Biochemical and Biophysical Research Communications
Regulation of adult hematopoiesis by the a disintegrin and metalloproteinase 10 (ADAM10)
Introduction
In recent studies emerging evidence for the crucial role of tightly controlled cell–cell communication between the bone marrow microenvironment and hematopoietic cells during hematopoiesis has been established. However the detailed molecular mechanisms regulating this process have remained elusive so far. A rapid and irreversible posttranslational modification of cell surface protein density can be achieved by an extracellular proteolytic cleavage step which is referred to as “ectodomain shedding” [1], [2], [3], [4]. ADAM10 is an ubiquitously expressed protein which is capable of cleaving numerous transmembrane proteins such as Notch receptors [5] that are implicated for proper regulation of hematopoietic development and homeostasis. In order to study the consequence of ADAM10 downregulation during adult hematopoiesis, we conditionally inactivated its expression using the inducible Mx-Cre strain [6] and a myeloid-specific deleter strain [7], respectively. The Mx promoter becomes activated by interferon response or synthetic double-stranded RNA in most cells of immunologically important tissues including bone marrow stroma cells [6]. A similarly induced knockout of the Notch1 receptor and the putative sole downstream signaling mediator Rbp-j, led to de novo ectopic B-cell development in the thymus from common lymphoid progenitors [8] thus underlining the importance of Notch receptor signaling for cell-fate decisions during lymphopoiesis.
In this study, similar to a recently published report [9], we show that the induced loss of ADAM10 leads to the development and progression of a MPD with concomitant loss of mature B- and T-lymphocytes in the bone marrow, spleen and peripheral blood. The expanded myeloid population mainly consists of CD11b+/Gr1+ cells with normal ADAM10 expression levels. Additionally bone marrow transplantation (BMT) experiments strongly support a non-cell autonomous origin of the MPD which is possibly triggered and maintained by the excessive secretion of G-CSF, TIMP-1 and IL-16.
Section snippets
Generation of inducible conditional ADAM10 (cKO) mice
The ADAM10flox/flox mice were crossed with Mx-Cre mice [10], K5-tTA-Cre mice [11] or LysM-Cre mice [7]. To induce Mx-mediated deletion of ADAM10 during adult hematopoiesis, 6-week old ADAM10flox/flox/Mx-Cre+/− (ADAM10−/−) and ADAM10flox/flox (Ctr) mice were injected three times in two-day intervals with 250 μg polyinosinic–polycytidylic acid (pI–pC).
Western blot of tissue lysates
Tissues were uniformly lysed (150 mM NaCl, 2 mM EDTA, 0.8 mM EGTA, 10 mM Tris–HCl, pH 7.4 and Complete Protease Inhibitors (1:25, Roche)). ADAM10 was
Lack of ADAM10 expression and activity in the adult hematopoietic system
We induced the deletion of the protease in ADAM10flox/flox; Mx1Cre mice and sacrificed mice 2 weeks after the last injection of double stranded RNA (pI–pC). Genomic deletion was observed in bone marrow, spleen, liver and skin (Fig. 1A). Immunoblot analysis revealed a strong downregulation of ADAM10 protein expression in bone marrow and spleen as well as of the proform of the protease in liver (Fig. 1B). Despite significantly downregulated ADAM10 protein expression (Fig. 1CI) in bone marrow
Discussion and conclusion
Since the metalloproteinase ADAM10 is a well-known control point to modulate cell–cell contacts [13], [14] we aimed to analyze if this protease plays a cell autonomous or a non-cell autonomous role in the hematopoiesis of adult mice. With the generation of conditional ADAM10-deficient mice under the control of the interferon-inducible Mx-promoter we were able to address this question. The different time points chosen for our analysis resemble the chronic and acceleration phases of MPD patients.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft SFB877. M.D. is supported by the Netherlands Heart Foundation (Dr. E. Dekker post-doctoral fellow Grant 2007T034 and 2012T079).
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Present address: Heart Research Centre Göttingen, Universitätsmedizin Göttingen, Department of Cardiology and Pneumology, University Göttingen, Göttingen, Germany.