TGF-β Signaling in Fetal Mesenchymal Progenitor Cells Plays an Essential Role in the Emergence of Bone Marrow Hematopoietic Niches

Abstract Hematopoietic stem/progenitor cells (HSPC) reside in a unique microenvironment within the bone marrow called the bone marrow hematopoietic niche. Mesenchymal stromal cells, including CXCL12-abundant reticular (CAR) cells, osteoblasts, arteriolar pericytes, and adipocytes are all important components of the niche. The development and maintenance of mesenchymal stromal cells in the bone marrow is not well characterized. A prior study suggested that these stromal cells are derived from two distinct types of mesenchymal stem/progenitor cells (MSPCs). Primitive MSPCs are present in fetal bone and are responsible for osteoblasts, CAR cells, and adipocytes through approximately 3 weeks after birth, and definitive MPSCs are present at birth and generate bone marrow mesenchymal stromal cells in adult mice. In this study, we abrogated transforming growth factor-b (TGF-β) signaling in MSPCs by deleting Tgfbr2in mesenchymal cells using a doxycycline-repressible Sp7(osterix)-Cre transgene (Osx-Cre).We previously reported that loss of TGF-βsignaling during fetal development results in a marked expansion of CAR cells and adipocytes in the bone marrow, while osteoblasts are significantly reduced. These stromal alterations are associated with significant defects in hematopoiesis, including a shift from lymphopoiesis to myelopoiesis. However, hematopoietic stem cell function is preserved. Interestingly, TGF-βsignaling is dispensable for the maintenance of mesenchymal cells in the bone marrow after birth under steady state conditions. These data show that TGF-βplays an essential role in the lineage specification of fetal but not definitive MSPCs and is required for the establishment of normal hematopoietic niches in fetal and perinatal bone marrow. Canonical TGF-bsignaling is dependent on SMAD4. To investigate whether MSPC lineage specification by TGF-bis dependent on SMAD4, we generated Osx-Cre Smad4Δ/Δmice. Osx-Cre Smad4Δ/Δmice are runted to a similar degree as Osx-CreTgfbr2Δ/Δmice secondary to a loss of mature osteoblasts. However, the magnitude of the increase in bone marrow adiposity is significantly reduced in Osx-Cre Smad4∆/∆mice compared to Osx-Cre, Tgfbr2Δ/Δmice. These data suggested that non-canonical signaling contributes to the suppressive effect of TGF-b on adipogenesis. To test this hypothesis, we generated cultures of mesenchymal stromal cells from wildtype neonatal bone marrow. As expected, in wildtype cultures, the addition of TGF-bpotently suppressed adipocyte formation. To assess the role of MAPK activation on the suppression of adipogenesis by TGF-b, we pharmacologically inhibited MAPK activation. Inhibition of MAPK alone did not suppress adipocyte formation. However, it completely blocked the suppressive effect of TGF-bon adipogenesis. Prior studies showed that phosphorylation of serine 82 of PPARgby MAPK decreases its transcriptional activity. Since PPARgis a master regulator of adipogenesis, we assessed the ability of TGF-b to induce PPARgphosphorylation. Indeed, the addition of TGF-b to the MSPC cultures resulted in reproducible PPARgphosphorylation. These data suggest that TGF-b suppresses adipocyte specification of MSPCs, in part, in a MAPK-dependent fashion through phosphorylation of PPARg. In summary, our data suggest that TGF-b plays a key role in the lineage specification of fetal MSPCs during development and is required for the proper development of fetal hematopoietic niches in the bone marrow. The contribution of TGF-b signaling in MSPCs to the stromal and hematopoietic response to different stressors is an active area of investigation. Disclosures No relevant conflicts of interest to declare.