Anchoring at an island to relieve stress

In this issue of Blood, Ulyanova and colleagues present a systematic analysis of the role of β₁ integrins in erythropoiesis and demonstrate that α₅β₁ integrin is a critical requirement for mounting successful stress erythropoiesis.¹ 

Stress erythropoiesis is increasingly recognized as more than simply an expansion of steady-state erythropoiesis. Rather, it is a process arising from unique progenitor cells with distinct quantitative and qualitative cytokine requirements. Increased erythropoietin (Epo) levels resulting from kidney hypoxia due to anemia, initiate the response of stress erythropoiesis. A progenitor, named stress burst-forming unit-erythroid (BFU-E) and found in the spleen but not in bone marrow of mice subjected to erythropoietic stress, has been identified as able to produce erythroid colonies when exposed to high Epo concentrations with no additional in vitro requirement for stem cell factor (SCF) or interleukin-3, in contrast to bone marrow BFU-E.²  Stress BFU-E are stimulated to expand by bone morphogenetic protein 4 (BMP4) produced in the spleen in response to hypoxia. Erythropoietic stress leads to mobilization of bone marrow progenitors which home to the spleen and differentiate into stress BFU-E, under the influence of BMP4 and Hedgehog signaling.³  Nevertheless, homeostatic erythropoietic pathways like SCF–c-kit and glucocorticoid receptor–mediated signaling are also vital for successful stress erythropoietic response in vivo.^4,5  Stress erythropoiesis has been shown to be impaired in gene-targeted mouse models where steady-state erythropoiesis is fairly normal, like in mice with deficiency of growth arrest–specific gene 6 (Gas6)⁶  or conditional deletion of focal adhesion kinase (FAK).⁷  On the other hand, stress erythropoiesis in the splenic microenvironment can be successful in cases where homeostatic erythropoiesis in the bone marrow is pathologic, like in deficiency of Rac1 and Rac2 GTPases.⁸ 

The interactions of erythroid progenitors and precursors with macrophages and extracellular matrix components such as fibronectin and laminin within erythroblastic islands are critical for optimal proliferation, survival, differentiation, and terminal maturation into red blood cells.⁹  α₄β₁ and α₅β₁ integrins are the main erythroid cell receptors that interact with fibronectin. Whereas α₄β₁ mediates adhesion to several sites on fibronectin, α₅β₁ binds predominantly to the Arg-Gly-Asp (RGD) domain. α₅ expression is noted to be higher in early erythroblasts, whereas α₄ is widely expressed throughout nucleated erythroid cells. VCAM-1 represents the major and preferred ligand for α₄β₁ in stroma cells. The role of these integrins and their ligands in erythropoiesis has been extensively studied by in vitro and in vivo assays, frequently with contradictory results. In this issue, Ulyanova and colleagues offer a resolution to these conflicts, using a systematic in vivo genetic approach to analyze the erythropoietic phenotype after conditional deletion of β₁, α₄, or VCAM-1 in hematopoietic tissues of adult mice.¹  β₁ deletion produces β₁^Δ/Δ mice which demonstrate deficiency of α₅β₁ and compensatory overexpression of α₄β₇ in erythroid cells. α₄^Δ/Δ mice are deficient in α₄β₁ and α₄β₇ (but not α₅β₁) in hematopoietic cells. β₁^Δ/Δ, α₄^Δ/Δ, and VCAM-1^Δ/Δ mice have normal homeostatic erythropoiesis. Ulyanova and colleagues proceed to examine their stress erythropoiesis response to phenylhydrazine (PHZ). The survival of β₁^Δ/Δ mice is severely compromised due to their inability to mount successful life-saving splenic stress erythropoiesis, despite having a robust response to PHZ in the bone marrow regarding progenitor cell expansion and mobilization to peripheral blood. BFU-E, stress BFU-E, and CFU-E are all drastically diminished in β₁^Δ/Δ spleen, associated with increased apoptosis in splenic erythroblasts. To determine whether the phenotype observed is due to β₁ integrin deficiency in erythroid or stroma cells, Ulyanova and colleagues used irradiated wild-type recipient mice transplanted with β₁^Δ/Δ donor cells. These mice demonstrate similar although slightly improved phenotypic response, indicating that the great majority of the effects seen in β₁^Δ/Δ animals are predominantly hematopoietic cell autonomous.

An additional ingenious contribution of this study is the assay used to dissociate homing/lodgment in spleen from subsequent expansion/engraftment. Wild-type mice were injected with control or β₁^Δ/Δ bone marrow cells. Twenty-four hours later, recipients were killed and spleen cells were plated in methylcellulose colony assays. Each colony produced was genotyped to determine the origin (host vs donor) and proportion of homed progenitors. There was no significant impairment of splenic homing of β₁^Δ/Δ cells. Engraftment was evaluated by CFU-S assessment, 11 days after transplantation of lethally irradiated wild-type recipients. Despite the normal homing, the development of CFU-S was significantly impaired in mice transplanted with β₁-deficient hematopoietic cells suggesting impairment in survival and/or expansion of homed progenitors. α₄-deficient mice demonstrate a sufficient, although delayed, mounting of stress erythropoiesis and α₄^Δ/Δ hematopoietic cells perform normally in the above assays, implicating that the absence of α₅β₁-mediated signaling within the splenic microenvironment is responsible for the decreased progenitor cell expansion and survival observed in the spleen of β₁-deficient animals. Both α₄^Δ/Δ and β₁^Δ/Δ mice have terminal maturation defects in both bone marrow and spleen, pointing to the important role of α₄β₁ in the late steps of erythroid differentiation.¹⁰  The authors propose that retention and subsequent expansion of erythroid cells in the spleen is mainly an RGD-dependent process mediated by the α₅β₁ integrin, in contrast to bone marrow, where other ligands in stroma cells or matrix components offer redundant signals to erythroid cells via receptors such as CD44, Lutheran glycoprotein (Lu), α₄β₁, and α₄β₇ integrins (see figure).

This study presents yet another unique requirement for stress erythropoiesis in contrast to homeostatic erythropoiesis, and raises several intriguing questions for future research. Is the structure of erythroblastic island different in extramedullary versus medullary erythropoiesis? Which are the signaling pathways triggered by β₁ integrins within the erythroid progenitors after interaction with ligands on the stroma cells and matrix components of the erythropoietic niche and what are the gene expression patterns they induce in combination or distinctly? Are the RGD-dependent interaction and α₅β₁-mediated signaling potential targets to decrease stress erythropoiesis in conditions where stress-induced extramedullary erythropoiesis may cause more harm than benefit, like in thalassemias? Answers to these questions would further our understanding of the molecular control of erythropoiesis in health and disease and direct these findings toward future clinical applications.