Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation
© Congote et al; licensee BioMed Central Ltd. 2009
Received: 24 April 2009
Accepted: 23 October 2009
Published: 23 October 2009
C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells.
We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin.
CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.
Erythropoietin (EPO) is the most important cytokine involved in the production of red cells . In endothelial cells, EPO stimulates the synthesis of proteins involved in red cell formation, such as thrombospondins (TSPs) 1 and 4 [2, 3]. TSPs are very large extracellular matrix glycoproteins with multiple functions . The biological activity of the C-terminal, amphipathic peptide of TSP-4 (C21) has been described only recently  and therefore nothing is known about putative membrane receptors for the peptide on the surface of target cells. Our results on the search for potential C21 receptors can be summarized in three points: (1) CD44 was identified as a C21-binding membrane protein in lysates of skin fibroblasts, but we were not able to demonstrate a direct involvement of CD44 in the mitogenic action of C21. (2) Binding studies with a recombinant CD44 indicated that osteopontin (OPN) competed with C21 for CD44 binding, suggesting a possible function of C21 as OPN antagonist. (3) C21 and OPN had opposite effects on colony formation in cultures of primitive hematopoietic stem cells. Therefore, we speculate that C21 binding to CD44 could indirectly stimulate hematopoietic stem cell proliferation by preventing the inhibitory action of osteopontin (OPN).
Cell cultures of human skin fibroblasts, 293T kidney epithelial cells and Trichoplusia ni insect cells (TN) were done as previously described [5, 6]. Cord blood CD133+ precursor cells (Lonza) were cultured in methylcellulose medium (Methocult H4536, Stem Cell Technologies) supplemented with 3 U/ml EPO in the presence of 1 μM C21  or 0.5 μg/ml recombinant human osteopontin (OPN, carrier-free, R&D Systems). The cells (5000/ml) were plated on 12-well plates (Costar, 0.5 ml/well). The plasmid pENTR containing the cDNA coding for transcript 4 of human CD44, CD44v4 (Ultimate ORF Clone Collection, ID IOH53593, Invitrogen) was modified to introduce a stop codon at the beginning of the extracellular section of the transmembrane domain of CD44 with the "QuikChange®" Site directed mutagenesis kit (Stratagene). The modified cDNA was integrated into the destination vector pIB/V5-His-DEST with clonase (Invitrogen Gateway™ technology) for insect cell production and the complete CD44 sequence was integrated using the same method into the pLenti6/V5-DEST plasmid for lentiviral transduction in 293T cells.
C21-affinity chromatography, gel electrophoresis and MS analysis were done as previously described .
Soluble CD44 was isolated from three day supernatants of cultures from transfected TN cells cultured in serum-free Excel 405 medium (Sigma). 100 ml of ice-cold medium were mixed with 2 M Tris-HCl, pH 8 to a final Tris concentration of 0.1 M and stirred for 10 min. The precipitated proteins were separated by centrifugation at 10,000 × g for 20 min. The supernatant was mixed with 6 g SM-2 Adsorbent beads (Bio-Rad) and further stirred on ice for 20 min. The beads were trapped with 4 layers of cheese cloth and the remaining medium was mixed with 43 g ammonium sulfate and stirred for 1 h. The precipitated proteins were eliminated by centrifugation (20 min, 10,000 × g) and the supernatant was filtered through Durapore PVDF membranes (0.22 μm, Cat. SCGVU02RE, Millipore). The filter was cut in pieces, gently shook on a nutator at 8°C for 20 min, first with 5 ml 0.3% (w/v) Zwittergent 3-16 (Calbiochem) and then with 5 ml 1% (v/v) Triflouroacetic acid (TFA). The two extracts were mixed, warmed up to room temperature and applied to a Vydac semi-preparative C4 column (10 × 250 mm, Cat 214TP1010, Grace/Vydac). The column was washed with 30 ml 0.1%TFA and the extract components separated with a gradient of acetonitrile . The collected fractions were immediately lyophilized, dissolved in 60 μl of the storage buffer . The fractions containing CD44 were identified by applying 2 μl on nitrocellulose paper. The dot blots were developed, scanned and measured as previously indicated . We used a monoclonal anti-CD44 antibody recognizing all CD44 isoforms (R&D Systems). Digestion of CD44 with peptide:N-glycosidase F (PNGase F, New England Biolabs) was done following the instructions of the manufacturer. Binding of biotinylated C21 (Biotin-C21) to CD44 on nitrocellulose membranes was done as previously described for ROD1 . Binding competition studies were done with the same OPN preparation utilized for cell culture as indicated above and with human umbilical cord hyaluronic acid (potassium salt, Calbiochem).
Results and discussion
Identification and potential significance of CD44 as a C21 binding protein
Biotin-C21 binding to CD44 is inhibited by hyaluronic acid and osteopontin
If CD44 is not implicated in the action of C21, it is still possible that C21-CD44 interactions may have an effect in the function of the membrane receptor in its capacity as a cell-adhesion molecule. The best known ligand of CD44 is hyaluronic acid (HA, hyaluronan), a ubiquitous cell matrix component, which plays an important role in hematopoiesis . Of particular importance is the HA of the endosteal bone marrow niche, which plays a role in primitive hematopoietic stem cell proliferation and homing of transplanted cells to the marrow [14, 15]. OPN, another CD44 ligand, is a negative regulatory protein of the hematopoietic stem cell niche [16, 17].
Opposite effects of C21 and OPN on colony formation of CD133+ cord blood cells
In conclusion, C21 could act on target cells by multiple mechanisms, which may involve competition with OPN for CD44 binding sites, ROD1-dependent signal transduction pathways involving proliferation and, probably, ROD1- independent effects on apoptosis, as previously discussed in .
We thank Drs. Leonid Kriazhev and Marcos Di Falco from the McGill University and Genome Quebec Innovation Centre for in-gel digestion and MS analysis. This work was supported by the Canadian Blood Services/CIHR blood utilization and conservation initiative.
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