Involvement of urokinase receptor in the cross-talk between human hematopoietic stem cells and bone marrow microenvironment

Abstract

Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and can be induced to mobilize into the circulation for transplantation. Homing and lodgement into BM of transplanted HSCs are the first critical steps in their engraftment and involve multiple interactions between HSCs and the BM microenvironment.uPAR is a three domain receptor (DIDIIDIII) which binds urokinase, vitronectin, integrins. uPAR can be cleaved and shed from the cell surface generating full-length and cleaved soluble forms (suPAR and DIIDIII-suPAR). DIIDIII-suPAR can bind fMLF receptors through the SRSRY sequence (residues 88-92).We previously reported the involvement of soluble uPAR in HSC mobilization. We now investigate its possible role in HSC homing and engraftment.We show similar levels of circulating full-length suPAR in healthy donors and in acute myeloid leukemia (AML) patients before and after the pre-transplant conditioning regimen. By contrast, levels of circulating DIIDIII-suPAR in AML patients are higher as compared to controls and significantly decrease after the conditioning.We found that suPAR and uPAR84-95, a uPAR-derived peptide which mimics active DIIDIII-suPAR, induce a significant increase in Long Term Culture (LTC)-Initiating Cells (ICs) and in the release of clonogenic progenitors from LTCs of CD34+ HSCs. Further, suPAR increases adhesion and survival of CD34+ KG1 AML cells, whereas uPAR84-95 increases their proliferation.Thus, circulating DIIDIII-suPAR, strongly increased in HSC mobilization, is indeed down-regulated by pre-transplant conditioning, probably to favour HSC homing. BM full-length suPAR and DIIDIII-suPAR may be involved in HSC lodgement within the BM by contributing to a suitable microenvironment.

Keywords: bone marrow microenvironment; hematopoietic stem cells; leukemia; uPAR; urokinase receptor.

Figure 1. Circulating cleaved forms of suPAR are lowered by chemotherapy-based conditioning regimen

Circulating forms of soluble uPAR forms were measured in healthy donors (n=4) and in AML patients (n=5) before and after the pre-transplantation chemotherapy-based conditioning regimen, using a time-resolved fluorescence immunoassay [31]. This technique allows to quantitate full-length suPAR and its cleavage products, DIIDIII-suPAR and DI-suPAR. (*) p≤0.05, as determined by the Student's t test.

Figure 2. Bone marrow stroma cells express uPAR and its ligands

Bone marrow stroma cells from ten healthy donors were cultured in long-term stem cell medium and then lysed. 50 μg of cell lysate was analyzed by Western blot with a monoclonal antibody able to recognize both full-length and cleaved uPAR (first inset), a polyclonal antibody directed to the uPAR_84–95 region (second inset), and specific antibodies for uPA and vitronectin (VN) (Panel A). Bone marrow stroma cells from two further healthy donors were cultured at subconfluence and incubated with serum-free culture medium additioned with 1 mg/ml BSA for 3 days at 37°C, 5% CO₂. Then, TCA precipitated incubation media and 50 μg of cell lysates were analyzed by Western blot with the uPAR specific monoclonal antibody (Panel B).

Figure 3. Soluble uPAR forms increase the number of LTC-ICs and clonogenic progenitors in long-term cultures of PB-CD34⁺ cells

CD34⁺ cells purified from PB of four donors were plated on BM-derived stroma, allowed to adhere, and cultured for 5 weeks with or without suPAR, uPAR_84-95 peptide or its scrambled version. At the end of the fifth week, adherent cells were harvested and cultured in methylcellulose for LTC-ICs calculation (Panel A). Output of clonogenic progenitors in culture medium was assayed by replating non-adherent cells in methylcellulose for short term cultures; myeloid and erythroid colonies were counted as colony forming cells (CFCs) (Panel B). (*) p≤0.05, as determined by the Student's t test.

Figure 4. Full-length suPAR increases adhesion of KG1 cells to fibronectin

Panel A: KG1 cells were resuspended in 1mg/ml BSA-RPMI at a density of 1×10⁶ cells/ml, pre-incubated with or without suPAR (20 nM), uPAR_84-95 or scrambled peptide (10 nM) for 30′ at 37°C and plated on plastic-bound fibronectin for 16 h at 4°C. Attached cells were fixed with 3% PFA and stained with crystal violet; stain was eluted and its absorbance at 540 nm was measured by a spectrophotometer. (*) p≤0.05, as determined by the Student's t test. Panel B: suPAR-treated or untreated KG1 cells were fixed with PFA, incubated with antibodies against total or active β1 integrin or with nonimmune mouse Ig and, then, with FITC anti-mouse IgG. Cells were finally analyzed by flow cytometry using a FACScan (MFI: Mean Fluorescence Intensity). (*) p≤0.05, as determined by the Student's t test. Panel C: Cell surface antigens of suPAR-treated or untreated KG1 cells were biotinylated with EZ-link sulfo-NHS-LC-biotin. Cells were lysed; 50 μg of cell lysates were analyzed by Western blot with an antibody against total β1 integrin, 400 μg of cell lysates were incubated with an antibody against active β1 integrin or nonimmune mouse Ig. Immunocomplexes were then precipitated by protein A-Sepharose, eluted and analyzed by Western blot with horseradish peroxidase-conjugated streptavidin.

Figure 5. uPAR_84-95 increases KG1 cell proliferation

KG1 cells were serum-starved overnight, plated on plastic-bound fibronectin (FN) and cultured with or without full-length suPAR (20nM) (■), uPAR_84-95 peptide (10 nM) or it scrambled version (10 nM) (▲) for 0, 24, 48, 72 hours or 6 days (144h). At indicated times, 20μl/well of CellTiter 96 AQueous One Solution Reagent was added. After incubation for 4 h at 37°C, 5% CO₂, the absorbance was determined by an ELISA reader (Bio-Rad) at a wavelength of 490 nm. Fold increase represents the ratio between values obtained by treated cells and values obtained by the corresponding control at each time. (*) p≤0.05, as determined by the Student's t test, compared to time 0.

Figure 6. Full-length suPAR protects KG1 cells from apoptosis

KG1 cells were serum starved for indicated times, in the absence (Panel A) or in the presence of full-length suPAR or diluent (Panel B). Cells were then lysed and analyzed by Western blot with antibodies directed to full-length/cleaved Caspase3 or Parp1; not starved KG1 cells and KG1 cells treated with etoposide were used as negative and positive apoptosis control, respectively. The graphs show the O.D. obtained by densitometric scanning of cleaved caspase/Parp1 bands normalized to the O.D. of corresponding tubulin/actin bands.