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First published online January 12, 2006
Stem Cells Vol. 24 No. 5 May 2006, pp. 1150 -1161
doi:10.1634/stemcells.2005-0426; www.StemCells.com
© 2006 AlphaMed Press

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STEM CELL GENETICS AND GENOMICS

Na+/H+ Exchanger Regulatory Factor-1 Is a Hematopoietic Ligand for a Subset of the CD34 Family of Stem Cell Surface Proteins

Poh C. Tana, Sebastian G.B. Furnessa, Helen Merkensa, Shujun Lina, Marcia L. McCoyb, Calvin D. Roskelleyb, Jürgen Kasta, Kelly M. McNagnya

a The Biomedical Research Centre and
b Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada

Key Words. Hematopoiesis • Adhesion • Differentiation • Stem cells • CD34 • Podocalyxin • Endoglycan • Sialomucin • Na+/H+ exchanger regulatory factor-1 • Na+/H+ exchanger regulatory factor-2

Correspondence: Kelly M. McNagny, Ph.D., The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Telephone: +1-604-822-7810; Fax: +1-604-822-7815; e-mail: kelly{at}brc.ubc.ca

Received August 30, 2005; accepted for publication January 6, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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– /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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD34, podocalyxin, and endoglycan comprise a family of hematopoietic and vascular-restricted sialomucins [1–3]. CD34 is the highest profile member of this family due to its widespread use as a marker of hematopoietic stem cells (HSCs) [4, 5]. The members of this family share several biochemical motifs, including a heavily glycosylated mucin domain, a disulfide-bonded globular domain, a membrane proximal stalk domain, a transmembrane region, and a highly conserved cytoplasmic tail of 70–80 amino acids (aa) with consensus phosphorylation sites for protein kinase C (PKC) and casein kinase II [2, 3]. Their classification as a gene family is supported by a similar exon/intron structure in their respective genomic loci and similar patterns of alternative splicing that give rise to mRNAs, encoding protein isoforms that lack or maintain the bulk of the cytoplasmic domain [2, 6, 7].

All three proteins are expressed on HSCs/progenitors, but their function on these cells has remained enigmatic [1, 4, 7–10]. It has been shown that when expressed by specialized endothelial cells in the lymph nodes, called high endothelial venules (HEVs) [1, 8, 11], CD34, podocalyxin, and endoglycan are modified with an unusual type of carbohydrate moiety that permits their recognition by a leukocyte-specific carbohydrate-binding receptor called L-selectin (reviewed by Rosen [12]). Leukocyte- or L-selectin is expressed by newly formed lymphocytes and is used by these cells to bind to the appropriately glycosylated CD34 family members expressed on HEVs. This initial binding is the first key step in a process that culminates in leukocyte migration into the peripheral lymph nodes [12]. 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.

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 [13]. Similarly, we have shown that podocalyxin expression is naturally up-regulated on a subset of invasive human breast carcinomas and that podocalyxin over-expression may play a role in disrupting epithelial architecture [14]. Finally, deletion of the podocalyxin-encoding gene in mice leads to increased adhesion between kidney podocytes, and this results in a lack of urine production, kidney failure, and peri-natal death [2].

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 [15] and that deletion of CD34 or the distantly related mucin, CD43, leads to enhanced aggregation of mast cells and impairment in mast cell homing due to enhanced adhesion [16]. This block in adhesion is reversible by the ectopic re-expression of CD34, and intriguingly, adhesion is blocked most effectively by the naturally occurring splice variant of CD34 that lacks most of the cytoplasmic domain [16] (although CD43 is a sialomucin, it lacks the genomic organization and additional motifs that would classify it as a CD34 family member). In support of this anti-adhesive role for CD34, cross-linking studies using antibodies directed against the mucin domain of CD34, but not the stalk/globular domain, enhance both homotypic [17] and heterotypic [18] cell adhesion. Subsequent studies reveal that this antibody cross-linking results in intracellular signaling that allows active relocalization of CD34 to a cap [19], a clearance that presumably allows cell-cell adhesion. Additionally, neuraminidase treatment causes similar cell-cell adhesion [17], suggesting that either cleavage or clearance of the negatively charged moieties enables increased adhesion. In summary, the data suggest that although CD34-type proteins can function as pro-adhesive molecules when appropriately glycosylated, under most conditions they function as molecular "Teflon" to block nonspecific adhesion and cell-cell junction formation. The data also suggest that the cytoplasmic domain may fine-tune this effect [16].

The 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 [20, 21]. It has also been shown that activation of vascular endothelial cells leads to the relocalization of CD34 on these cells [22, 23]. Similarly, it has been shown that ectopic expression of podocalyxin in MDCK cells leads to apical localization of the molecule, activation of RhoA, and polymerization of actin at the sites of expression [24]. These effects are likely to be regulated by cytosolic binding proteins. To date, however, the only known ligands for members of this family are the cytoplasmic adaptor protein CrkL, which binds to the membrane proximal domain of CD34 [25], and the podocyte-specific PSD-95/Drosophila Discs large/ZO-1 (PDZ) and Ezrin/Radixin/Moesin (ERM) domain-containing protein, NHERF-2, which binds to the tail of podocalyxin [26–28].

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.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Media
Factor-dependent continuous cell line, Paterson Laboratories 1 (FDC-P1) cells [29] 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.

Antibodies
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.

Peptides
Peptides were as follows:

  1. Podo-1–73 (C-terminal 73 aa, avian thrombomucin; GenBank accession number CAA74311 [GenBank] [7]): HQRFSQKKSQQRLT- EELQTMENGYHDNPTLEVMETGSEMQEKKVNLNG-ELGDSWIVPLDTIMKEDLEEEDTHL.
  2. Podo-15–73 (C-terminal 58 aa): ELQTMENGYHDNPTLE-VMETGSEMQEKKVNLNGELGDSWIVPLDTIMKEDL-EEEDTHL.
  3. Podo-53–73 (C-terminal 20 aa): WIVPLDTIMKEDLEEED-THL.
  4. Podo-53–72 (C-terminal 20 aa, less the terminal leucine): WIVPLDTIMKEDLEEEDTH.
  5. Endo (C-terminal 26 aa, murine podocalyxin-like 2; GenBank accession number AAH33384 [GenBank] [30]): SSWSALMGSKRDP-EDSDVFEEDTHL.
  6. CD34 (C-terminal 72 aa, murine CD34; GenBank accession number NP_598415 [30]): RRSWSPTGERLGEDPYYTE-NGGGQGYSSGPGASPE TQGKANVTRGAQENGTGQ-ATSRNGHSARQHVVADTEL.

Phage Screens
Phage screens for podocalyxin-binding proteins were performed essentially as described previously [32]. Peptides Podo-1–73, Endo, and CD34 were used to probe an avian early hematopoietic progenitor library [7, 33, 34]. 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 8–12 hours. Filters were washed four times for 20 minutes each (0.1% Triton-X/PBS), and blocked overnight at 4°C (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°C 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).

Confocal Microscopy and Flow Cytometry
Immunofluorescent staining was performed as described previously [7]. For intracellular staining, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed four times (1% bovine serum albumin [BSA]-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).

Cell Stimulation, Counting, and Analysis
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 100–250 cells each were made for every time point, and the average percentage of each cell phenotype was obtained.

Affinity Purification and Mass Spectrometry
All washes and buffers were at 4°C, 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).

Immunoblotting, Affinity Purification, and Immunoprecipitation
Immunoblotting was conducted using standard protocols [15] 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.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Cytoplasmic Podocalyxin Ligands
Cytoplasmic domains of CD34-type proteins exhibit a remarkably high degree of sequence conservation, which could reflect the binding of highly conserved intracellular ligands [2, 5, 7] (supplemental online Fig. 1AGo). To identify these potential ligands, we screened an early hematopoietic progenitor cDNA expression library cloned into {lambda}-phage [34] for binding partners to the cytoplasmic tail of podocalyxin. Briefly, a 73-aa peptide was synthesized with an N-terminal biotin affinity tag and incubated with immobilized proteins from {lambda}-ZAP phage plaques, and filter-bound peptides were detected using SAP and a chromogenic substrate as described previously [32] (Fig. 1Go). In our initial screen of 1 x 106 clones we identified several reactive phage plaques, but only one was consistently reactive after several rounds of screening. This clone was sequenced and found to encode the C-terminal 232 aa of the tandem PDZ domain-containing protein, NHERF-1 [35] (Fig. 1Go).


Figure 1
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  		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.

 
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. 1CGo). 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. 1BGo). This is also true of the very C-terminal sequence of the proteins, which contains a putative docking site for NHERF-type PDZ domains [28, 32] (supplemental online Fig. 1AGo). In podocalyxin and endoglycan, this C-terminal docking sequence is DTHL, whereas in CD34 it is DTEL.

CD34-Type Proteins Bind NHERF-1 from Hematopoietic Progenitor Cell Extracts
As 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 [29] and expresses high endogenous levels of podocalyxin (unpublished results). Binding proteins from FDC-P1 lysates were affinity-purified using synthetic peptides corresponding to the cytoplasmic tails of CD34, podocalyxin, and endoglycan. Affinity-purified proteins were eluted and separated by SDS-PAGE, and resolved bands of interest were excised and subjected to ingel tryptic digestion followed by LC-MS/MS. Table 1Go shows the resulting mass spectrometry analyses of two excised bands shown in Figure 2AGo. Both bands (22 and 16 independent peptide analyses in two separate experiments; Fig. 2BGo) correspond to murine NHERF-1, confirming that NHERF-1 interacts specifically with both podocalyxin and endoglycan but not CD34. Since NHERF-1 has been reported to undergo cell-cycle-dependent phosphorylation [36] and contains numerous potential phosphorylation sites, one possible difference between the higher and lower molecular weight isoforms that we detected is that they correspond to phosphorylated and nonphosphorylated forms of the protein. This notion is supported by mass spectrometry data that reveals a mass sequence consistent with phosphorylation of Ser275 (Table 1Go; Fig. 2BGo), previously reported in human fibroblasts [37] but not previously reported in mouse. Phosphorylation of this residue was confirmed by tandem MS/MS sequencing (Fig. 2CGo). 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.


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  		Table 1. Peptide sequences obtained by LC-MS/MS and matched to murine NHERF-1

 

Figure 2
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  		Figure 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 1Go. (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.

 
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. 2DGo) [28, 32]

NHERF-1 Associates with Podocalyxin In Vivo
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 3Go, 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. 4Go). 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. 4AGo, 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 30–60 minutes.


Figure 3
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  		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.

 

Figure 4
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  		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.

 
PKC phosphorylation of NHERF-1 has been shown to regulate its oligomerization [37], and PKC activation is one of the pathways downstream of IL-3R signaling. We therefore tested whether cytokine-dependent relocalization of podocalyxin could be mimicked by PMA-induced activation of PKC pathways in FDC-P1 cells [38]. 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. 4BGo). 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. 4CGo). No NHERF-1 was co-precipitated from the podocalyxin-negative murine myeloid cell line, FD5.

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.

NHERF-1 Is Expressed by Mature Hematopoietic Cells and by Cells with an HSC Phenotype
Although the expression of NHERF-1 by kidney cells and epithelial cells has been described previously [39], its hematopoietic distribution has never been examined. We therefore performed a detailed flow cytometric survey of hematopoietic tissues for NHERF-1 expression by staining permeabilized cells (Fig. 5Go). The MDA-231 cell line, which lacks significant levels of NHERF-1 [39, 40], served as a negative control (not shown). NHERF-1 was broadly expressed by essentially all cells in hematopoietic tissues (bone marrow, thymus, spleen, and peripheral lymph nodes), and the highest levels were observed in T cell precursors in the thymus (Fig. 5AGo). Two peaks of expression were found in spleen and mesenteric lymph nodes, one bright and corresponding to the frequency of T cells in these tissues (30% and 50%, respectively) and one with lower intensity corresponding to the frequency of B lineage cells. Consistent with the flow cytometric analyses, immunoblotting revealed NHERF-1 expression by all hematopoietic tissues and the highest levels in thymocyte lysates (Fig. 5BGo). Bone marrow, which showed the lowest levels of NHERF-1 expression by flow cytometry, was also found to have the lowest expression levels by immunoblot; to obtain near-equivalent detection of NHERF-1, approximately 13-fold more bone marrow protein extracts had to be loaded per lane (Fig. 5BGo). Again, specificity of the antibody reactivity for NHERF-1 was confirmed by blotting extracts from kidney and MDA-231 breast cancer cells, which express or lack NHERF-1, respectively [39, 40].


Figure 5
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  		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.

 
We 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 5CGo).

Previously, it has been reported that podocalyxin is expressed by human cells with an HSC phenotype [9], and we have shown that murine podocalyxin is expressed by the c-kit+/lineage marker– /Sca-1+ (KLS) fraction of cells in bone marrow and that these cells can reconstitute all hematopoietic lineage cells in lethally irradiated recipients. To confirm that NHERF-1 is co-expressed by HSCs, we performed multicolor flow cytometric analyses to identify this rare KLS population and look for co-expression of NHERF-1. NHERF-1 was expressed by all cells bearing Sca-1 and c-kit on their surface and lacking expression of lineage-restricted markers (Fig. 6Go). Since these cells have previously been shown to contain all HSC activity [41] and to express podocalyxin, we conclude that NHERF-1 is a bone fide ligand for podocalyxin in HSCs.


Figure 6
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  		Figure 6. NHERF-1 distribution on Lin–, 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– (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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Until 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.

Function of CD34-Type Molecules
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 [42, 43]. The recent discovery of two additional CD34-related proteins (podocalyxin and endoglycan) suggests that the lack of defects in these mice may be due, in part, to functional redundancy with these new family members [1, 2, 7, 8]. A corollary to this hypothesis is that the most profound defects in mice lacking these molecules would be in tissues where they are expressed singly and lack the capacity for functional compensation. With this concept in mind, we (and others) have characterized defects in cell types where these molecules are aberrantly expressed or in mice where the encoding genes have been disrupted. Consistently, we have found that these molecules play an important role in blocking cell adhesion and cell-cell contact. For example, we have shown that mast cells lacking CD34 show an increased propensity to aggregate and adhere in vitro and that they exhibit impaired homing and migration in vivo [16]. This enhanced adhesion is reversible by the ectopic re-expression of CD34 and, intriguingly, is more potently reversed by the expression of the naturally occurring short form of CD34 lacking most of the cytoplasmic domain. Likewise, we have found that in mice lacking podocalyxin on their kidney podocytes (where it is normally abundantly expressed), there is a striking increase in cell-cell adherens and tight junctions, which leads to a block in urine production and perinatal death [2]. Finally, it has been shown that ectopic expression of podocalyxin in epithelial cells leads to decreased cell-cell adhesion and that up-regulation in tumors correlates with metastatic behavior [13, 14, 44]. In summary, these data suggest that the principle function of CD34-type molecules is to block cell adhesion and increase invasiveness.

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 [16, 45].

CD34-Family Ligands
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 [25]. Although its role in CD34 function has not been resolved, CrkL has been implicated in linking a number of extracellular signaling pathways to cytoskeletal rearrangements, cell migration, and differentiation. Thus, it is one likely candidate for regulating the localization of CD34 during adhesion. Similarly, two groups have identified the PDZ and ERM domain-containing protein, NHERF-2, as a cytoplasmic ligand for podocalyxin in kidney podocytes [28, 32] (reviewed in Weinman [46]). Moreover, it has been shown that NHERF-2 and podocalyxin colocalize with ezrin and actin in an apical domain of kidney podocytes and that loss of this complex correlates with the pathological loss of foot processes in disease models [47]. It is unlikely, however, that NHERF-2 is a hematopoietic ligand of CD34-type proteins in HSCs and vascular endothelia since its expression is relatively restricted to podocytes and other rare cell types in nonhematopoietic tissues [39]. 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).

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 [32, 47]. In addition, there is reason to believe that there may be differences in the function of NHERF-1 and NHERF-2. Although they share an overall sequence identity of 50% (supplemental online Fig. 1CGo), there are a number of significant differences between these proteins, including a much greater number of potential phosphorylation sites in NHERF-1. It has also been noted that in proximal tubules in the kidney (one of the few cell types in which these molecules are co-expressed) these molecules display differences in their subcellular localization, with NHERF-1 residing in the apical regions of microvilli and NHERF-2 more closely associated with the vesicle-rich domain at the base of microvilli [39]. Thus, there may be functional heterogeneity within the NHERF family.

It 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.

Functional Significance of CD34 Family Proteins and NHERF-1
Although 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 [48]. NHERF-1 has been shown to bind to the C-terminus of a large number of cytosolic proteins and trans-membrane receptors via its two tandem PDZ domains, including ß2-adrenergic receptors (ß2-AR), cystic fibrosis transmembrane conductance regulator (CFTR), platelet-derived growth factor receptor (PDGF-R), purinergic receptor (P2Y), transient receptor potential-4 and -5 (Trp4, Trp5), and phospholipase-C-ß isoforms. These are then linked to the cytoskeleton by virtue of the ability of NHERF-1 to oligomerize and to bind members of the ezrin/radixin/moesin family of cytoskeletal proteins [48]. With regard to the CD34 family, we speculate that their ability to block hematopoietic adhesion may be intimately associated with their degree of clustering in the plasma membrane by NHERF-like proteins. Previously, we noted that podocalyxin is selectively upregulated in a subset of human breast carcinomas and that its expression strongly correlates with poor patient outcome in vivo and with a loss of tumor cell polarity in vitro [14]. Strikingly, the cells with the greatest loss of polarity and highest metastatic behavior also show a loss of NHERF-1 expression [40, 49] (C. D. Roskelley, D. G. Huntsman, K. M. McNagny, unpublished results). Taken together, these data would suggest that upregulation of podocalyxin and loss of NHERF-1 are required for a dominant loss of cell contact/adhesion and that the ability to interact with NHERF-1 affords cells the ability to clear these antiadhesins from pro-adhesive molecules and establish apical and basolateral domains. This is further supported by a recent report showing that podocalyxin is involved in establishing the apical (nonadhesive) domain on epithelial cells and that this is critically dependent on the C-terminal PDZ-docking site and correlates with a co-localization of NHERF-2 [50]. 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.

Previously, 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 [16, 45]. By analogy with their documented role in epithelial cell polarization [50], we propose that in hematopoietic cells, NHERF-1 serves as a potent regulator of this phenomenon by actively redistributing with podocalyxin to nonadhesive domains, thereby permitting cell adhesion. This is consistent with our observation that in a subset of FDC-P1 cells, podocalyxin and NHERF-1 show the most dramatic colocalization on cells exhibiting a polarized cap of podocalyxin and that this colocalization is enhanced with IL-3 stimulation. This active redistribution would permit rapid changes in the adhesive properties of cells in the absence of a need for de novo protein synthesis. In this light, it is noteworthy that in a recent survey of gene allelic variants that most closely correlated with HSC turnover, the IL-3R locus was found to be one of the four most tightly linked quantitative trait loci associated with this behavior [51]. IL-3R-mediated signaling through various pathways (e.g., PI3K/Akt, Jak/STAT, Ras/MAPK, PKC) (reviewed by Rane et al. [52] and Brose et al. [53]) plays a role in survival of hematopoietic progenitor cells (HPCs) and stimulates their proliferation and differentiation [54] (reviewed by Ivanovic [55]). It has also been shown that IL-3 signaling enhances adhesive interactions between HPCs and bone marrow stromal cells [56]. On the other hand, PMA stimulation is generally (but not completely) restricted to PKC signaling pathways. On its own, PMA is not able to maintain cell proliferation of FDC-P1 cells and therefore only activates a small fraction of the pathways stimulated by IL-3 [57]. 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.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

DISCLOSURES
The authors indicate no potential conflicts of interest.


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 Materials and Methods
 Results
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 References
 

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