Molecular mechanisms involved in murine bone marrow erythropoietic response to acute anaemia by bleeding

Abstract

The underlying interactions among apoptosis, eiythroid proliferation and differentiation involved in bone marrow eiythropoietic response after an acute blood-loss have not yet been elucidated in detail. We hypothesized that Erythropoietin receptor, Bax, caspase-3, cytochrome c, Smac/DIABLO and Bcl-xL molecules play important roles at time of acute eiythropoietic need to ameliorate the hypoxic stress. Experiments were performed using in vivo murine model of anaemia induced by blood-loss in a time course study of 15 days. Haematological parameters and bone marrow cellularities were determined. Bone marrow apoptotic assays included: double fluorescent staining (acridine orange/ethidium bromide) and TUNEL. Bone marrow clonogenic assays were performed for evaluating eiythroid colony forming unif s expansión. The Eiythropoietin receptor, Bax, Bcl-xu Smac/DIABLO, caspase-3 and cytochrome c expressions were assessed by immunoblottings. Caspase-3 activity was determined with a colorimetric assay kit. Bleeding induces bone marrow apoptosis from 1 to 3 days, concomitant with Bax over-expression and Bcl-xL decrease. The mitochondrial dysfunction caused cytochrome c and Smac/DIABLO release to cytosol and caspase-3 activation. Eiythropoietic recoveiy was associated with Eiythropoietin receptor over expression from the third day, concomitant with the eiythroid progenitors and Bcl-xL/Bax ratio enhancements. Eiythropoiesis after bleeding depends on a delicate balance among proapoptotic (Bax, caspase-3, cytochrome c, Smac/DIABLO) and prosurvival proteins (Eiythropoietin receptor, Bcl-xL), as the crucial regulators in bone marrow eiythroid recoveiy. These findings provide new insights into the homeostatic mechanisms which promote eiythropoietic response post-bleeding.