Abstract
During cytokine- or chemotherapy-induced hematopoietic stem cell (HSC) mobilization, a highly proteolytic microenvironment can be observed in the bone marrow that has a strong influence on adhesive and chemotactic interactions of HSC with their niches. The increase of proteases during mobilization goes along with a decrease of endogenous protease inhibitors. Prominent members of the proteases involved in HSC mobilization belong to the families of matrix metalloproteinases and cathepsins, which are able to degrade chemokines/cytokines, extracellular matrix components, and membrane-bound adhesion receptors. To determine the functional activity of different proteolytic enzymes, zymographic analyses with different substrates and pH conditions can be employed. An involvement of cysteine cathepsins can be determined by the “active site labeling” technique using a modified inhibitor irreversibly binding to the active center of the enzymes. Intact or degraded chemokines and cytokines, which fall into the range between 1000 and 20,000 Da, can readily be detected by MALDI-TOF analysis. These three methods can help to detect proteolytic activities directly involved in the mobilization process.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells. Nature 505:327–334
Crane GM, Jeffery E, Morrison SJ (2017) Adult haematopoietic stem cell niches. Nat Rev Immunol 17(9):573–590
Wei Q, Frenette PS (2018) Niches for hematopoietic stem cells and their progeny. Immunity 48(4):632–648
Gattazzo F, Urciuolo A, Bonaldo P (2014) Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta 1840(8):2506–2519
Klamer S, Voermans C (2014) The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment. Cell Adhes Migr 8(6):563–577
Greenbaum AM, Link DC (2011) Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization. Leukemia 25(2):211–217
Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS (1995) The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 92(21):9647–9651
Greenbaum A, Hsu YM, Day RB, Schuettpelz LG, Christopher MJ, Borgerding JN, Nagasawa T, Link DC (2013) CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495(7440):227–230
Levesque J-P, Hendy J, Takamatsu Y, Williams B, Winkler IG, Simmons PJ (2002) Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment. Exp Hematol 30(5):440–449
Marquez-Curtis L, Jalili A, Deiteren K, Shirvaikar N, Lambeir AM, Janowska-Wieczorek A (2008) Carboxypeptidase M expressed by human bone marrow cells cleaves the C-terminal lysine of stromal cell-derived factor-1alpha: another player in hematopoietic stem/progenitor cell mobilization? Stem Cells 26:1211–1220
Levesque J-P, Takamatsu Y, Nilsson SK, Haylock DN, Simmons P (2001) Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood 98(5):1289–1297
Staudt ND, Maurer A, Spring B, Kalbacher H, Aicher WK, Klein G (2012) Processing of CXCL12 by different osteoblast-secreted cathepsins. Stem Cells Dev 21(11):1924–1935
Overall CM (2002) Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol 22(1):51–86
Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8(3):221–233
Janowska-Wieczorek A, Marquez LA, Dobrowsky A, Ratajczak MZ, Cabuhat ML (2000) Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines. Exp Hematol 28(11):1274–1285
Steinl C, Essl M, Schreiber TD, Geiger K, Prokop L, Stevanovic S, Pötz O, Abele H, Wessels JT, Aicher WK, Klein G (2013) Release of matrix metalloproteinase-8 during physiological trafficking and induced mobilization of human hematopoietic stem cells. Stem Cells Dev 22(9):1307–1318
Shirvaikar N, Marquez-Curtis LA, Shaw AR, Turner AR, Janowska-Wieczorek A (2010) MT1-MMP association with membrane lipid rafts facilitates G-CSF—induced hematopoietic stem/progenitor cell mobilization. Exp Hematol 38(9):823–835
Golan K, Vagima Y, Goichberg P, Gur-Cohen S, Lapidot T (2011) MT1-MMP and RECK: opposite and essential roles in hematopoietic stem and progenitor cell retention and migration. J Mol Med (Berl) 89(12):1167–1174
Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM, Pham C, Link D (2004) Characterization of hematopoietic progenitor mobilization in protease-deficient mice. Blood 104:65–72
Brix K, Dunkhorst A, Mayer K, Jordans S (2008) Cysteine cathepsins: cellular roadmap to different functions. Biochimie 90(2):194–207
Reiser J, Adair B, Reinheckel T (2010) Specialized roles for cysteine cathepsins in health and disease. J Clin Invest 120(10):3421–3431
Staudt ND, Aicher WK, Kalbacher H, Stevanovic S, Carmona AK, Bogyo M, Klein G (2010) Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts. Haematologica 95:1452–1460
Lapidot T, Petit I (2002) Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 30:973–981
Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220
Inanc S, Keles D, Oktay G (2017) An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13. BioTechniques 63(4):174–180
Yasumitsu H (2017) Serine protease zymography: low-cost, rapid, and highly sensitive RAMA casein zymography. Methods Mol Biol 1626:13–24
Fonović M, Bogyo M (2007) Activity based probes for proteases: applications to biomarker discovery, molecular imaging and drug screening. Curr Pharm Des 13(3):253–261
Cho YT, Su H, Wu WJ, Wu DC, Hou MF, Kuo CH, Shiea J (2015) Biomarker characterization by MALDI-TOF/MS. Adv Clin Chem 69:209–254
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Maurer, A., Klein, G., Staudt, N.D. (2019). Assessment of Proteolytic Activities in the Bone Marrow Microenvironment. In: Klein, G., Wuchter, P. (eds) Stem Cell Mobilization. Methods in Molecular Biology, vol 2017. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9574-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9574-5_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9573-8
Online ISBN: 978-1-4939-9574-5
eBook Packages: Springer Protocols