A Novel Mechanism of Megakaryopoiesis from Pre-Adipocytes: Involvement of Transferrin/ CD71/ TPO Pathways

Platelets are released from terminally differentiated megakaryocytes (MKs) through lineage commitment, although the underlying mechanisms of megakaryopoiesis and subsequent thrombopoiesis are incompletely understood. MKs were reportedly generated from not only hematopoietic stem cells (HSCs), but also pre-adipocytes, without any gene transfer in an in vitro culture system (Matsubara et al, 2012 Methods Mol Biol). Because of high efficiency of platelet production from pre-adipocytes, much interest has been attracted to clarify the mechanisms of megakaryopoiesis from pre-adipocytes. Here we demonstrate a novel mechanism that megakaryopoiesis from pre-adipocytes is regulated by the inducible production of endogenous TPO via transferrin receptor CD71. NF-kB pathway in pre-adipocytes might modulate the TPO production through the stimulation of transferrin.

Previously, we reported that pre-adipocyte cell line OP9 has the gene expressions of thrombopoietin (TPO) and its receptor c-MPL. This study first examined the TPO protein levels in supernatant from human (Cell Applications) and mouse pre-adipocytes cultured with MK lineage induction (MKLI) media (Ono et al, 2012 Blood, Matsubara et al, 2012 Methods Mol Biol) in the absence of recombinant TPO (rTPO). The TPO levels were 36.6 ± 9.2 pg/mL in mouse pre-adipocytes on Day 8 and 36.1 ± 5.8 pg/mL in human pre-adipocytes on Day 8, and undetectable levels of TPO were observed in both cells before MK induction. Differentiation efficiency (approximately 20%) of MKs from pre-adipocytes cultured with rTPO was similar to that without rTPO. The function of pre-adipocyte-derived platelets cultured with rTPO was similar to that without rTPO when assessed by incorporation of platelets into thrombus formation on the collagen-coated surface under flow conditions. The number of MKs produced from pre-adipocytes was markedly decreased in the presence of anti-c-MPL blocking antibody, AMM2. Regarding the MK differentiation from HSCs, it is well known that HSCs do not differentiate into MKs in the absence of rTPO. We did not observe the TPO secretion from human bone marrow CD34(+) cells and mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells in a culture condition using MKLI media in the absence of rTPO. The observations suggest that the MK differentiation from pre-adipocytes has a distinct mechanism of megakaryopoiesis from HSCs. These findings indicate the unique mechanism of megakaryopoiesis from pre-adipocytes; the endogenous TPO stimulation via c-MPL in pre-adipocytes has activity for promoting MK differentiation and subsequent production of functional platelets.

To elucidate the molecular mechanism of TPO production during MK differentiation from pre-adipocytes, we examined which components of MKLI media were responsible for TPO production. We found iron-saturated transferrin, but not apo-transferrin, included in MKLI media is the critical factor to induce TPO production and MK differentiation from pre-adipocytes. The effects of transferrin were not observed when pre-adipocytes were cultured in the presence of anti-CD71 blocking antibody, 8D3, or deferoxamine mesilate, an iron-chelating agent. The effects of transferrin on TPO production and MK differentiation were also abolished in pre-adipocytes transfected with siRNA-CD71, and the TPO levels were 44.8 ± 15.9 pg/mL for siRNA-control and undetectable levels for siRNA-CD71 on Day 6. We next investigated a mechanism of inducing TPO production via CD71 in pre-adipocytes. The 5’ region of human TPO gene contains transcription factor-binding sequences, such as GATA1, GATA2 and NF-kB. Since NF-kB is a downstream target of CD71, we then examined whether NF-kB involved in MK differentiation derived from pre-adipocytes. Nuclear translocation of NF-kB in pre-adipocytes was observed when cells were stimulated with transferrin. Both TPO secretion and MK production were decreased in the presence of a peptide to inhibit nuclear translocation of NF-kB. Taken together, it is concluded that NF-kB pathway in pre-adipocytes modulates megakaryopoiesis through endogenous TPO production via CD71. The present findings suggest that pre-adipocytes have a unique mechanism of megakaryopoiesis through lineage commitment.