Abstract
Hematopoietic stem cells reside in the bone marrow (BM) niches in the endosteum region, in close proximity to bone-forming osteoblasts and bone-resorbing osteoclasts (Ocl). Tightly regulated SDF-1/CXCR4 interactions retain stem and progenitor cells within their stromal microenvironment. G-CSF induced mobilization impairs this balance by inducing SDF-1 secretion, its proteolytic degradation, and CXCR4 upregulation.
Increased Ocl activity in the BM of G-CSF treated mice was documented, however, no active role for Ocl in mobilization was revealed. Ocl secrete the mobilizing chemokines IL-8 and SDF-1, the cytokine HGF, and the metalloprotease MMP-9, which participate in stress-induced mobilization. We hypothesized that in addition to their role in physiological bone remodeling, Ocl are also involved in stem cell mobilization.
Ten daily injections of either G-CSF, Pamidronate (Ocl apoptotic drug used to treat osteoporotic patients) or both, led to mobilization of murine progenitor cells, accompanied by Ocl activation in the endosteum region (assayed by TRAP staining for activated Ocl). Interestingly, Pamidronate treatment induced two waves of mobilization on days 5 and 10, correlating with increased levels of TRAP+ multinucleated Ocl in the endosteum and increase in SDF-1 and HGF mRNA in the BM. Stress-inducing conditions, which are also known to mediate bone remodeling, namely LPS stimulation (mimicking bacterial infection) and controlled bleeding (mimicking injury), also triggered progenitor mobilization and CXCR4 upregulation. Of note, LPS-induced mobilization was dependent on functional CXCR4 upregulation and MMP2/9 secretion, was accompanied by Ocl activation and reduction in BM SDF-1 levels. Treatment of primary murine osteoblast/ Ocl precursor cultures with G-CSF, SDF-1 and HGF, increased dose dependently the formation of TRAP+ multinucleated Ocl, suggesting that SDF-1 and HGF are important regulators of the cross talk between Ocl activation and stem cell mobilization. Mobilization was documented in mice receiving five daily injections of either SDF-1 (10ug), or HGF (1.5ug) together with increased levels of Ocl precursors in the blood and BM, as well as number and size of TRAP+ Ocl in the endosteum region. Unexpectedly, Pamidronate also activated SDF-1 expression in cultures of primary mouse osteoblasts, suggesting Ocl activation prior to apoptosis by this drug.
Finally, the effect of the major Ocl proteinase, cathepsin K (CTK) as a candidate regulator of stem cell mobilization was investigated. Unexpectedly, recombinant human CTK inactivated human SDF-1 and abolished its chemotactic activity in vitro. This process was completely abrogated by a broad range proteinase inhibitor. We identified N-terminal cleavage and a dose dependent degradation of SDF-1 by CTK. These results suggest that CTK produced and secreted by activated Ocl also participates in the impairment of the steady state homeostatic balance of SDF-1/CXCR4 interactions, inducing local degradation of SDF-1 in the endosteum, thus facilitating stem cell mobilization. Our findings indicate that molecules involved in stem and progenitor cell anchorage, migration, and mobilization are also involved in Ocl activation. In summary, our results add mechanistic insight to the osteoblast/Ocl endosteal interactions and molecular pathways that regulate stem cell mobilization, which is of relevance for pathological stem cell malignancies and clinical mobilization, particularly for patients with poor mobilization.
Heparanase regulates retention and proliferation of primitive Sca-1+/c-Kit+/Lin− cells via modulation of the bone marrow microenvironment
