The expression of extracellular matrix protein periostin (POSTN) was attenuated in Med1−/− mouse embryonic fibroblasts (MEFs), which exhibited a decreased capability to support hematopoietic progenitor cells (HPCs) in vitro. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN antibody, the growth of BM cells and number of long-term culture-initiating cells (LTC-ICs) were attenuated. When BM cells were cocultured with Med1−/− MEFs in the presence of recombinant POSTN, the growth of BM cells and the number of LTC-ICs were restored. Moreover, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the growth and cobblestone formation, a leukemic stem cell feature, of stromal cell-dependent MB-1 myeloblastoma cells. POSTN was expressed both in BM cells and variably in different BM stromal cells. Expression in the latter cells was increased by physical interaction with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. The addition of recombinant POSTN to BM stromal cells induced intracellular signaling downstream of integrin αvβ3. These results suggest that stromal cell POSTN supports both normal HPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner.

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin Ctreated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

Periostin (POSTN), the fasciclin family extracellular matrix protein also known as osteoblast-specific factor 2 (OSF-2), was previously reported to be required for optimal B lymphopoiesis in vitro. Now, our study first demonstrates the proof that POSTN might be a bona fide niche factor for both normal and malignant myelopoiesis, indicating that it is a niche molecule for hematopoietic stem cells and diverse hematopoietic precursor cells. The Mediator, composed of about 31 subunits, is a master transcriptional coregulator complex that is essential for global transcription governed by RNA polymerase II. Among the Mediator subunits, MED1 acts as a specific coactivator for activators that include nuclear receptors and GATA1. We previously reported that Med1−/− mouse embryonic fibroblasts (MEFs) have a decreased capability to support hematopoietic stem/progenitor cells (HSPCs) relative to wild-type MEFs in vitro, and that the attenuated expression of full-length osteopontin and FGF7 in Med1−/− MEFs is responsible for the observed phenotype. The microarray analyses, showing that the expression of POSTN was also suppressed in Med1−/− MEFs, prompted us to study the role for POSTN in support of both normal and malignant HSPCs in our in vitro niche model. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN blocking antibody, the mitogenicity and growth of BM cells were attenuated. The number of long-term culture-initiating cells (LTC-ICs), i.e., number of both granulo-monocytic and erythroid colonies, was also decreased. When BM cells were cocultured with Med1-/- MEFs in the presence of recombinant POSTN, the mitogenicity and growth of BM cells and the number of LTC-ICs were restored. These results suggest that POSTN mediates mitogenicity of BM cells and HSPCs support. The MB-1 myeloblastoma cell line, originally established from a patient with myeloid crisis chronic myeloid leukemia, is a mesenchymal stromal cell-dependent cell line. These cells are unique in that they grow by forming cobblestone areas in the presence of niche cells but die of apoptosis when detached from stromal cells, thus faithfully conforming to a stochastic model of leukemic stem cells in vitro. Intriguingly, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the mitogenicity and growth, as well as the cobblestone formation, a leukemic stem cell feature, of MB-1 myeloblastoma cells. Therefore, it appears that niche cell-derived POSTN supports niche-dependent MB-1 myeloblastoma cells. While POSTN was expressed both in BM cells and variably in different BM stromal cells, expression in the latter cells was markedly increased by tactile interaction with hematopoietic cells. Specifically, POSTN was robustly induced 6 hours after BM stromal cells were cocultured with BM cells or MB-1 myeloblastoma cells, and the induction sustained for as long as 24 hours. However, POSTN expression was not enhanced when BM cells were cocultured but physically separated from MS-5 or OP-9 cells using transwell culture wells. Therefore, the major source of POSTN in the coculture appears to be the BM stromal cells associated with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. Although β3 mRNA was especially prominent in both BM cells and MB-1 cells, in accordance with a previous report that integrin β3/CD61 marks HSPCs, western blot analysis showed that αv and β3 expression levels were below the detection level on BM cells. Hence, integrin αvβ3 is scarce on BM cells compared to BM stromal cells, although it does not exclude the possibility that functional integrin αvβ3 might be enriched on HSPCs as suggested previously. When an excess amount of exogenous POSTN was added to MS-5 or OP-9 BM stromal cells after 24-h serum starvation, FAK (the immediate target of integrin αvβ3) and MAP kinases ERK1/ERK2 (the intermediate hub of various intracellular signals) were robustly phosphorylated as early as 10 min, and the phosphorylation was sustained for over 60 min. Thus, POSTN effectively activates integrin αvβ3 and subsequent intracellular signaling in BM stromal cells. These results suggest that stromal cell POSTN supports both normal HSPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. Disclosures No relevant conflicts of interest to declare.