Abstract
BackgroundHematopoietic stem cell (HSC) maintenance requires a specific microenvironment. HSC niches can be activated by tissue damaging chemotherapeutic drugs and various cell signaling molecules such as SDF-1 and FGF, which might also result in bone marrow stress. Recent research has insufficiently shown that endosteal osteolineage cells and other niche constituents recover after marrow injury.MethodsWe investigated the role of FGF2 in the osteoblastic niche cells during hematopoietic homeostasis recovery after bone marrow injury. Mice were treated with 5-fluorouracil (5FU) to eliminate actively cycling cells in the bone marrow. Primary osteoblasts were isolated and subjected to cell culture. Real-time PCR, western blot and immunohistochemical staining were performed to study niche-related genes, osteoblast markers, and FGF2 signaling. Proliferation rate were analyzed by marker gene Ki67 and colony formation assay. Also, osterix-positive osteoprogenitor cells were isolated by FACS from Osx-GFP-Cre mice after 5FU treatment, and subjected to RNA-sequencing and analyzed for Fgf receptors and niche markers.ResultsThe endosteal osteolineage cells isolated from 5FU-treated mice showed increased expression of the niche-related genes Sdf-1, Jagged-1, Scf, N-cad, Angpt1 and Vcam-1 and the osteoblast marker genes Osx, Opn, Runx2, and Alp, indicating that BM stress upon 5FU treatment activated the osteoblastic niche. Endosteal osteoblast expanded from a single layer to several layers 3 and 6 days after 5FU treatment. During the early recovery phase in 5FU-activated osteoblastic niches increased FGF2 expression and activated its downstream pERK. FGF2 treatment resulted in increased proliferation rate and the expression of niche marker genes in 5FU-activated osteoblastic niche cells. RNA-seq analysis in Osterix-positive osteoprogenitor cells isolated from 5FU-treated Osx-GFP mice showed significantly increased expression of Fgf receptors Fgfr1, 2 and 3. Although osteoblastic niche cells were damaged by 5FU treatment in the beginning, the increased number of OB layers in the recovery phase may be derived from resident osteoprogenitor cells by FGF2 activation under stress.ConclusionsTaken together, FGF2 signaling can regulate osteoblastic niche cells to support HSC homeostasis in response to bone marrow damage.
Highlights
Hematopoietic stem cell (HSC) maintenance requires a specific microenvironment
Fgfrs subtype expression in endosteal osteoblasts We have shown that fibroblast growth factor 2 (FGF2) supports osteoblastic niche cells, and increases HSC niche-related gene expression and the proliferation rate of OBs from 5FU-treated mice to allow for the recovery of bone marrow (BM) suppression
These conclusions are based on evidences that (1) HSC niche-related gene expression increased in osteoblastic niche cells isolated from 5FUtreated mice, (2) FGF2 increased proliferation in damaged osteoblastic niche cells, and (3) Fibroblast growth factors (FGFs) receptor signaling increased in osteoblastic niche cells, especially Osx + osteoprogenitor cells, following 5FU treatment
Summary
HSC niches can be activated by tissue damaging chemotherapeutic drugs and various cell signaling molecules such as SDF-1 and FGF, which might result in bone marrow stress. Hematopoietic stem cells (HSCs) are cells capable of differentiating into all blood cell lineages, and they are tightly regulated by the local niche to maintain homeostasis. HSC niches can be compromised by several different cell types, including endosteal osteolineage cells and vascular sinusoidal endothelial cells in the bone marrow. The majority of HSCs are quiescent but can be activated to proliferate and differentiate after suppression by infection, radiation, or chemotherapeutic drug treatment, such as cyclophosphamide and 5fluorouracil (5FU) [3,4,5,6]. Chemotherapeutic drug treatment for hematological diseases induce damage to the bone marrow (BM) microenvironment through multiple BM stress responses. Under BM stress, cycling cells are eliminated and surviving quiescent long-term hematopoietic stem cells (LT-HSCs) are activated to expand via a complex mechanism [7,8,9]
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