|
  
- 积分
- 0
- 威望
- 0
- 包包
- 483
|
作者：Poh C. Tana, Sebastian G.B. Furnessa, Helen Merkensa, Shujun Lina, Marcia L. McCoyb, Calvin D. Roskelleyb, Jrgen Kasta, Kelly M. McNagnya * x2 r* w# e* T: l
6 K8 U. s0 c% |5 y3 O 8 \, K% ?+ l; n5 y: c& b2 H4 f
+ d2 ` x0 ~$ Z2 R) E
6 s+ @& Z0 k8 f/ @: J& P: U- c& ~6 V+ k
2 |. w( G$ h6 v 5 r: B2 z& ^/ X2 c8 V
2 E' ^9 L. r8 ^1 B( x( z, |5 v1 c
; S8 e' i \6 W( m1 o
+ F) q9 v3 {8 r0 m0 n, U9 t 1 E. p1 @& q* `' V
U8 G' f8 d8 j" z0 U3 g5 J
9 n+ f4 l2 R& f1 r/ ? j 【摘要】+ r' F/ H7 g6 C6 [. C
CD34 and its relatives, podocalyxin and endoglycan, comprise a family of surface sialomucins expressed by hematopoietic stem/progenitor cells and vascular endothelia. Recent data suggest that they serve as either pro- or antiadhesion molecules depending on their cellular context and their post-translational modifications. In addition, their ability to function as blockers of adhesion may be further regulated by their subcellular localization in membrane microdomains via activation-dependent linkage with the actin cytoskeleton. To gain further insights into the function and regulation of CD34-type molecules, we sought to identify the intracellular ligands that govern their localization. Using both genetic and biochemical approaches, we have identified the Na /H exchanger regulatory factor-1 (NHERF-1) as a selective ligand for podocalyxin and endoglycan but not for the closely related CD34. Furthermore, we show that NHERF-1 is expressed by all c-kit /lineage marker¨C /Sca-1 cells, which are known to express podocalyxin and have long-term repopulating abilities. Finally, we show that these proteins relocalize and colocalize in response to cytokine signaling. The results suggest that this cytosolic adaptor protein may be important for mobilization of CD34-type proteins in the plasma membrane and may thereby regulate their ability to block or enhance hematopoietic cell adhesion. ! x4 O1 u% O% j2 V( J+ N
【关键词】 Hematopoiesis Adhesion Differentiation Stem cells CD Podocalyxin Endoglycan Sialomucin Na /H exchanger regulatory factor- Na /H exchanger regulatory factor-
4 _' v0 q& U, z INTRODUCTION
& l9 I0 t4 E6 \$ u; m; Y+ I
' K2 F8 \6 e( bCD34, podocalyxin, and endoglycan comprise a family of hematopoietic and vascular-restricted sialomucins .
) \2 z! C3 I) j# o
$ \$ C% Q6 R1 }1 @ ~" H$ qAll three proteins are expressed on HSCs/progenitors, but their function on these cells has remained enigmatic . Although this is a well-documented function for CD34-type proteins on HEVs, several observations suggest that this proadhesive function is an important exception rather than a general rule. Most notably, the binding of CD34-type proteins by L-selectin is highly dependent on the modification of these proteins with HEV-specific carbohydrate moieties. These modifications, however, have not been detected on virtually any other vascular endothelial cell type or on hematopoietic cells.0 y$ C& A7 J# d: f8 _: I. k. x5 d, t: I
' y! c; c1 K5 Z. n( O
In contrast to their proadhesive function on HEVs, it has been demonstrated that ectopic expression of podocalyxin in Chinese hamster ovary or Madin-Darby canine kidney cells leads to a block in cell aggregation and in cell-cell junction formation, respectively .
9 P* z1 s9 s& s3 l) d& g7 f% n% n- ^6 f1 L) v; v. n. a
On hematopoietic cells, too, there is recent evidence that these molecules may function as antiadhesins. We have shown that CD34 is a selective marker of murine mast cells .# A; T/ A F% x, K6 g9 b
' p& |: e% U7 D. B, u1 YThe observation that isoforms of CD34-type proteins lacking most of the cytoplasmic domain are more effective in blocking cell adhesion has led us to speculate that the members of this family can regulate their antiadhesive properties dynamically by association with cytoskeletal elements that enhance or inhibit their localization at the sites of cell-cell or cell-matrix attachment. A number of observations in the literature are consistent with this hypothesis. It has been demonstrated that activation of the PKC pathway in cells leads to a rapid phosphorylation of the cytoplasmic tail of CD34 .
5 \. e1 K5 L& M8 n- `& c1 e& X% p2 F, K: _# r
As a first step toward understanding the role of cytoplasmic binding proteins in the regulation of CD34-type protein function in hematopoietic cells, we have used genetic screens and biochemical approaches to identify hematopoietic ligands for the cytoplasmic tail of podocalyxin. Here we identify NHERF-1, a homologue of NHERF-2, as a binding protein for podocalyxin and endoglycan but not CD34. We show that podocalyxin and NHERF-1 are co-expressed in normal HSCs and that they colocalize upon podocalyxin clustering. Furthermore, this clustering is enhanced by cytokine treatment. Our results suggest that NHERF-1 is a ligand for a subset of CD34-type proteins and that it may play a role in regulating their antiadhesive properties in HSCs.
0 S) w0 s7 n4 z, y/ y9 i# d3 z
" S3 v5 @/ e- \: FMATERIALS AND METHODS
% D4 @, f- C# L1 R! ^2 f0 p- R! B" D4 R+ M) _; Q- l9 S6 N
Cells and Media( o8 \7 f; C# c- x3 M
/ j a- B) H+ b; t
Factor-dependent continuous cell line, Paterson Laboratories 1 (FDC-P1) cells were maintained in RPMI (Gibco, Grand Island, NY, http://www.invitrogen.com), 2 mM L-glutamine, penicillin/streptomycin, 10% fetal bovine serum, and Walter and Eliza Hall Institute-3B (WEHI-3B) conditioned media containing interleukin-3. Bone marrow cells were obtained by flushing femurs and tibias of 8- to 10-week-old C57BL/6 mice with room-temperature phosphate-buffered saline (PBS) using a 25-gauge needle. Thymus, spleen, Peyer¡¯s patches, and lymph nodes were dispersed into single-cell suspensions by passing through a 45-µm nylon cell strainer.2 i3 L0 O, ?4 @( H1 c
( w3 s2 U0 @8 X2 w+ @9 f6 TAntibodies& o- |* E1 C3 f8 B& M0 [* c
0 A5 p0 y4 q$ \Rabbit anti-NHERF-1 antibody ab3452 (Abcam, Cambridge, U.K., http://www.abcam.com) was used for all fluorescent assays, and rabbit anti-NHERF-1 antibodies APZ-006 (Alomone Laboratories, Jerusalem, http://www.alomone.com) and ab3452 were used for immunoblot analyses. Rat anti-mouse podocalyxin antibody MAB1556 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was used for all staining, immunoprecipitation, and immunoblot studies. Secondary antibodies were goat anti-rabbit AlexaFluor 488 (Molecular Probes Inc., Burlington, ON, Canada, http://probes.invitrogen.com), goat anti-rat AlexaFluor 568 (Molecular Probes), goat anti-rat PE (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), goat anti-rabbit HRP (DAKO, Carpenteria, CA, http://www.dako.com), and goat anti-rat HRP (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Isotype controls were rabbit IgG (H&L; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and rat IgG2a (Cedar Lane, Hornby, ON, Canada, http://www.cedar-lanelabs.com). Directly conjugated lineage-specific antibodies Ter119, CD3, Gr-1, Mac-1, and B220; Sca-1; and c-kit were purchased from R&D Systems. All antibodies were titrated to optimal concentrations prior to each type of assay.
0 V# ]' i3 w0 q1 J: I6 G- Y3 I: O1 V. X" _; L" I$ P
Peptides- f% {5 v2 y* Q, i9 t
2 j! r! i S' O+ ]
Peptides were as follows:$ w3 B: T/ x( [" |3 H" b
1 E, I) V! P$ R/ X
Podo-1¨C73 (C-terminal 73 aa, avian thrombomucin; GenBank accession number CAA74311 ): HQRFSQKKSQQRLT- EELQTMENGYHDNPTLEVMETGSEMQEKKVNLNG-ELGDSWIVPLDTIMKEDLEEEDTHL.2 b, f" N* O) f V
5 p1 Z( u/ X& ^& G# P" L( R- [Podo-15¨C73 (C-terminal 58 aa): ELQTMENGYHDNPTLE-VMETGSEMQEKKVNLNGELGDSWIVPLDTIMKEDL-EEEDTHL.& `1 o: T. H6 Q! i! ?
) K3 {2 U7 o K5 h
Podo-53¨C73 (C-terminal 20 aa): WIVPLDTIMKEDLEEED-THL.
/ {. Z1 |% m" C/ H# ^
$ e i+ ]: S" j9 VPodo-53¨C72 (C-terminal 20 aa, less the terminal leucine): WIVPLDTIMKEDLEEEDTH.1 _5 {0 b1 m5 j# v9 s7 i
6 T2 x3 n m3 i. ]' s2 B3 l- kEndo (C-terminal 26 aa, murine podocalyxin-like 2; GenBank accession number AAH33384 ): SSWSALMGSKRDP-EDSDVFEEDTHL.; T e4 u. D7 ^9 x4 P4 T
# d5 R* y- P0 b
CD34 (C-terminal 72 aa, murine CD34; GenBank accession number NP_598415 ): RRSWSPTGERLGEDPYYTE-NGGGQGYSSGPGASPE TQGKANVTRGAQENGTGQ-ATSRNGHSARQHVVADTEL.
$ W: b( x; b! Q* P% q- b* h' ~' j5 D$ W& l9 L% _% L5 v
Phage Screens
( C. X" X- a. B D0 I
7 B+ {5 K/ j+ E* g5 N7 NPhage screens for podocalyxin-binding proteins were performed essentially as described previously . Briefly, phage-infected XL-1 Blue MRF Escherichia coli were plated on 30-mm Luria-Bertani 0.7% plates at a density of 20,000 plaques per plate. As visible plaques appeared, isopropyl ß-D-thiogalactopyranoside (Fermentas, ON, Canada, www.fermentas.com)-soaked nitro-cellulose membranes (Bio-Rad, ON, Canada, http://www.bio-rad.com) were overlaid and allowed to incubate for 8¨C12 hours. Filters were washed four times for 20 minutes each (0.1% Triton-X/PBS), and blocked overnight at 4¡ãC (2% bovine serum albumin-PBS-0.02% sodium azide). Biotinylated peptides (25 pmol/ml in wash buffer) were complexed with streptavidin-alkaline phosphatase (SAP; 1 µg/ml) for 20 minutes prior to incubation with filters in blocking buffer. Biotin/SAP complexes (1 µg/µl) were added as a nonspecific blocking reagent. After overnight incubation at 4¡ãC on an orbital shaker, the filters were washed four times for 15 minutes each prior to 5 minutes of incubation with SAP developing buffer (Roche Diagnostics, QC, Canada, http://www.roche-applied-science.com). Filters were dried on Whatman 3MM paper, positive plaque lifts were aligned, and phage plugs were removed and transferred to microcentrifuge tubes containing 500 µl of SM buffer and 4% chloroform. Each plug was subcloned and rescreened, and purified phagemids were excised in vivo using ExAssist protocols recommended by the manufacturer (Stratagene, La Jolla, CA, http://www.stratagene.com), prior to automated sequencing (Lone Star Labs, Houston, TX, http://www.lslabs.com).
7 J. D5 d% R' [# g
0 @- \5 F" I/ L! t, j/ i- iConfocal Microscopy and Flow Cytometry* z* o+ ~. r. A! H5 e9 c
# ^6 h# Y! N* Q' B8 ^
Immunofluorescent staining was performed as described previously -PBS), permeabilized with 0.1% Triton-PBS for 15 minutes, washed four times, and blocked (1% BSA-10% goat serum-PBS) for 30 minutes. These were then incubated with secondary antibody alone, isotype control, or primary antibody for 30 minutes, washed four times, incubated with FACS buffer or secondary antibody for 30 minutes, washed four times, and analyzed by flow cytometry (FACSCalibur, Beckton Dickinson). For confocal microscopy, cells were resuspended in Fluormount G (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) prior to imaging on a confocal microscope (Bio-Rad Radiance 2000; Nikon Eclipse TE300 microscope with MaiTai Sapphire laser, x60 objective, x2.5 zoom, 166 lines/second; Bio-Rad Lasersharp 2000 software) or on an Olympus FluoView 1000 system (Olympus IX81 microscope, x60 objective, x1.7 zoom, 10 µs per pixel; FluoView 1000 software).3 n3 N! \. N8 K, b1 p, {
! ~% X# p( E$ k. tCell Stimulation, Counting, and Analysis
; a" D) S: r/ {+ c; A% {- w" G1 ]" {& [) ]8 [3 F) K; e
FDC-P1 cells were interleukin-3 (IL-3)-starved for 2 hours before stimulation with IL-3 or 100 nM phorbol 12-myristate 13-acetate (PMA) for 10, 20, 30, 60, or 120 minutes prior to staining. After staining, eight random fields were captured from each time point to assess podocalyxin distribution in the plasma membrane. Three cell phenotypes were present: punctate, capped, and global staining of podocalyxin. Three counts of 100¨C250 cells each were made for every time point, and the average percentage of each cell phenotype was obtained.- x5 a+ `; k: ^. L" h6 b1 T# B
5 Z6 n$ {# b( VAffinity Purification and Mass Spectrometry
1 V" A* T# }4 m; |4 ~2 \6 Z
+ p: y5 U5 V) G0 U& n% t; d0 ^( aAll washes and buffers were at 4¡ãC, and centrifuge steps were conducted at 2000 rpm for 30 seconds in a benchtop microcentrifuge (Hoeffer). Streptavidin-Sepharose resin (Amersham Biosciences, QC, http://www.amersham.com), 25-µl bed volume, was pre-equilibrated in TBS wash buffer (25 mM Tris, pH 7.9, 138 mM NaCl, 2.7 mM KCl) plus 0.15% NP-40, 6% glycerol, 66 nM ethylenediaminetetraacetic acid (EDTA), 500 nM MgCl2, 1 mM PMSF, 1x protease inhibitors (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Twenty-five micrograms of each peptide were incubated with 25 µl of streptavidin-Sepharose (Amersham Biosciences) on a rotator for 30 minutes in 1.5 ml of this buffer. Whole cell extract (3.5 mg) (TBS, 0.5% NP-40, 20% glycerol, 200 nM EDTA, 1.5 mM MgCl2, 1 mM PMSF, 1x protease inhibitors) was diluted to the same concentration as wash buffer with TBS and precleared for 30 minutes against 25 µl of streptavidin-Sepharose. Unbound peptide was removed with 500-µl washes (three times) of wash buffer. Precleared lysate was added to peptide binding reactions and incubated for 120 minutes on a rotator. Nonspecific proteins were removed by washing the resin bed seven times with wash buffer. Bound material was eluted by boiling in SDS-loading buffer, and specific interactors were identified by Coomassie staining of SDS-polyacrylamide gel electrophoresis (PAGE), in-gel tryptic digest, and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
. g) H2 e% X; u6 n, l$ T5 w7 i) J- }/ a" Z) N0 c
Immunoblotting, Affinity Purification, and Immunoprecipitation6 a+ h3 Q1 y$ j/ O
/ ]. b3 E; o! f" @$ tImmunoblotting was conducted using standard protocols with antibodies at 1 µg/ml in 1% BSA (ab3542) or 5% nonfat milk (MAB1556) in TBS-T overnight. Affinity purification was carried out essentially as above except that 5 µg of peptide was used for each purification along with 200 µg of whole cell extract. Immunoprecipitation was performed using standard techniques.0 W; p# _0 m' b' g
, u& ]4 J% a7 G2 yRESULTS
+ ^ L Q: p/ R( ~7 J) D8 E
- f. p/ R$ Z( |Identification of Cytoplasmic Podocalyxin Ligands5 R* x! B8 i4 E" m0 h( l
7 @( b- b0 a9 JCytoplasmic domains of CD34-type proteins exhibit a remarkably high degree of sequence conservation, which could reflect the binding of highly conserved intracellular ligands (Fig. 1).: p- i; A2 j: n8 P
# t" n+ Q% c6 _7 W* C! O6 e
Figure 1. Phage screen to identify NHERF-1 as a binding protein for a subset of CD34-type proteins. (A): Schematic of library screening strategy for identification of podocalyxin-binding proteins. Biotinylated peptides (*) corresponding to the intracellular domain of avian podocalyxin were complexed with SAP and incubated with phage-expressed cDNAs immobilized on nylon filters (see Materials and Methods). Positive plaques were isolated and purified, and cDNA inserts were sequenced. Sequencing data suggest that one clone, 15b-2, is a 5'-truncated version of avian NHERF-1 encoding 232 of the 333 aa full length NHERF-1. (B): Schematic structure of the murine NHERF-1 gene. NHERF-1 contains six exons (large boxes) and is made up of two tandem PDZ binding domains and an ERM domain. White boxes represent coding sequences, whereas black boxes denote untranslated exonic sequences. The gray line indicates the homologous region of NHERF-1 contained within the avian cDNA clone of phage 15b-2). (C): NHERF-1 binds podocalyxin and endoglycan but not CD34. Purified phage were plated and screened for the ability to bind to the C-terminal sequences of CD34, podocalyxin, endoglycan, or a control peptide encoding the SDF-1 molecule. Abbreviations: DTXL, C-terminal amino acid sequences for CD34 family proteins; Endo, endoglycan; ERM, Ezrin/Radixin/Moesin; NHERF, Na /H exchanger regulatory factor; PDZ, PSD-95/Drosophila Discs large/ZO-1; Podo, podocalyxin; SAP, streptavidin-coupled alkaline phosphatase.( I4 C* ]) G. o: t+ v- n
$ f: K$ n. k/ G' Y5 q7 A
To determine whether this clone also had affinity for other members of the CD34 family, we tested its ability to bind peptides corresponding to the cytoplasmic domains of CD34 and endoglycan or to stromal-derived factor-1 (SDF-1) as an irrelevant control (Fig. 1C). NHERF-1-expressing phage plaques bound to the tails of podocalyxin and endoglycan but, interestingly, not to CD34. In this regard, it is noteworthy that the tails of endoglycan and podocalyxin show a much higher degree of sequence similarity to each other than they do to CD34 (supplemental online Fig. 1B). This is also true of the very C-terminal sequence of the proteins, which contains a putative docking site for NHERF-type PDZ domains (supplemental online Fig. 1A). In podocalyxin and endoglycan, this C-terminal docking sequence is DTHL, whereas in CD34 it is DTEL.
# T3 ~6 P2 P8 @) G9 X c7 M: K' n7 \7 I% f( n* k
CD34-Type Proteins Bind NHERF-1 from Hematopoietic Progenitor Cell Extracts
8 z& V) F$ {7 f$ Z6 [# f
7 g( D2 S1 x7 X: a) c2 tAs an independent approach to search for additional interactors that may not have been identified in the phage screen, we affinity-purified CD34-family binding proteins from progenitor cell extracts and identified them via mass spectrometry. As a source of binding proteins, we chose the IL-3-dependent hematopoietic murine progenitor cell line FDC-P1 since this line has been well studied but not previously reported in mouse. Phosphorylation of this residue was confirmed by tandem MS/MS sequencing (Fig. 2C). Although this phosphorylation is naturally occurring, it is not required for the binding of NHERF-1 to podocalyxin and endoglycan peptides since these molecules bind NHERF-1 in phage plaques, which lack these modifications. In summary, both phage screens and biochemical analyses suggest that NHERF-1 is a bona fide hematopoietic ligand for a subset of CD34-type proteins.
' b! a5 `( j5 L; s
4 M% ?$ E- @; m& o; j; v0 MTable 1. Peptide sequences obtained by LC-MS/MS and matched to murine NHERF-1
, b8 Y! V5 T1 R& W
3 d& U, E; ^$ v0 y9 t6 AFigure 2. Endogenous NHERF-1 specifically interacts with podocalyxin and endoglycan but not CD34. (A): Whole cell lysates from FDC-P1 were affinity-purified using biotinylated CD34 family peptides on streptavidin-Sepharose resin. After extensive washing, proteins were eluted by boiling in SDS load buffer and resolved by SDS-polyacryl-amide gel electrophoresis (SDS-PAGE). The bands indicated (1 and 2) were excised, subjected to in-gel tryptic digestion, and analyzed by LC-MS/MS. (B): Schematic showing the NHERF-1 protein and the peptides covering the corresponding regions identified by mass spectrometry. Indicated by * (and in dark gray) is the phosphorylated peptide identified by mass spectrometry, shown in Fig. 2C and Table 1. (C): Partial spectra from tandem MS/MS sequencing of the triple-charged phosphorylated peptide EALVEPASESPRPALAR (m/z 624.98) show the phosphorylated residue to be Ser275. Double-charged y ion series containing DHA in position 275 indicates a 98 Da, ß-elimination, neutral loss of phosphoric acid. The boundary of the neutral loss is coincident with phosphorylated Ser275 and unmodified Ser273. (D): Specificity of NHERF-1 for CD34 family members. Proteins that bind to the CD34 family C-terminal peptides were affinity-purified, resolved by SDS-PAGE, and transferred to nitrocellulose membranes, and NHERF-1-reactive proteins were identified via immunoblotting. Different lengths of C-terminal tails from CD34, podocalyxin, and endoglycan were used (see Materials and Methods). These are indicated schematically with wavy lines followed by the very C-terminal amino acids corresponding to the PDZ docking motif. Abbreviations: DHA, dehydroalanine; Endo, endoglycan; ERM, Ezrin/Radixin/Moesin; NHERF, Na /H exchanger regulatory factor; PDZ, PSD-95/Drosophila Discs large/ZO-1; Podo, podocalyxin.. [; L* S0 _$ i3 w
: r$ _1 |' X8 G- _5 W
To further characterize the interaction between NHERF-1 and the cytoplasmic tails of the CD34 family members, biotinylated peptides corresponding to the final 20 aa of podocalyxin, as well as these final 20 aa minus the C-terminal leucine, were evaluated for their ability to bind NHERF-1 from FDC-P1 extracts by small-scale affinity purification and anti-NHERF-1 immunoblotting. The C-terminal 20 aa of podocalyxin and endoglycan were sufficient for NHERF-1 binding, whereas truncation of the C-terminal leucine residue ablated binding of NHERF-1, consistent with previous reports suggesting the importance of this residue in the recognition of proteins by PDZ domains (Fig. 2D)
( [, T9 S" }7 q. L/ k! c: `1 w, N! @! M& U6 ]
NHERF-1 Associates with Podocalyxin In Vivo
) [8 G( J2 h6 r3 N# E1 X7 j1 \- Z4 Q# j8 g7 Y- S# S7 ?
The interaction of NHERF-1 with podocalyxin was confirmed using two additional methods: colocalization via confocal microscopy and direct co-immunoprecipitation from cell lysates. FDC-P1 cells were surface-stained for podocalyxin and then cytoplasmically stained for NHERF-1. As shown in Figure 3, although most FDC-P1 cells express both podocalyxin (panel I) and NHERF-1 (panel II), only a subset of these cells exhibited strong colocalization of these antigens (panel III). Interestingly, within the subset of these cells, the strongest colocalization correlated with polarized capping of podocalyxin on the cell membrane; cells uniformly expressing podocalyxin on their surface showed only weak colocalization, whereas cells displaying asymmetric localization of podocalyxin on their surface showed high overlap with NHERF-1. Since the survival and proliferation of FDC-P1 cells are cytokine-dependent, capping of podocalyxin on a subset of these cells may be a consequence of variable IL-3R signaling. To test this hypothesis, we performed confocal analyses of podocalyxin distribution in the plasma membrane of IL-3-starved or in IL-3-stimulated cells (Fig. 4). The majority of IL-3-starved cells displayed a uniform "halo" distribution of podocalyxin, and only rarely could cells be found with podocalyxin capped on one pole (Fig. 4A, 0 hours). Interestingly, within minutes of IL-3 stimulation, podocalyxin capping increased and this correlated with NHERF-1 co-localization and reached a steady-state maximum at 30¨C60 minutes.
1 {$ H4 C5 [$ [% O( a3 E! L) ?8 M) X, }
Figure 3. NHERF-1 co-localization with podocalyxin in early progenitor cells. FDC-P1 cells were fixed, stained with NHERF-1 and podocalyxin antibodies, and detected with AlexaFluor 488 (green) and Al-exaFluor 568 (red) secondary antibodies, respectively, prior to confocal microscopy analysis. All pictures were taken with a x60 oil objective with x2.5 zoom. Open arrows indicate cells with global expression of podocalyxin, and closed arrows show more localized expression of podocalyxin on one side of the cell. Scale bars, 10 µm. Abbreviations: NHERF, Na /H exchanger regulatory factor; Podo, podocalyxin.3 ], j; p! @7 {
8 ]% q0 l5 t1 t" X$ ~; q* j F0 G: ^
Figure 4. IL-3 and PMA differentially regulate localization of podocalyxin and NHERF-1 in FDC-P1. (A, B): Graphs show the kinetics of podocalyxin and NHERF-1 relocalization in response to IL-3 and PMA. Three cell phenotypes were scored for each time point: 1) punctate, 2) capped, and 3) uniform surface expression of podocalyxin. Cells were starved for 2 hours (time = 0 hour) prior to IL-3 or PMA stimulation. Blue lines, uniform expression of podocalyxin on cells; orange lines, capped/clustered staining; red lines, punctate staining. Three counts of x100 cells each were averaged at each time point. Graphs are representative of two independent experiments. Representative pictures of these phenotypes are shown. (C): Immunoblot of immunopurified podocalyxin complexes from FDC-P1 cells. Cells were starved of IL-3 and either left untreated or treated for 1 hour with IL-3 and PMA as indicated prior to immunoprecipitation. The FD5 cell line (podocalyxin-negative) was used as a negative control. Upper blot, anti-NHERF-1. Lower blot, anti-podocalyxin. Abbreviations: IB, antibody used for immunoblotting; IL, interleukin; IP, antibody used for immunoprecipitation; LC, light chain; NHERF, Na /H exchanger regulatory factor; PMA, phorbol 12-myristate 13-acetate; Podo, podocalyxin.2 A+ {9 w: Y4 k, R! P
% B5 ]+ N7 a7 d, {! s2 g/ W, aPKC phosphorylation of NHERF-1 has been shown to regulate its oligomerization . Strikingly, PMA failed to induce strong capping of podocalyxin, and instead, PKC activation led to the redistribution of podocalyxin into a more punctate or speckled pattern on the surface of these cells (Fig. 4B). This reached a maximum after 30 minutes and then gradually declined to the background levels. In addition, by using an anti-podocalyxin antibody, we found that endogenous NHERF-1 co-precipitated weakly with podocalyxin in IL-3-starved cells and this interaction was only mildly enhanced by PMA treatment but was enhanced by stimulation with IL-3 (Fig. 4C). No NHERF-1 was co-precipitated from the podocalyxin-negative murine myeloid cell line, FD5.$ f& S- A6 V8 w- B/ b
1 o/ O# ~: ]" I) c; P
Cytokine stimulation leads to a very distinctive capping of podocalyxin on the surface of cells, and this correlates with the formation of a complex with NHERF-1; activation of the PKC pathway alone results in only minimal capping of podocalyxin and a correspondingly lower association with NHERF-1.
, e) T( f) d) }3 E+ O# L! m: _
2 V* t" m0 i5 N3 w2 NNHERF-1 Is Expressed by Mature Hematopoietic Cells and by Cells with an HSC Phenotype
2 k9 ?5 J6 M2 Y3 |8 O A
8 B1 u1 k* v4 H# _* x4 R0 {Although the expression of NHERF-1 by kidney cells and epithelial cells has been described previously .
* b' l+ m, B, v* o. n; R" D7 [: g, U) B- ~5 w' [
Figure 5. Hematopoietic distribution of NHERF-1. (A): Single-cell suspensions from the indicated tissues or cell lines were isolated, fixed, stained with NHERF-1 (blue) or control antibodies (red), and subjected to flow cytometric analyses. Note that the thymus has the highest and whole bone marrow lysates the lowest expression for endogenous NHERF-1 proteins. (B): Immunoblot analysis of whole cell lysates for NHERF-1 expression or actin as a control. In the case of whole bone marrow lysates, approximately 13 times more protein extract was loaded. MDA-231 and murine kidney cells were used as negative and positive controls, respectively. (C): Two-color flow cytometric analysis of NHERF-1 expression by bone marrow cells. Bone marrow suspensions were surface stained for the markers indicated (Phycoerythrin-conjugates) and cytoplasmically stained for NHERF-1 with a fluorescein isothiocyanate-conjugated secondary antibody. In each profile, cells were gated to show only the lineage marker-positive fraction indicated. Abbreviations: 13x BM, bone marrow lysates with 13 times more protein extract; LN, lymph nodes; NHERF, Na /H exchanger regulatory factor; SP, spleen; Thy, thymus.
- y" D+ y- c l4 c2 t( D
) z+ \0 V; U7 D9 a _% h2 aWe also performed a detailed two-color immunofluorescence analysis of NHERF-1 expression by lineage-restricted precursors in the bone marrow. Cells stained for lineage-specific markers B220 (B lineage cells), CD3 (T lineage cells), Mac-1 (myelomonocytic cells), Gr-1 (granulocytes), and Ter119 (erythroid cells) were fixed and stained for cytoplasmic NHERF-1. NHERF-1 was detected in the majority of cells expressing lineage-restricted markers, although it was low to negative on distinct subsets of B lineage and erythroid lineage cells (B220 and Ter119 stains, Fig 5C).
+ C- Y& ^9 i, P) @. a" }7 M. u r- g. N5 O- n/ l3 _9 [
Previously, it has been reported that podocalyxin is expressed by human cells with an HSC phenotype and to express podocalyxin, we conclude that NHERF-1 is a bone fide ligand for podocalyxin in HSCs. |2 K2 R, B/ Z K* B6 Z5 ^
- n' U8 ], A* b& F, K' M
Figure 6. NHERF-1 distribution on Lin¨C, c-kit , and Sca-1 cells. Single-cell suspension of bone marrow were stained and analyzed by flow cytometry. (A): Flow cytometry results of gated cells that were c-kit high and Lin¨C (2.42% of the total bone marrow). These cells were further gated for Sca-1 cells. (B, C): Bone marrow cells were stained with all three hematopoietic markers and AlexaFluor 488 antibody alone (B) or with a rabbit isotype control (C). Profile shows only those cells that were positive for c-kit and negative for lineage markers. (D): Same as (C) but stained with NHERF-1-specific antibodies. Abbreviation: NHERF, Na /H exchanger regulatory factor.' m; e* D' ~9 a9 C$ O' j2 I
9 V, R) R! e! t; ^" |. {
DISCUSSION
% }5 I7 o! I" n* R6 s: ` V0 T
2 a! f8 }# q; i6 V4 eUntil recently, the role of CD34-type proteins in hematopoiesis and development has remained obscure. Attempts to link their surface expression to intracellular signaling pathways has led a number of groups to search for intracellular ligands for these molecules. Here we describe the identification of the cytosolic protein NHERF-1 as a hematopoietic ligand for a subset of CD34-type proteins and show that it is expressed by cells with an HSC phenotype. These results have important implications for the function of CD34-type molecules in hematopoiesis and in a variety of nonhematopoietic cell types.5 }- X! a4 d5 w- F! r
G, Y" t, u: Y: E( s% z2 I+ A
Function of CD34-Type Molecules
, t9 B1 Z( v; v/ g( K6 r! ?/ d0 ~2 |; t' B6 D, c/ l
Despite the widespread use of CD34 over the past 20 years as a clinical marker of human HSCs, its functional role on hematopoietic lineage cells has remained enigmatic. Although there has been speculation that this antigen may play a role in blocking hematopoietic cell maturation or enhancing proliferation or act as a homing receptor, there are only minor defects in mice lacking this protein . In summary, these data suggest that the principle function of CD34-type molecules is to block cell adhesion and increase invasiveness.+ q. q4 c8 ]* V/ g
0 C+ T& q" u0 @$ f3 w7 i4 Z8 S+ v
One important caveat to this antiadhesive hypothesis is that under many circumstances, the cells that normally express this family of molecules are able to adhere to basement membranes and substrates. In this regard, it is noteworthy that all of the models showing antiadhesive roles for these molecules involve either the deletion of the encoding genes (complete loss of function) or high-level ectopic overexpression of these molecules (potent gain of function). Thus, the ability of normal cells to overcome the antiadhesive properties of CD34-type proteins may reflect a tight control over the levels of expression of these antiadhesins or the ability to tightly regulate their subcellular localization in an activation-dependent manner. The latter model correlates well with our observation that a naturally occurring splice variant encoding a cytoplasmically truncated form of the CD34 (which, presumably, has lost the ability to be actively redistributed in the plasma membrane) is a dominant blocker of adhesion .
% L: Q4 k" x6 v" h' X6 [0 X* @+ \
) t) c2 l) H$ Q$ ]: R; zCD34-Family Ligands2 W& V, ]" j& `, s; h# K7 @+ S
" H0 A' p: U6 N5 ]6 J8 Q3 B* |As a first step toward revealing the mechanisms underlying this latter hypothesis, several groups have pursued intracellular ligands for this family of molecules. For example, the adapter molecule CrkL was recently identified as a cytoplasmic ligand for CD34 . Correspondingly, we have failed to detect NHERF-2 as a ligand in hematopoietic tissues and cells by either mass spectrometry or functional screens (data not shown).. n* E5 B+ n1 ^! J
% o9 d+ s' i* s" [( g, ^Instead, we have identified a close relative of NHERF-2, NHERF-1, as a hematopoietic ligand for these proteins and shown that NHERF-1 has specificity for podocalyxin and endoglycan but not CD34. Although NHERF-1 has been postulated to be a ligand for podocalyxin, based on its similarity to NHERF-2, to our knowledge, our results are the first to demonstrate a naturally occurring interaction between podocalyxin and NHERF-1 . Thus, there may be functional heterogeneity within the NHERF family.
8 e1 z5 F) A7 O6 l7 j* o! Q( Z
5 K$ F0 V3 F! ]# BIt is intriguing that NHERF-1 has strong affinity for podocalyxin and endoglycan but not CD34. The cytoplasmic domains of podocalyxin and endoglycan show a much higher degree of sequence similarity to each other than to CD34, and this includes an amino acid substitution in the C-terminal PDZ domain-docking site from DTHL (podocalyxin and endoglycan) to DTEL (CD34). This is the first clear demonstration of functional heterogeneity in this family of sialomucins and may indicate the existence of an independent PDZ domain-docking protein for CD34.: l$ Q* }( G- y! i. [
: M9 k; W2 C( t9 z/ R# ^/ c+ bFunctional Significance of CD34 Family Proteins and NHERF-1
9 u- A+ W6 Q5 S! U: L" s
2 L. ?, p l3 b& MAlthough NHERF-1 was first described as a specific regulator of transmembrane Na /H exchangers, it is thought to act as a broad-based scaffolding protein for linking membrane proximal proteins with the actin cytoskeleton, thereby regulating their subcellular localization and, potentially, their stability and internalization . The fact that this domain is established prior to cell adhesion is consistent with a model in which podocalyxin clustering permits establishment of a podocalyxin-free and adhesion-molecule-rich basolateral domain.) @' L! _5 z% V) l# v* c7 @ a
, l; R/ Z- G' s! u |: O* E/ I- KPreviously, we have shown that podocalyxin and CD34 play a role in blocking hematopoietic cell adhesion in vivo and in vitro and that truncation of the cytoplasmic domain increases the effectiveness of this block . An interesting possibility is that podocalyxin, as a downstream modulator of adhesion, may be a mediator of IL-3R-dependent HSC mobilization or turnover. Clarification of this model will be facilitated by the expression of dominant-negative forms of NHERF-1 in HSCs and the generation of null backgrounds for assessing their function./ x# p. ?7 y" C) Y2 X' Q! D0 @/ i# e
& [: v- J# _) Z: v- @# x, cACKNOWLEDGMENTS
D+ w( V5 [, W. |
8 `0 o/ N; ]/ y$ |We thank Andy Johnson and Dr. Stephane Corbel for assistance in this work and the University of British Columbia FACS facility for assistance in flow cytometry analysis and Phil Owen of The Biomedical Research Centre peptide synthesis facility for all peptides. We also thank Dr. Michael Hughes for critical reading and helpful discussions of the manuscript. P.C.T. is funded by a Graduate Trainee Award from Stem Cell Network and Canadian Institutes for Health Research, Heart and Stroke Foundation of Canada, and the Centre for Blood Research. M.L.M. is funded by the Canadian Breast Cancer Research Alliance. K.M.M. is a Scholar of the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research. This work was funded by a Canadian Institutes of Health Research grant (MT-15477; to K.M.M.), by a Grant in Aid from the Heart and Stroke Foundation of British Columbia & Yukon.5 J0 K5 {0 L& N8 r
1 Z ~" \- a4 I- h1 H1 i! {DISCLOSURES9 c/ ]9 l& N& c- I( c& j
- C) f3 `' ?: t; C8 G5 QThe authors indicate no potential conflicts of interest.2 [# X! A" f: G# v0 p; d$ I8 x( s
【参考文献】
" C* V: p5 L4 ?5 w1 Z
1 y# _; D+ q9 B2 ]7 z' \2 w- U% z3 `2 J4 u- V8 M: Q9 W- P
Sassetti C, Van Zante A, Rosen SD. Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins. J Biol Chem 2000;275:9001¨C9010.. k: f4 _8 Y4 A! u
. H5 a/ r4 r$ w4 T) U: M; v
Doyonnas R, Kershaw DB, Duhme C et al. Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin. J Exp Med 2001;194:13¨C27.: p3 [5 D. C# I) g; Q, |; Q
3 O$ b8 l7 s1 H$ O/ H: `1 HNielsen JS, Doyonnas R, McNagny KM. Avian models to study the transcriptional control of hematopoietic lineage commitment and to identify lineage-specific genes. Cells Tissues Organs 2002;171:44¨C63.0 _: V( x. Y! S& S# |
: N a0 s9 q2 ]5 ^Krause DS, Fackler MJ, Civin CI et al. CD34: Structure, biology, and clinical utility. Blood 1996;87:1¨C13.
; d* x5 p3 u2 h4 D; `1 w
9 U ~; n' R0 K- C8 FLanza F, Healy L, Sutherland DR. Structural and functional features of the CD34 antigen: An update. J Biol Regul Homeost Agents 2001;15: 1¨C13.9 a/ k3 S# A; r- H- G5 f4 X
' B3 r4 E! I" z: }0 Y! _, JNakamura Y, Komano H, Nakauchi H. Two alternative forms of cDNA encoding CD34. Exp Hematol 1993;21:236¨C242.% c$ Z2 A0 d3 s6 \% o' h* _; c
+ F, T7 N# o1 Z) cMcNagny KM, Pettersson I, Rossi F et al. Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors. J Cell Biol 1997;138:1395¨C1407.0 T& t, y8 X7 O7 P$ \
# Y6 I9 [* l8 o5 h* E; j2 z# ?+ tSassetti C, Tangemann K, Singer MS et al. Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: Parallels to CD34. J Exp Med 1998;187:1965¨C1975.
6 Y: v9 z5 l. r8 M
% Q9 |0 Z0 N' B2 |8 p5 n1 UKerosuo L, Juvonen E, Alitalo R et al. Podocalyxin in human haematopoietic cells. Br J Haematol 2004;124:809¨C818.. Q- q) f$ \4 s/ i8 {
, A& ^" b% B' bHara T, Nakano Y, Tanaka M et al. Identification of podocalyxin-like protein 1 as a novel cell surface marker for hemangioblasts in the murine aorta-gonad-mesonephros region. Immunity 1999;11:567¨C578.+ ?$ z( z5 h [' i9 |
4 m9 Y0 D' m: j" }2 ^* n; y8 uBaumheter S, Singer MS, Henzel W et al. Binding of L-selectin to the vascular sialomucin CD34. Science 1993;262:436¨C438.: F4 x1 C$ l6 L5 \) e' J1 ~- J" m
! C9 ~) W) @8 S0 d5 p3 l1 o# U- `4 G; Q
Rosen SD. Ligands for L-selectin: Homing, inflammation, and beyond. Annu Rev Immunol 2004;22:129¨C156.
& X+ Q0 G& z2 W" _% r( W7 {
: S% T$ ~4 T2 a$ z2 Q- R9 ~5 G9 nTakeda T, Go WY, Orlando RA et al. Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells. Mol Biol Cell 2000;11:3219¨C3232.
3 Z+ U$ u" p5 G! t, E0 h& r- w1 a) r: Y( o' Y) C6 c
Somasiri A, Nielsen JS, Makretsov N et al. Overexpression of the anti-adhesin podocalyxin is an independent predictor of breast cancer progression. Cancer Res 2004;64:5068¨C5073.
3 O6 ], F3 N% P5 s$ e0 V5 ?7 p! ` q1 s8 v1 B( @4 u
Drew E, Merkens H, Chelliah S et al. CD34 is a specific marker of mature murine mast cells. Exp Hematol.. Oct 2002;30:1211.# y% g( N6 u4 p: P
) y [3 S& m MDrew E, Merzaban JS, Seo W et al. CD34 and CD43 inhibit mast cell adhesion and are required for optimal mast cell reconstitution. Immunity 2005;22:43¨C57.
+ j8 j9 Q- m- f! U5 R* v" P1 f1 ?; C
Majdic O, Stockl J, Pickl WF et al. Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34. Blood 1994;83:1226¨C1234.1 v: n' j, Q" @+ K' z8 F
" ? |# }6 U( S# T& h6 hHealy L, May G, Gale K et al. The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion. Proc Natl Acad Sci U S A 1995;92:12240¨C12244.
P7 B# x& R H" e
4 p0 J8 G( e4 ? p2 j6 k: m' bTada J, Omine M, Suda T et al. A common signaling pathway via Syk and Lyn tyrosine kinases generated from capping of the sialomucins CD34 and CD43 in immature hematopoietic cells. Blood 1999;93:3723¨C3735.
5 y8 ?% e* D4 a* V0 P7 W( M! j
6 o5 _% G- M8 b; J( s4 K, rFackler MJ, Civin CI, Sutherland DR et al. Activated protein kinase C directly phosphorylates the CD34 antigen on hematopoietic cells. J Biol Chem 1990;265:11056¨C11061.( E; N. ~, ~- p, w
& G: c, @% I5 |7 L' J
Sutherland DR, Fackler MJ, May WS et al. Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells. Leuk Lymphoma 1992;8:337¨C344.
/ j l k, e) C% ~3 S6 A
+ X X' ^: p( s Z; g) D& }6 B/ W# ZFackler MJ, Civin CI, May WS. Up-regulation of surface CD34 is associated with protein kinase C-mediated hyperphosphorylation of CD34. J Biol Chem 1992;267:17540¨C17546." k, Y( D. J% D a7 \, c
& {+ Q$ S4 R, x& `! k
Delia D, Lampugnani MG, Resnati M et al. CD34 expression is regulated reciprocally with adhesion molecules in vascular endothelial cells in vitro. Blood 1993;81:1001¨C1008.9 K. Y6 k5 X" `, M+ K j& ]
F/ T7 J8 x5 v8 \5 v: rSchmieder S, Nagai M, Orlando RA et al. Podocalyxin activates RhoA and induces actin reorganization through NHERF1 and Ezrin in MDCK cells. J Am Soc Nephrol 2004;15:2289¨C2298.
6 t" h2 w3 G" [6 E+ ~3 s% {2 K7 D
Felschow DM, McVeigh ML, Hoehn GT et al. The adapter protein CrkL associates with CD34. Blood 2001;97:3768¨C3775.. ?- c3 U& O8 x8 z- t* T& r
7 `6 }0 ?# N$ V7 `$ jSabolic I, Herak-Kramberger CM, Ljubojevic M et al. NHE3 and NHERF are targeted to the basolateral membrane in proximal tubules of colchicine-treated rats. Kidney Int 2002;61:1351¨C1364.
$ H" X$ j+ w4 f4 E+ T, a/ K; F% c0 n. c4 u8 ~
Liedtke CM, Raghuram V, Yun CC et al. Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1. Am J Physiol Cell Physiol 2004;286:C1037¨CC1044.
7 W% I& w- k5 V! Y+ d c/ ]1 Y" v5 }7 O0 O
Takeda T, McQuistan T, Orlando RA et al. Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 2001;108:289¨C301.% ^3 F4 ?' s D/ M, @' p
) A. c3 I3 j) H
Dexter TM, Garland J, Scott D et al. Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med 1980;152:1036¨C1047.
- C. z% `1 p, h. W
+ u. E% f. c# X( W3 lStrausberg RL, Feingold EA, Grouse LH et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci U S A 2002;99:16899¨C16903.$ ~$ y" N# x: I( _1 ~
4 e4 q7 x! E8 Z4 H; F$ p- t
Brown J, Greaves MF, Molgaard HV. The gene encoding the stem cell antigen, CD34, is conserved in mouse and expressed in haemopoietic progenitor cell lines, brain, and embryonic fibroblasts. Int Immunol 1991;3:175¨C184.$ f) u4 ^" x$ h$ d7 `3 G/ o
# U7 E! G% G- k9 `- V
Li Y, Li J, Straight SW et al. PDZ domain-mediated interaction of rabbit podocalyxin and Na( )/H( ) exchange regulatory factor-2. Am J Physiol Renal Physiol 2002;282:F1129¨CF1139.
3 t1 E& u) h" t1 t3 Z/ a$ q/ n; _, k# L( \, P0 d0 Q8 m) X
Graf T, McNagny K, Brady G et al. Chicken "erythroid" cells transformed by the Gag-Myb-Ets-encoding E26 leukemia virus are multipotent. Cell 1992;70:201¨C213.
6 w4 S8 j5 O$ z& @ p% B$ E. W9 \! N; R8 z- {
McNagny KM, Rossi F, Smith G et al. The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin. Blood 1996;87:1343¨C1352.
' i. r: e* p4 T; Z0 F$ ~( P; { {) u. V9 ^
Weinman EJ, Shenolikar S. The Na-H exchanger regulatory factor. Exp Nephrol 1997;5:449¨C452.
. m T% }' j4 s v9 e( }- O: U8 y7 j t: e6 G) |) C& n2 _0 J' _
He J, Lau AG, Yaffe MB et al. Phosphorylation and cell cycle-dependent regulation of Na /H exchanger regulatory factor-1 by Cdc2 kinase. J Biol Chem 2001;276:41559¨C41565.' i9 Y3 J g) F5 I% i7 z
+ m( Q6 {! o0 _, [: E
Fouassier L, Nichols MT, Gidey E et al. Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50. Exp Cell Res 2005;306:264¨C273.
7 j( ? d" X' d0 q+ @) G# o* S( x* z; D! {: Z3 u
McCubrey JA, Steelman LS, Sandlin G et al. Effects of phorbol esters on an interleukin-3-dependent cell line. Blood 1990;76:63¨C72.
* _" r- N4 X7 H# P1 m+ j7 \) M
# z5 q! W, j* W; h ~8 C3 i7 L5 NWade JB, Liu J, Coleman RA et al. Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell Physiol 2003;285:C1494¨CC1503.
9 F& z# t/ h t5 c+ Q+ V; r
3 \6 u6 r( J) Y# aEdiger TR, Kraus WL, Weinman EJ et al. Estrogen receptor regulation of the Na /H exchange regulatory factor. Endocrinology 1999;140: 2976¨C2982.# D1 @; j& e+ B7 `5 E
: R% R4 O9 b% o& `) O! d0 D( Z; q9 ySpangrude GJ, Smith L, Uchida N et al. Mouse hematopoietic stem cells. Blood 1991;78:1395¨C1402.
% w) `3 }9 ?5 H4 U6 V. A
+ f* h0 y5 ~# {. |! wCheng J, Baumhueter S, Cacalano G et al. Hematopoietic defects in mice lacking the sialomucin CD34. Blood 1996;87:479¨C490.0 {: P9 s2 v P \
+ u3 g9 R- ?& u5 h
Suzuki A, Andrew DP, Gonzalo JA et al. CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein. Blood 1996;87:3550¨C3562.
) P9 z6 b; t- G% |5 P) j2 P, x" B6 |* }
Schopperle WM, Kershaw DB, DeWolf WC. Human embryonal carcinoma tumor antigen, Gp200/GCTM-2, is podocalyxin. Biochem Biophys Res Commun 2003;300:285¨C290.* r; U$ ]8 Y/ m: E' I4 X
+ L+ u5 j1 }2 A7 `9 |! `Doyonnas R, Nielsen JS, Chelliah S et al. Podocalyxin is a CD34-related marker of murine hematopoietic stem cells and embryonic erythroid cells. Blood 2005;105:4170¨C4178.
$ b. `* d/ m* M9 [0 t
( T" D, o" D: x6 ^+ aWeinman EJ. New functions for the NHERF family of proteins. J Clin Invest 2001;108:185¨C186./ ?2 F. W2 C/ c+ O& f( L. J" j% d7 d
& b R9 R" q' b5 _8 V) i* I, B
Takeda T. Podocyte cytoskeleton is connected to the integral membrane protein podocalyxin through Na /H -exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol 2003;7:260¨C269.
' G0 b; M! N: B
: i' ?* `) E9 Q+ o6 tVoltz JW, Weinman EJ, Shenolikar S. Expanding the role of NHERF, a PDZ-domain containing protein adapter, to growth regulation. Oncogene 2001;20:6309¨C6314.$ G; z2 }. Y* I
' R7 ^& Q0 A8 z0 v( Q, fDai JL, Wang L, Sahin AA et al. NHERF (Na /H exchanger regulatory factor) gene mutations in human breast cancer. Oncogene 2004;23: 8681¨C8687.
4 B( V- ^' k+ Q- k9 H7 Y, e
5 |4 O) ~% O8 u, E+ x1 S6 p4 pMeder D, Shevchenko A, Simons K et al. Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells. J Cell Biol 2005;168:303¨C313.
: q0 s4 s6 H4 O! m
6 Y: w5 j+ s2 y5 s2 e+ J5 \Bystrykh L, Weersing E, Dontje B et al. Uncovering regulatory pathways that affect hematopoietic stem cell function using ¡®genetical genomics¡¯. Nat Genet 2005;37:225¨C232.+ Z, |/ O0 v% \( B4 b9 B0 _
% O# Q: [3 i% u0 B0 hRane SG, Reddy EP. Janus kinases: Components of multiple signaling pathways. Oncogene 2000;19:5662¨C5679.
# Q9 U1 X! f7 k
5 v" G8 I& m3 _. k/ f5 tBrose N, Rosenmund C. Move over protein kinase C, you¡¯ve got company: Alternative cellular effectors of diacylglycerol and phorbol esters. J Cell Sci 2002;115:4399¨C4411.% R$ v& n+ s' y/ N! ~
3 K9 X$ n/ X6 V
Yanai T, Sugimoto K, Takashita E et al. Separate control of the survival, the self-renewal and the differentiation of hemopoietic stem cells. Cell Struct Funct 1995;20:117¨C124.
, h; T9 o) o! a! S' \$ n
: }" b7 [9 h. d" P, mIvanovic Z. Interleukin-3 and ex vivo maintenance of hematopoietic stem cells: Facts and controversies. Eur Cytokine Netw 2004;15:6¨C13." H' R, O0 s; v
0 i- }' P4 H2 b! l& _7 U/ Y2 F
Oostendorp RA, Dormer P. VLA-4-mediated interactions between normal human hematopoietic progenitors and stromal cells. Leuk Lymphoma 1997;24:423¨C435.4 I9 q; s" Y/ g0 O( e6 z0 T
8 }) O( o* E( a( s$ y# a' ^
Schrader W. Interleukin-3. In: Thompson A, ed. The Cytokine Handbook. London: Academic Press Limited, 1994:81¨C98. |
|
发表于 2009-3-5 00:07
发表于 2015-6-9 19:59
