Translin, a highly conserved DNA/RNA binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses a broad variety of functions, including RNA processing and DNA repair. Recent studies have reported that Translin is involved in mesenchymal cell physiology. Thus, here we analyzed the intrinsic role of Translin in mesenchymal cell proliferation and differentiation. Translin-deficient E11.5 mouse embryonic fibroblasts showed enhanced growth. Translin-deficient bone marrow-derived mesenchymal stem cells showed substantial expansion in vivo and enhanced proliferation in vitro. These cells also showed enhanced osteogenic and adipocytic differentiation. Histological analyses showed adipocytic hypertrophy in various adipose tissues. Translin knockout did not affect the growth of subcutaneous white adipose tissue-derived stem cells, but enhanced adipocytic differentiation was observed in vitro. Contrary to previous reports, in vitro-fertilized Translin-null mice were not runted and exhibited normal metabolic homeostasis, indicating the fragility of these mice to environmental conditions. Together, these data suggest that Translin plays an intrinsic role in restricting mesenchymal cell proliferation and differentiation.

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.

The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. Disclosures No relevant conflicts of interest to declare.

Translin, a ubiquitous RNA/DNA-binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses endoribonuclease activity and plays a physiological role in restricting the size and differentiation of mesenchymal precursor cells. However, the precise role of Translin in epithelial cells remains unclear. Here, we show evidence that Translin restricts the growth of pubertal mammary epithelial cells. The mammary epithelia of Translin-null females exhibited retarded growth before puberty, but highly enhanced growth and DNA synthesis with increased ramification after the onset of puberty. Primary cultures of Translin-null mammary epithelial cells showed augmented DNA synthesis in a ligand-independent and ligand-enhanced manner. Translin-null ovariectomized mice implanted with slow-release estrogen pellets showed enhanced length and ramification of the mammary glands. Mammary epithelial growth was also observed in ovariectomized Translin-null mice implanted with placebo pellets. Luciferase reporter assays using embryonic fibroblasts from Translin-null mice showed unaltered estrogen receptor α function. These results indicate that Translin plays a physiological role in restricting intrinsic growth, beyond mesenchymal cells, of pubertal mammary epithelial cells.