HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0725v1 26/5/1211    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Marquez-Curtis, L. Articles by Janowska-Wieczorek, A. Search for Related Content PubMed PubMed Citation Articles by Marquez-Curtis, L. Articles by Janowska-Wieczorek, A.

THE STEM CELL NICHE

Carboxypeptidase M Expressed by Human Bone Marrow Cells Cleaves the C-Terminal Lysine of Stromal Cell-Derived Factor-1: Another Player in Hematopoietic Stem/Progenitor Cell Mobilization?

^aResearch and Development, Canadian Blood Services, Edmonton, Alberta, Canada;
^bDepartment of Medicine, University of Alberta, Edmonton, Alberta, Canada;
^cLaboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium

Key Words. Carboxypeptidase M • Stromal cell-derived factor-1 • CXCR4 • Granulocyte-colony-stimulating factor • Mobilization • Bone marrow

Correspondence: Correspondence: Anna Janowska-Wieczorek, M.D., Ph.D., Department of Medicine, Faculty of Medicine and Dentistry, CBS Building, 8249–114th Street, Edmonton, Alberta T6G 2R8, Canada. Telephone: 780-431-8761; Fax: 780-702-8622; e-mail: anna.janowska{at}blood.ca

Received on August 30, 2007; accepted for publication on February 15, 2008.

First published online in STEM CELLS EXPRESS  February 21, 2008.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Carboxypeptidase M (CPM) is a membrane-bound zinc-dependent protease that cleaves C-terminal basic residues, such as arginine or lysine, from peptides/proteins. We examined whether CPM is expressed by hematopoietic and stromal cells and could degrade stromal cell-derived factor (SDF)-1, a potent chemoattractant for hematopoietic stem/progenitor cells (HSPC). We found that (a) CPM transcript is expressed by bone marrow (BM) and mobilized peripheral blood CD34⁺ cells, myeloid, erythroid, and megakaryocytic cell progenitors, mononuclear cells (MNC), polymorphonuclear cells (PMN), and stromal cells, including mesenchymal stem cells; and that (b) granulocyte-colony-stimulating factor (G-CSF) significantly increases its expression at the gene and protein levels in MNC and PMN. Moreover, we found that recombinant CPM cleaves full-length SDF-1 (1–68) rapidly, removing the C-terminal lysine and yielding des-lys SDF-1 (1–67). We demonstrated that such CPM treatment of SDF-1 reduced the in vitro chemotaxis of HSPC, which, however, was preserved when the CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. Thus, we present evidence that CPM is expressed by cells occurring in the BM microenvironment and that the mobilizing agent G-CSF strongly upregulates it in MNC and PMN. We suggest that cleavage of the C-terminal lysine residue of SDF-1 by CPM leads to attenuated chemotactic responses and could facilitate G-CSF-induced mobilization of HSPC from BM to peripheral blood.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Carboxypeptidase M (CPM) belongs to the family of zinc-dependent enzymes that catalyze the hydrolysis of the C-terminal peptide bond in peptides and proteins. They have been implicated in protein digestion, modulation of hormone activities, thrombosis, hemostasis, and inflammation. On the basis of structural arrangement, substrate specificity, and biological function, carboxypeptidases (CP) are broadly classified into a digestive/pancreatic subfamily (CPA1–CPA6, CPB, and CPU) and a regulator subfamily (CPE/H, CPN, CPD, CPZ, CPX1, CPX2, and CPM). CPM, CPE/H, CPN, CPD, CPZ, CPB, and CPU cleave C-terminal arginine or lysine, albeit with different specificities [1, 2]. Unique to CPM is its binding via a glycosylphosphatidylinositol (GPI) glycan anchor [3] to the plasma membranes of various cell types (lung endothelial cells, alveolar macrophages, ovarian follicles, trophoblasts, placental and kidney microvilli, blood vessels, and nerves), although a soluble form of CPM has also been demonstrated (seminal plasma, urine, and amniotic fluid) [4, 5]. The physiological role of CPM has not been clearly defined; however, its presence on macrophages and endothelial and epithelial cells [6–8] makes it a prime candidate for the regulation or processing of peptides generated during inflammation, such as complement anaphylatoxins (C3a, C4a, and C5a) and kinins (also described as substrates for CPN [9] and possibly CPU [10]). ProCPU and CPN are not membrane-bound and are produced in the liver and secreted into the bloodstream [11]. In this work we focused on CPM because this membrane-bound CP is ideally situated to elicit a more targeted activity at local tissue sites [12] than soluble CPN or CPU. CPM is known to cleave C-terminal lysine or arginine of peptides and proteins [4], and therefore we investigated whether the chemokine stromal cell-derived factor (SDF)-1 (CXCL12), which has lysine at the carboxy (C) terminus, is a substrate for CPM. In the bone marrow (BM) microenvironment, SDF-1 is produced mainly by endothelial cells, fibroblastic cells, and osteoblasts. It provides a potent retention signal for hematopoietic stem/progenitor cells (HSPC), mediated via the CXCR4 receptor expressed on these cells [13, 14]. Until recently, research focused on two human SDF-1 isoforms, SDF-1 (1–68) and SDF-1β (1–72), with SDF-1β having an additional four amino acid residues at the C terminus. Human SDF-1 is composed of three distinct structural regions: an N terminus, a central core region of three antiparallel β-sheets, and a C-terminal helix. Previous studies have demonstrated the essential role of the N terminus, especially the first nine residues, as the major site for direct interaction with the CXCR4 receptor and subsequent signal transduction [15]. However, the functional role of the C terminus has been much less defined and appreciated. When exposed to human serum, full-length SDF-1 (1–68) undergoes rapid processing at the C terminus to produce des-lys SDF-1 (1–67) [16, 17]. This C-terminal cleavage reduces the ability of SDF-1 to bind to heparin and cells and to stimulate chemotaxis [17]. The N-terminal cleavage of SDF-1 is effected by various enzymes such as matrix metalloproteinases (MMPs) [18], CD26/dipeptidylpeptidase (DPP) IV [19], and neutrophil serine proteases [20] or elastases [21], generating distinct truncated forms that are functionally inactive. These enzymes are thought to be released during granulocyte-colony-stimulating factor (G-CSF)-induced HSPC mobilization, leading to a highly proteolytic microenvironment in the BM [22, 23]. It has been postulated that disruption of SDF-1/CXCR4 signaling is a key step in the release of HSPC from the BM to peripheral blood (PB) during mobilization [24, 25]. Nevertheless, mice deficient in neutrophil proteases exhibit normal HSPC mobilization in response to G-CSF [26], and this may be partly explained by the existence of other proteases that have compensatory activities. In this work we investigated whether (a) CPM is expressed by various cells of the BM, (b) the mobilizing agent G-CSF upregulates it, and (c) CPM catalyzes the cleavage of SDF-1.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Cell Lines and Hematopoietic and Stromal Cells
The hematopoietic cell lines THP-1 (monocytic), KG-1, KG-1a, HL-60 (all myeloid), Jurkat, and Raji (both lymphoid) were obtained from the American Type Culture Collection (Rockville, MD, http://www.atcc.org) and grown in RPMI medium supplemented with 10% fetal calf serum (FCS). BM samples were collected from healthy, unrelated bone marrow donors, mobilized peripheral blood (mPB) samples from patients diagnosed with non-Hodgkin's lymphoma who had undergone G-CSF mobilization at the Cross Cancer Institute (Edmonton, AB, Canada), and cord blood (CB) samples from healthy, full-term newborn infants (with the mothers' informed consent), all in accordance with the guidelines approved by the University of Alberta Health Research Ethics Board. Light-density mononuclear cells (MNC) were separated by centrifugation using a 60% Percoll density gradient (1.077 g/ml; Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com). Polymorphonuclear cells (PMN) were obtained from whole blood after density gradient centrifugation using Lympholyte-poly (1.113 g/ml; CedarLane, Hornby, ON, Canada, http://www.cedarlanelabs.com) according to the manufacturer's instructions.

CD34⁺ cells were isolated from the light-density MNC interphase using the Miltenyi MACS system (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) according to the manufacturer's instructions and as we described previously [27]. For some experiments, CD34⁺ cells were ex vivo expanded in a serum-free liquid culture system, supplemented with the appropriate recombinant human (rh) cytokines and growth factors, into myeloid (colony forming unit-granulocyte/macrophage [CFU-GM[, using granulocyte-macrophage colony-stimulating factor and interleukin [IL]-3), erythroid (blast-forming unit-erythroid [BFU-E], using erythropoietin and stem cell factor), and megakaryocytic (colony forming unit-megakaryocyte [CFU-Meg], using thrombopoietin and IL-3) lineages, as we described previously [28]. Ex vivo expansion cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO₂ for up to 11 days. Cells were analyzed by flow cytometry, confocal microscopy, and gel-based reverse transcription (RT)-polymerase chain reaction (PCR) for CPM expression, as described below.

Mesenchymal stem cells (MSC) were established from BM or CB as we previously described [29], and colony-forming unit-fibroblast (CFU-F) cultures were from BM buffy coat cells, as we also described [30]. Human umbilical vein endothelial cells (HUVEC) were cultured on gelatin-coated flasks in medium consisting of M199, 10 mM L-glutamine, 250 IU/ml penicillin-streptomycin, 20% FCS (all from Invitrogen, Burlington, ON, Canada, http://www.invitrogen.com), and endothelial cell growth supplement (Collaborative Biomedical, Bedford, MA, http://www.bdbiosciences.com). Cells were grown almost to confluence, incubated for 24 hours in serum-free Iscove's modified Dulbecco's medium (IMDM) at 37°C in 5% CO₂, trypsinized, and analyzed by RT-PCR for CPM expression.

In some experiments, CD34⁺ cells, MNC, PMN, and CFU-F stromal cells were incubated in serum-free IMDM with or without 100 ng/ml rhG-CSF (Filgrastim; Amgen, Thousand Oaks, CA, http://www.amgen.com) for 24–48 hours at 37°C. After the incubation period, some cells were set aside for flow cytometry, and the remaining cell pellets were used for RNA isolation and RT-PCR or lysed and analyzed by Western blot for CPM expression.

RT-PCR Analysis of CPM Expression
RNA was extracted from cell pellets using TRIZOL (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), and RT-PCRs were carried out using the following primer sequences for human CPM [31] (Integrated DNA Technologies, Inc., San Diego, http://www.idtdna.com): CPM sense, 5'-GCCTGATGATGATGTTTTTC-3'; CPM antisense, 5'-TGGTGATGTGGGTTGAGTTT-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal mRNA control. For all samples, PCR was run for 10 cycles (heat denaturation step at 94°C for 45 seconds, primer annealing step at 55°C for 45 seconds, and primer extension at 72°C for 1 minute), after which GAPDH primers were added and PCR was continued for 30 more cycles. PCR products (306 base pairs [bp] for GAPDH and 715 bp for CPM) were electrophoresed through 2.0% agarose gels containing 0.1 µg/µl ethidium bromide, and gels were visualized under UV light and photographed using a Kodak DC120 digital camera (Kodak, Rochester, NY, http://www.kodak.com) or the FluorChem imaging system (Alpha Innotech, San Leandro, CA, http://www.alphainnotech.com). Densitometric analysis was carried out using the AlphaEase FCr image analysis software (Alpha Innotech).

Fluorescence-Activated Cell Sorting Analysis
Ex vivo-expanded hematopoietic precursors were evaluated by flow cytometry for expression of differentiation markers glycophorin A (BFU-E), CD33 (CFU-GM), and CD41 (CFU-Meg) using antibodies from Beckman Coulter (Jersey City, NJ, http://www.beckmancoulter.com) or BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml). Hematopoietic cell lines (THP-1, KG-1, KG-1a, HL-60, Jurkat, and Raji), BM and PB CD34⁺ cells, MNC, PMN, and hematopoietic precursor cells were stained with a 1:250 dilution of mouse monoclonal anti-CPM antibody (NCL-CPMm; Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk) for 45 minutes at 4°C followed by staining with a 1:200 dilution of secondary antibody goat anti-mouse IgG (Alexa Fluor-488, A11001; Invitrogen) for 30 minutes at 4°C. The percentage of stained cells was compared with that of a mouse IgG1 isotype-matched control (Dako, Mississauga, ON, Canada, http://www.dako.com) using the FACScan flow cytometer and analyzed with FCS Express v3 software (De Novo Software, Thornhill, ON, Canada, http://www.denovosoftware.com).

Confocal Analysis
Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton. The cells were then incubated with the 1:300 dilution CPM primary antibody (mouse anti-human CPM antibody, NCL-CPMm; Novocastra), washed, and stained with 1:300 dilution goat anti-mouse Alexa Fluor-488 (Invitrogen). Finally, they were mounted in ProLong antifade reagent (Invitrogen) and examined using an LSM510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Western Blot
Cell pellets were sonicated in lysis buffer (1% [vol/vol] Triton, 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.5 µg/ml pepstatin A, pH 7.3; all reagents from Sigma-Aldrich, Oakville, ON, Canada, http://www.sigmaaldrich.com), and the lysates were clarified by centrifugation at 14,000 rpm for 10 minutes at 4°C. Protein concentration was determined using the Bio-Rad protein assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA, http://www.bio-rad.com), and equal amounts of proteins were loaded alongside molecular weight markers (Precision Plus Protein Standards; Bio-Rad). Samples were resolved in a 10% polyacrylamide gel under reducing conditions and transferred to a polyvinylidene fluoride membrane. Following blockage with 5% fat-free dried milk (in Tris-buffered saline and 0.05% Tween 20), the membrane was probed with monoclonal mouse CPM antibody (NCL-CPMm; Novocastra) at 1:200 dilution and then with a 1:2,000 dilution of secondary antibody (Immunopure goat anti-mouse, peroxidase-conjugated IgG; Pierce, Rockford, IL, http://www.piercenet.com). Chemiluminescence detection was performed using the Supersignal West Pico Chemiluminescence system (Pierce), and densitometric analysis of the blots was carried out using the AlphaEase FCr image analysis software (Alpha Innotech).

Mass Spectrometric Analysis of CPM-Mediated Hydrolysis of SDF-1 (1–68)
The recombinant CPM used in this study was purified from the supernatant of Pichia pastoris yeast strain X33 expressing CPM, which was cloned using the pPICZ expression vector as previously described [32]. To study the kinetics of CPM-mediated hydrolysis, SDF-1 (1–68) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), in a final concentration of 5 µM, was mixed with CPM (final concentration, 15 nM) and incubated at 37°C in the presence of 100 mM HEPES at pH 7.4. At certain time intervals, aliquots were withdrawn and quenched with 1% solution of trifluoroacetic acid. C18 Zip Tips (Millipore, Billerica, MA, http://www.millipore.com) were used to desalt the samples. Elution was performed stepwise with 20 µl of 30% and 10 µl of 50% acetonitrile in 0.1% acetic acid. The mixture of this combined elution was introduced to an Esquire ESI Ion Trap mass spectrometer (Bruker, Bremen, Germany, http://www.bruker.com) using a syringe pump at 3 µl/minute. The instrument was used in a normal range (620–1,200 m/z) and resolution setting (1 mass unit at 3,000 m/z), optimized on an m/z value near the most abundant ion of the intact peptide. The spectra were deconvoluted, and the concentrations of the intact and truncated peptides were calculated from their relative abundance.

Chemotaxis Assay
Cells (1.5 x 10⁶ cells per milliliter of IMDM containing 0.1% bovine serum albumin [BSA]) were loaded in the upper compartments of Boyden chambers and incubated (at 37°C and 5% CO₂) for 3 hours. The compartments were separated by polycarbonate membrane filters (13-mm diameter, 8-µm pore size; VWR, Mississauga, ON, Canada, http://www.vwrcanlab.com). The lower compartments contained serum-free medium with or without 10, 20, or 200 ng/ml rh SDF-1 or 10 ng/ml rh SDF-1β (both from Peprotech), which had been treated or not with recombinant CPM. Preincubation of 5 µM SDF-1 with 20 nM CPM was carried out for 1 hour at 37°C. For some experiments CPM was preincubated with a 5 µM concentration of the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGETPA; EMD Biosciences, San Diego, http://www.emdbiosciences.com) for 10 minutes at 37°C prior to the chemotaxis assay. In another set of experiments, cells highly expressing CPM (THP-1 cells) were preincubated with 5 µM MERGETPA (for 30 minutes at 37°C prior to the assay) and allowed to migrate toward 3 ng/ml full-length SDF-1 (1–68) or synthetic des-lys SDF-1 (1–67) (Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada, http://www.brc.ubc.ca). Percentage of migration was calculated from the ratio of the number of cells recovered from the lower compartment to the total number of cells loaded. Each experiment was carried out at least three independent times in triplicate or quadruplicate. Arithmetic means and SDs were calculated, and statistical significance was defined as p .05 using the nonparametric Mann-Whitney test.

Competitive Binding Assay with the Anti-CXCR4 Antibody 12G5
To compare the binding efficacy to CXCR4 of SDF-1 (1–68) and CPM-treated SDF-1 (1–67), a competitive binding assay was used [33]. Briefly, cells highly expressing CXCR4 (Jurkat cells) were washed three times with cold buffer (phosphate-buffered saline, 0.1% BSA), and 4 x 10⁵ cells were incubated with 15 µl of anti-CXCR4-PE (clone 12G5; BD Pharmingen) and various concentrations (0, 20, 200, and 2,000 ng/ml) of SDF-1 (1–68) or CPM-treated SDF-1 (for 45 minutes at 4°C in the dark). For the CPM treatment, 5 µM SDF-1 (1–68) was incubated with 50 U/l of recombinant CPM for 1 hour at 37°C, under which conditions full-length SDF-1 is almost fully degraded to des-lys SDF-1, as described below. The stained cells were then washed three times and immediately analyzed by fluorescence-activated cell sorting (FACS). Mean fluorescence intensity of bound antibody (12G5) was plotted versus SDF-1 concentration.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
CPM Is Expressed in Hematopoietic Cell Lines
As surface CPM had earlier been shown to be expressed in B-lineage malignant primary cells and cell lines [34], we decided to investigate its expression at the gene level in other hematopoietic cell lines using semiquantitative gel-based RT-PCR. These cell lines showed variable CPM gene expression, with the monocytic THP-1 cell line expressing the highest, followed by the myeloid KG-1 cell line and the lymphoid Jurkat T-cell line. Expression of CPM transcript was absent in the lymphoid Raji and the myeloid HL-60 cell lines (Fig. 1A). Surface protein expression of CPM in these cell lines (as examined by flow cytometry and confocal microscopy; Fig. 1B) was consistent with their gene expression. Particularly in THP-1 cells, which strongly express CPM, confocal microscopy demonstrated its localization on the plasma membrane surface. Interestingly, KG-1a cells, which are morphologically, cytochemically, and functionally less mature than their parental KG-1 cells, expressed hardly any CPM on their surface (Fig. 1C).

View larger version (28K): [in this window] [in a new window]   Figure 1. CPM expression in hematopoietic cell lines. (A): Reverse transcription-polymerase chain reaction analysis of CPM transcripts (715 bp) in hematopoietic cell lines. GAPDH (306 bp) was used as the internal mRNA loading control. Images are representative of 2–4 independent experiments. (B): Surface protein expression of CPM. Flow cytometric analysis of CPM expression (upper row) is shown as a red line, and the solid blue area represents the isotypic control. Confocal microscopic analysis of hematopoietic cell lines (lower row) showing surface localization of CPM (original magnification, x40). (C): Flow cytometric analysis showing higher CPM surface expression in more mature KG-1 cells. Abbreviations: bp, base pairs; CPM, carboxypeptidase M; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (38K): [in this window] [in a new window]   Figure 2. CPM expression in primary hematopoietic and non-hematopoietic cells. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CPM in MNC from BM and mPB; CD34+ cells from mPB, BM, and CB; MSC established from CB MNC from passage 4 (early) or passage 13 (late); HUVEC; and BM-derived CFU-F. GAPDH was used as the internal mRNA loading control, and THP-1 was used as the positive control. Images are representative of 3–10 independent experiments except for BM MNC (n = 1) and BM CD34+ (n = 2). (B): RT-PCR analysis of CPM in ex vivo-expanded E, GM, and Meg from CB, mPB, and BM CD34+ cells at days 9 and 10 of expansion. Images are representative of three separate experiments except for BM CD34+ cells (n = 2). (C): Surface protein expression of CPM. Flow cytometric analysis and confocal micrograph showing surface localization of CPM (original magnification, x40) in BM CD34+ cells and in fibroblastic cells (CFU-F). (D): Flow cytometric analysis of CPM and lineage markers in ex vivo expanded cells. Erythroid (BFU-E), granulocyte-macrophage (CFU-GM), and megakaryocytic (CFU-Meg) progenitors were expanded from CB CD34+ cells, and CPM expression was measured on days 0, 6, and 11 of expansion. Lineage markers GPA (BFU-E), CD33 (CFU-GM), and CD41 (CFU-Meg) were measured on day 11 of expansion. CPM expression is shown as a red line, and the solid blue area represents isotypic control. Abbreviations: BFU-E, blast forming unit-erythroid; BM, bone marrow; CB, cord blood; CFU-F, colony-forming unit-fibroblasts; CPM, carboxypeptidase M; E, erythroblasts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM, granulocyte-macrophages; GPA, glycophorin A; HUVEC, human umbilical vein endothelial cells; Meg, megakaryocytes; MNC, mononuclear cells; mPB, mobilized peripheral blood; MSC, mesenchymal stem cells.

View larger version (42K): [in this window] [in a new window]   Figure 3. Effect of granulocyte-colony-stimulating factor (G-CSF) on gene and protein expression of CPM. Gene expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) using GAPDH as internal mRNA loading con, lysate protein expression was examined by Western blot, and surface protein expression was examined by flow cytometry. (A): MNC from mPB (n = 4) and CB samples (n = 3; shown here are two samples, CB1 and CB2) were incubated without (con) or with 100 ng/ml G-CSF (+G) for 24 hours at 37°C in a humidified atmosphere with 5% CO2. THP-1 was used as positive con in the Western blot, and equal amounts of lysate proteins were loaded as determined by Bio-Rad assay. (B): Similarly, PMN isolated from the whole blood of normal donors (n = 5) were incubated without (con) or with 100 ng/ml G-CSF (+G) for 24–48 hours at 37°C in a humidified atmosphere with 5% CO2, and the expression of CPM transcript was examined by RT-PCR. The numbers at the bottom of the gel indicate fold increase in expression relative to the con. Surface expression of CPM in PMN is shown by flow cytometry. The red line represents con cells, the blue line represents cells stimulated with G-CSF, and the gray area represents isotypic con. (C): RT-PCR analysis of CPM expression in CD34+ cells from BM (n = 2), mPB (n = 7), and CB (n = 2) and BM-derived CFU-F (n = 6) incubated without (con) or with 100 ng/ml G-CSF (+G) for 24 hours at 37°C. The numbers at the bottom indicate fold increase in expression relative to the con. For fluorescence-activated cell sorting analysis, mPB CD34+ cells (n = 3) were labeled after incubation with or without 100 ng/ml G-CSF. The red line in the histograms corresponds to con cells, the blue line to cells treated with G-CSF, and the shaded gray area to the isotype con. (D): CPM expression was also analyzed in the granulocyte population of two representative leukapheresis products (G-CSF-mobilized patients; n = 4). The red line corresponds to CPM expression and the shaded blue area to the isotype con. Abbreviations: BM, bone marrow; CB, cord blood; CFU-F, colony-forming unit-fibroblasts; con, control; CPM, carboxypeptidase M; +G, with 100 ng/ml granulocyte-colony-stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MNC, mononuclear cells; mPB, mobilized peripheral blood; PMN, polymorphonuclear cells.

SDF-1 Is Cleaved by CPM

View larger version (22K): [in this window] [in a new window]   Figure 4. Cleavage of SDF-1 by CPM. (A): Mass spectra of intact (1–68) and des-lys SDF-1 (1–67). Left panel: Ion trap mass spectra of intact (bottom) and carboxypeptidase M (CPM)-cleaved SDF-1 (top) showing the m/z values of the +7 to +12 charged ions. Right panel: Overlay of the deconvoluted masses of the +1 charged species. The y-axis represents the intensity in arbitrary units. Apart from the expected mass, both spectra contain a 97-unit larger component (presumably due to a bound phosphate counter ion) that is also cleaved by CPM. The peaks with m/z 7,962 and 8,059 represent SDF-1 (1–68); 7,833 and 7,930 belong to SDF-1 (1–67), the difference in mass matching that of a lysine residue. The SDF-1 (1–67) spectrum was obtained after incubation of 5 µM SDF-1 (1–68) with 20 nM CPM for 1 hour at 37°C. Under these conditions SDF-1 (1–68) was completely converted, and apart from SDF-1 (1–67), no other degradation product was observed. (B): Decay of SDF-1 (1–68) after incubation with CPM. The data are shown for a CPM concentration of 15 nM. This figure shows a representative example of two separate experiments, each performed in duplicate, with slightly different enzyme concentrations. Abbreviations: min, minutes; SDF, stromal cell-derived factor.

Cleavage of SDF-1 by CPM Abrogates Chemotaxis

View larger version (20K): [in this window] [in a new window]   Figure 5. The effect of CPM on the chemotaxis of cells toward SDF-1. (A): CD34+ cells from BM (n = 1) and CB (n = 4) were allowed to cross bare filters toward media alone or toward SDF-1 (1–68) (200 ng/ml) pretreated or not with CPM. CB CD34+ cells were allowed to migrate toward SDF-1 (1–68) (200 ng/ml) treated or not with CPM that had been preincubated or not with MERGETPA. (B): Chemotaxis of Jurkat cells toward 10 ng/ml SDF-1 or SDF-1β, pretreated or not with CPM. (C): Effect of CP inhibitor (MERGETPA) on chemotaxis of CPM-expressing THP-1 cells. Cells were preincubated with MERGETPA, and chemotaxis toward full-length SDF-1 (1–68) and truncated SDF-1 (1–67) was evaluated. The percentage of migration was calculated from the ratio of the number of cells that crossed over into the lower compartments to the total number of cells loaded onto the top compartments of Boyden chambers at the start of the chemotaxis assay. p .05 indicates significant statistical differences in percentage of migration using the nonparametric Mann-Whitney test. Abbreviations: BM, bone marrow; CB, cord blood; CPM, carboxypeptidase M; MERGETPA, DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid; SDF, stromal cell-derived factor.

Furthermore, to examine whether inhibition of CPM on the surface of cells migrating toward SDF-1 alters their chemotactic response, we incubated THP-1 cells highly expressing CPM with MERGETPA. We found that cell migration toward SDF-1 (1–68) increased by a factor of 1.4 ± 0.1 (p = .05), whereas no effect on chemotaxis toward already cleaved SDF-1 (1–67) was observed (Fig. 5C). The effect on migration was more pronounced when nonsaturating concentrations of SDF-1 (1–68) were used (data not shown).

CPM-Cleaved SDF-1 Interacts Differently with CXCR4
In our attempt to understand why chemotactic response toward cleaved SDF-1 (1–67) is lower compared with full-length SDF-1 (1–68), we performed a competitive binding assay using phycoerythrin-conjugated anti-CXCR4 monoclonal antibody (MoAb) (12G5). It was previously shown that the 12G5 clone competes with SDF-1 in binding to its receptor CXCR4 [33]. For this set of experiments we used Jurkat cells, which highly express CXCR4, and exposed them to increasing doses of native full-length SDF-1 (1–68) and CPM-treated SDF-1 (1–67) at 4°C (to prevent internalization) and using a constant concentration of 12G5 MoAb. The amount of 12G5 bound to cell surface was measured by FACS analysis. Native full-length SDF-1 (control) and CPM-treated SDF-1 (1–67) were able to compete with 12G5, resulting in reduced binding of 12G5 to the cell surface in a dose-dependent manner (Fig. 6A). However, the binding of 12G5 to the cell surface was significantly higher with CPM-treated SDF-1 (Fig. 6B), suggesting that truncated SDF-1 (1–67) did not compete as efficiently for CXCR4. This may provide some explanation of the reduced chemotactic response shown in Figure 5A.

View larger version (21K): [in this window] [in a new window]   Figure 6. Binding of full-length and CPM-treated SDF-1 with CXCR4. (A): Flow cytometric analysis of CXCR4 expression on Jurkat cells exposed to decreasing concentrations of SDF-1, namely, 2,000 (yellow line), 200 (purple line), 20 (blue line), and 0 ng/ml (red line), before and after CPM treatment. The shaded black area represents isotype control. (B): Plot of mean fluorescence intensity of 12G5 binding versus concentration of SDF-1. Statistical analysis was carried out on the basis of data from three independent experiments; *, p .05. Abbreviations: CPM, carboxypeptidase M; SDF, stromal cell-derived factor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

As CPM was first identified and named for its "membrane-bound" characteristic [36], we also show here the cell surface localization of this enzyme. In view of its membrane distribution and subcellular localization, CPM may be ideally situated to modulate the activity and potentially participate in the degradation of extracellular proteins and peptides in the BM microenvironment. One of the most abundant peptides in the BM is the chemokine SDF-1; in fact, BM stromal cells constitutively produce approximately 0.8 µg/ml (0.1 µM) SDF-1, as measured from cultured cells [14]. Endogenous SDF-1 provides a potent retention signal for HSPC, and it has been postulated that degradation of SDF-1 in the BM facilitates the mobilization of these cells to the PB [22–25]. The isoform of SDF-1 possesses a lysine C-terminal residue, which makes it a potential substrate for basic carboxypeptidases. Here we show for the first time that CPM cleaves the C-terminal lysine of SDF-1. Recently, another carboxypeptidase, CPN, identified in human plasma and serum, was also reported to specifically remove the C-terminal lysine of SDF-1 [16, 17].

We found that cleavage of its C-terminal lysine resulted in a significant decrease in the chemoattractant activity of SDF-1 (1–68). The role of the C terminus in chemotaxis has been investigated in a recent study demonstrating that a mutated form of SDF-1 with a defective C-terminal -helix (but a normal N terminus) failed to activate cellular signaling and chemotaxis even though the binding affinity was retained [37]. In addition to SDF-1 and SDF-1β, four other human SDF-1 isoforms have recently been identified, all of which stimulated cell migration to various degrees and whose amino acid sequences differed from that of SDF-1 only by a few amino acids in the C terminus [38]. Taken together, these results suggest that the C terminus of SDF-1 plays an important role in eliciting a chemotactic response.

The presence of CPM in the BM microenvironment takes on greater significance with our finding that the mobilization agent G-CSF strongly upregulates CPM in MNC and PMN. Several other proteases (e.g., elastase, cathepsin G, and MMP-9) are known to be released in large amounts in the BM, particularly by neutrophils, during mobilization induced by G-CSF [22, 23]. Recently, the serine protease DPPIV (CD26) was also shown to be induced during mobilization of murine HSPC [39]. Furthermore, short-term in vitro G-CSF treatment upregulated CD26, resulting in downregulation of the functional ability of CB CD34⁺ HSPC to respond to an SDF-1 gradient [40].

We suggest that CPM present on the cell surface of hematopoietic and stromal cells may participate in a more subtle way to alter the SDF-1 gradient that keeps the HSPC within the BM niches. This idea is strengthened by our observations that inhibition of carboxypeptidase activity on cells expressing CPM (THP-1 cells) enhanced cell migration toward SDF-1 (1–68). As it has been suggested that the C-terminal lysine of SDF-1 contributes to its binding with heparin on the cell surface, preserving the activity of SDF-1 [17], we propose that CPM cleavage of lysine could facilitate the release of SDF-1 from the cell surface and its further degradation by various proteases. Because SDF-1 is constitutively expressed by BM stromal cells, its controlled degradation becomes even more important in regulation of its biological functions.

To explain the abrogated chemotactic response of des-lys SDF-1, we investigated whether there is a difference in the nature of the interaction of full-length (1–68) and CPM-treated SDF-1 (1–67) with CXCR4. Binding of SDF-1 to CXCR4 involves several parts of the receptor: the N-terminal region and the extracellular loops (ECL) 2 and 3. The N-terminal domain and ECL3 are determinants for ligand binding, whereas ECL2 is considered important for receptor signaling. The MoAb 12G5 recognizes a conformational epitope of ECL2 that is present on CXCR4 and disappears when SDF1- binds [41, 42]. Therefore, our results suggest that following the initial binding, there is an effect of the C-terminal lysine on conformational changes related to receptor triggering that is reflected in the chemotaxis results.

	SUMMARY

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
We show in this work that (a) CPM is widely expressed by cells of the BM microenvironment, (b) G-CSF strongly upregulates its expression in MNC and PMN, and (c) CPM cleaves the C-terminal lysine of SDF-1, which abrogates the chemotactic response. Thus, CPM may participate with other proteases in modulating the SDF-1/CXCR4 axis and may therefore be implicated in HSPC mobilization.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
This work was supported by a Canadian Blood Services-CIHR Blood Utilization and Conservation Initiative grant (to A.J.-W.). It was also supported by the InterUniversity Attraction Poles Program-Belgian Science Policy and the Fund for Scientific Research of Flanders (FWO-Vlaanderen, Belgium). K.D. is a research assistant of FWO-Vlaanderen. We are grateful for helpful discussions with Dr. Paul Proost (Rega Institute, University of Leuven), for the technical assistance of Jencet Montaño and April Xu, and to Prof K. Augustyns (University of Antwerp, Antwerp, Belgium) for the use of the mass spectrometer. A.J. and K.D. contributed equally to this work.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 

1. Barrett AJ, Rawlings ND, Woessner JF, eds. Handbook of Proteolytic Enzymes. 2nd ed. London: Academic Press, 1998.

2. Reznik SE, Fricker LD. Carboxypeptidases from A to Z: Implications in embryonic development and Wnt binding. Cell Mol Life Sci 2001;58:1790–1804.[CrossRef][Medline]

3. Deddish PA, Skidgel RA, Kriho VB et al. Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. J Biol Chem 1990;265:15083–15089.[Abstract/Free Full Text]

4. Skidgel RA. In Hooper NM, ed. Zinc Metalloproteases in Health and Disease. Structure and function of mammalian zinc carboxypeptidases. London: Taylor & Francis Ltd, 1996:241-283.

5. Fujiwara H, Higuchi T, Sato Y et al. Regulation of human extravillous trophoblast function by membrane-bound peptidases. Biochim Biophys Acta 2005;1751:26–32.[Medline]

6. Krause SW, Rehli M, Andreesen R. Carboxypeptidase M as a marker of macrophage maturation. Immunol Rev 1998;161:119–127.[CrossRef][Medline]

7. Schremmer-Danninger E, Offner A, Siebeck M et al. B1 bradykinin receptors and carboxypeptidase M are both upregulated in the aorta of pigs after LPS infusion. Biochem Biophys Res Commun 1998;243:246–252.[CrossRef][Medline]

8. Forbes B, Wilson CG, Gumbleton M. Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: Implications for study of peptide absorption. Int J Pharm 1999;180:225–234.[CrossRef][Medline]

9. Bokisch V, Müller-Eberhard H. Anaphylatoxin inactivator of human plasma: Its isolation and characterization as a carboxypeptidase. J Clin Invest 1970;49:2427–2436.[Medline]

10. Myles T, Nishimura T, Yun TH et al. Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation. J Biol Chem 2003;278:51059–51067.[Abstract/Free Full Text]

11. Sato T, Miwa T, Akatsu H et al. Pro-carboxypeptidase R is an acute phase protein in the mouse, whereas carboxypeptidase N is not. J Immunol 2000;165:1053–1058.[Abstract/Free Full Text]

12. Reverter D, Maskos K, Tan F et al. Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity. J Mol Biol 2004;338:257–269.[CrossRef][Medline]

13. Aiuti A, Webb IJ, Bleul C et al. The chemokine SDF-1 is a chemoattractant for human CD34(+) hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34(+) progenitors to peripheral blood. J Exp Med 1997;185:111–120.[Abstract/Free Full Text]

14. Bleul CC, Fuhlbrigge RC, Casasnovas JM et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101–1109.[Abstract/Free Full Text]

15. Crump MP, Gong JH, Loetscher P et al. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J 1997;16:6996–7007.[CrossRef][Medline]

16. Davis DA, Singer KE, Sierra MD et al. Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1 alpha in the circulation. Blood 2005;105:4561–4568.[Abstract/Free Full Text]

17. De La Luz Sierra MD, Yang FQ, Narazaki M et al. Differential processing of stromal-derived factor-1 alpha and stromal-derived factor-1 beta explains functional diversity. Blood 2004;103:2452–2459.[Abstract/Free Full Text]

18. McQuibban GA, Butler GS, Gong JH et al. Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1. J Biol Chem 2001;276:43503–43508.[Abstract/Free Full Text]

19. Lambeir AM, Proost P, Durinx C et al. Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family. J Biol Chem 2001;276:29839–29845.[Abstract/Free Full Text]

20. Delgado MB, Clark-Lewis I, Loetscher P et al. Rapid inactivation of stromal cell-derived factor-1 by cathepsin G associated with lymphocytes. Eur J Immunol 2001;31:699–707.[CrossRef][Medline]

21. Valenzuela-Fernández A, Planchenault T, Baleux F et al. Leukocyte elastase negatively regulates stromal cell-derived factor-1 (SDF-1)/CXCR4 binding and functions by amino-terminal processing of SDF-1 and CXCR4. J Biol Chem 2002;277:15677–15689.[Abstract/Free Full Text]

22. Lévesque JP, Hendy J, Takamatsu Y et al. Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment. Exp Hematol 2002;30:440–449.[CrossRef][Medline]

23. Velders GA, Fibbe WE. Involvement of proteases in cytokine-induced hematopoietic stem cell mobilization. Ann NY Acad Sci 2005;1044:60–69.[CrossRef][Medline]

24. Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3:687–694.[CrossRef][Medline]

25. Winkler IG, Levesque JP. Mechanisms of hematopoietic stem cell mobilization: When innate immunity assails the cells that make blood and bone. Exp Hematol 2006;34:996–1009.[CrossRef][Medline]

26. Levesque JP, Liu F, Simmons PJ et al. Characterization of hematopoietic progenitor mobilization in protease-deficient mice. Blood 2004;104:65–72.[Abstract/Free Full Text]

27. Janowska-Wieczorek A, Marquez LA, Nabholtz JM et al. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood 1999;93:3379–3390.[Abstract/Free Full Text]

28. Majka M, Janowska-Wieczorek A, Ratajczak J et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner. Blood 2001;97:3075–3085.[Abstract/Free Full Text]

29. Son BR, Marquez-Curtis LA, Kucia M et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. STEM CELLS 2006;24:1254–1264.[Abstract/Free Full Text]

30. Mayani H, Guilbert LJ, Clark SC et al. Composition and functional integrity of the in vitro hematopoietic microenvironment in acute myelogenous leukemia: Effect of macrophage colony-stimulating factor. Exp Hematol 1992;20:1077–1084.[Medline]

31. Yoshioka S, Fujiwara H, Yamada S et al. Membrane-bound carboxypeptidase-M is expressed on human ovarian follicles and corpora lutea of menstrual cycle and early pregnancy. Mol Hum Reprod 1998;4:709–717.[Abstract/Free Full Text]

32. Deiteren K, Surpateanu G, Gilany K et al. The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over. Biochim Biophys Acta 2007;1774:267–277.[Medline]

33. Fedyk ER, Ryyan DH, Ritterman I et al. Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1). J Leukoc Biol 1999;66:667–673.[Abstract]

34. de Saint-Vis B, Cupillard L, Pandrau-Garcia D et al. Distribution of carboxypeptidase M on lymphoid and myeloid cells parallels the other zinc-dependent proteases CD10 and CD13. Blood 1995;86:1098–1105.[Abstract/Free Full Text]

35. Liu TM, Martina M, Hutmacher DW et al. Identification of common pathways mediating differentiation of bone marrow- and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. STEM CELLS 2007;25:750–760.[Abstract/Free Full Text]

36. Skidgel RA, Davis RM, Tan F. Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones. J Biol Chem 1989;264:2236–2241.[Abstract/Free Full Text]

37. Tan Y, Du J, Cai SX et al. Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal alpha-helix of SDF-1 alpha plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity. Exp Hematol 2006;34:1553–1562.[CrossRef][Medline]

38. Yu L, Cecil J, Peng SB et al. Identification and expression of novel isoforms of human stromal cell-derived factor 1. Gene 2006;374:174–179.[CrossRef][Medline]

39. Christopherson KW, Cooper S, Broxmeyer HE. Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells. Blood 2003;101:4680–4686.[Abstract/Free Full Text]

40. Christopherson KW, Uralil SE, Porecha NK et al. G-CSF- and GM-CSF-induced upregulation of CD26 peptidase downregulates the functional chemotactic response of CD34(+)CD38(–) human cord blood hematopoietic cells. Exp Hematol 2006;34:1060–1068.[CrossRef][Medline]

41. Zhou N, Luo Z, Luo J et al. Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 coreceptor by mutagenesis and molecular modeling studies. J Biol Chem 2001;276:42826–42833.[Abstract/Free Full Text]

42. Kucia M, Reca R, Miekus K et al. Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: Pivotal role of the SDF-1—CXCR4 axis. STEM CELLS 2005;23:879–894.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: 2007-0725v1 26/5/1211    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Marquez-Curtis, L. Articles by Janowska-Wieczorek, A. Search for Related Content PubMed PubMed Citation Articles by Marquez-Curtis, L. Articles by Janowska-Wieczorek, A.