7 N7 s+ _/ K" W* I) N- f' m; h9 S* m7 o; \+ c4 `& v
Saxton TM, Henkemeyer M, Gasca S et al. Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. EMBO J 1997;16:2352¨C2364. ( A& U2 e1 N* j. R/ N G. z" u D6 b" P7 E! l
Zhang EE, Chapeau E, Hagihara K et al. Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc Natl Acad Sci U S A 2004;101:16064¨C16069. ) r. ~6 s6 w' ~- e 6 d# B. P6 `; ?: YQu CK, Feng GS. Shp-2 has a positive regulatory role in ES cell differentiation and proliferation. Oncogene 1998;17:433¨C439. ' k. w$ ^# o' ?; D; P " x" @% L! Q2 `/ u( r+ V; P7 H' qQu CK, Yu WM, Azzarelli B et al. Biased suppression of hematopoiesis and multiple developmental defects in chimeric mice containing Shp-2 mutant cells. Mol Cell Biol 1998;18:6075¨C6082. 3 o; @+ E2 v* L( B 7 G7 e; ^4 F5 p9 s& z/ CQu CK, Nguyen S, Chen J et al. Requirement of Shp-2 tyrosine phosphatase in lymphoid and hematopoietic cell development. Blood 2001; 97:911¨C914. 4 v0 l7 i7 j2 G& C1 A1 t) w- l" S4 t5 v; L( W
Chan RJ, Johnson SA, Li Y et al. A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis. Blood 2003;102:2074¨C2080. , r: g2 H8 c( n- v' r R; u8 b 8 R5 C$ O, M4 r/ z! D% KZou GM, Wu W, Chen J et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells. Biol Cell 2003;95: 365¨C371.* h) C. [+ U2 m4 y
6 J5 K4 s5 N9 K- Z1 p+ n8 r
Choi K, Kennedy M, Kazarov A et al. A common precursor for hematopoietic and endothelial cells. Development 1998;125:725¨C732. * C/ N! {6 d5 s: r, I: l% J y' K3 \( H& F% R2 @Kennedy M, Firpo M, Choi K et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 1997;386:488¨C493.. G; L" H3 V- T4 k9 T. X
; ]9 N$ v' { Z: [2 N
Olson MC, Scott EW, Hack AA et al. PU. 1 is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. Immunity 1995;3:703¨C714.9 g. @8 u, P, l0 d* S( T3 Y
( J; o) H9 m+ t+ m
Hadland BK, Huppert SS, Kanungo J et al. A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development. Blood 2004;104:3097¨C3105.' U. [" n: B+ t4 R5 p% M0 i
~/ i+ ]1 M$ s4 m L" ~
Deng CX, Wynshaw-Boris A, Shen MM et al. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev 1994;8:3045¨C3057. 2 ]4 i$ }7 n) u1 }8 V3 f% X! G5 \3 b* b
Yamaguchi TP, Harpal K, Henkemeyer M et al. fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev 1994;8:3032¨C3044.2 T# k- C* C, z: @
0 T! M6 ]6 [# q
Saxton TM, Ciruna BG, Holmyard D et al. The SH2 tyrosine phosphatase shp2 is required for mammalian limb development. Nat Genet 2000;24:420¨C423. - ?! |& e- ~! I3 `0 \# } 1 _. v' d" i& a$ L# W$ S wSaxton TM, Pawson T. Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2. Proc Natl Acad Sci U S A 1999;96:3790¨C3795.作者: 红旗 时间: 2015-5-22 09:33