The E3 Ligase Itch Is a Regulator of Bone Marrow Mesenchymal Stem Cells and the Hematopoietic Stem Cell Niche

Abstract

Hematopoietic stem cells (HSCs) reside in a complex cellular microenvironment within the bone marrow. Bone marrow mesenchymal stromal/stem cells (BM-MSCs) have emerged as an important cellular component of this hematopoietic stem cell niche. BM-MSCs have been implicated in physiological hematopoiesis as well as in hematological malignancies. The intracellular regulatory network that governs BM-MSC homeostasis and their function within the HSC niche remains poorly understood. Posttranslational modification of proteins through ubiquitylation has been proven an integrative component in the regulation of quiescence, proliferation, self-renewal and differentiation of stem cells. We have previously demonstrated that the E3 ubiquitin ligase Itch is a negative regulator of HSC homeostasis and function (Rathinam et al., Nat.Immunol., 2011). Notably, patients suffering from a rare ITCH deficiency syndrome display morphologic abnormalities with dysmorphic features, suggesting a role for this E3 ligase within the skeletal tissue and mesenchymal lineage cells (Lohr et al., Am.J.Hum.Genet., 2010).In the present study, we identify Itch as a critical regulator of BM-MSCs. Itch-/- mice exhibit an at least two-fold reduction of MSCs (immunophenotypically identified as CD45-TER119-CD31-PDGFRα+Sca-1+CD90+) in the BM. Consistently, Colony Forming Unit-Fibroblasts (CFU-F) assays revealed a remarkable reduction of MSCs in the BM (p=0.0392). Moreover, in-vitro culture studies identified a hyperproliferative phenotype of Itch mutant MSCs. However, further studies revealed that Itch deficiency leads to reduced differentiation of MSCs into osteoblasts, both in-vivo and in-vitro, and therefore resulting in osteopenic bone phenotype. Reciprocal and Serial Bone marrow transplantation studies suggested an alteration of the niche for HSCs in Itch deficient BM. Interestingly, Itch-/- recipients exhibited a five-fold increase in their donor (wild-type) hematopoietic stem and progenitor cell compartment. Molecular and biochemical studies documented an increase of signals mediated by Notch in Itch deficient MSCs and co-localization of Itch and Notch-1 intracellular domain. Of note these findings gain importance, as Notch signaling has been previously implicated in the regulation of MSC differentiation into osteoblasts, as well as in the regulation of the HSC niche.In summary, our studies identified Itch as a positive regulator of BM-MSC homeostasis. Our results suggest that the Itch-Notch axis within the HSC niche may serve as a potential pharmaceutical target, especially in the setting of bone marrow transplantation in humans. Treatment of the transplant recipient with an Itch inhibitor may avoid graft failure or rescue poor hematopoietic recovery. DisclosuresSchünemann:German Academic Exchange Service: Other: scholarship; Boehringer Ingelheim Fonds: Other: travel grant.