BMP-6 inhibits human bone marrow B lymphopoiesis--upregulation of Id1 and Id3.

OBJECTIVE: In mammals, factors produced by bone marrow (BM) stromal cells are instrumental in orchestrating the developmental process of B lymphocytes. Bone morphogenetic proteins (BMPs) are multifunctional cytokines previously found to regulate hematopoietic stem cells. In the present study, we have explored the role of BMP-6 in human B progenitor cells. MATERIALS AND METHODS: In vitro B lymphopoiesis of CD10(+) B progenitor cells from human BM was evaluated in the presence or absence of BMP-6 in short- or long-term coculture on MS-5 stromal cells, by tracking CFSE-labeled CD10(+) B progenitor cells or by quantification of CD19(+) cells. DNA synthesis in the pre-B cell line Nalm-6 was measured by (3)H-thymidine incorporation. BMP-6-induced phosphorylation of Smad1/5/8 was determined by Western blot analysis, whereas elevation of Id1-Id4 mRNA levels and basal BMP-6 mRNA levels were measured by real-time and conventional RT-PCR, respectively. RESULTS: By in vitro coculture of CD10(+) B progenitor cells or monoculture of Nalm-6 cells, we found that BMP-6 inhibited B lymphopoiesis by impeding cell proliferation. Furthermore, in CD10(+) B progenitors as well as in Nalm-6 cells, BMP-6 rapidly induced phosphorylation of Smad1/5/8, followed by an upregulation of Id1 and Id3 mRNA levels. Finally, we demonstrated that human bone marrow stromal cells express BMP-6 mRNA whereas B progenitor cells did not. CONCLUSIONS: We suggest that BMP-6, produced by the BM, may participate to fine-tune the balance between proliferation, apoptosis, and differentiation in human B progenitor cells during BM B lymphopoiesis.

ALK2/ALK3-BMPR2/ACVR2A Mediate BMP2-Induced Downregulation of Pentraxin 3 Expression in Human Granulosa-Lutein Cells.

Bone morphogenetic protein 2 (BMP2) belongs to the transforming growth factor-ß superfamily and plays a critical role in regulating ovarian follicle function. Currently, the role of BMP2 during cumulus expansion remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of pentraxin 3 (PTX3) expression (the major component of cumulus expansion) and the underlying mechanisms in human granulosa-lutein (hGL) cells. Both primary and immortalized hGL cells were used as research models. Our results showed that treatment with BMP2 significantly suppressed the basal and luteinizing hormone-induced upregulation of PTX3. In addition, BMP2 stimulated the phosphorylation of SMAD1/5/8, and this effect was abolished by the addition of BMP type I receptor inhibitors, dorsomorphin homolog 1, and dorsomorphin but not SB431542. Moreover, the knockdown of activin receptorlike kinase 2/3 or BMP receptor type II/activin receptor type IIB receptors completely reversed the BMP2-induced phosphorylation of SMAD1/5/8 and restored PTX3 expression. Similarly, the knockdown of SMAD4 completely reversed the suppressive effect of BMP2 on the expression of PTX3. These results improve our understanding of the molecular mechanisms of BMP2 signaling. Our findings suggest that BMP2 may be involved in the regulation of cumulus expansion during the periovulatory stage.

Activin B Induces Noncanonical SMAD1/5/8 Signaling via BMP Type I Receptors in Hepatocytes: Evidence for a Role in Hepcidin Induction by Inflammation in Male Mice.

Induction of the iron regulatory hormone hepcidin contributes to the anemia of inflammation. Bone morphogenetic protein 6 (BMP6) signaling is a central regulator of hepcidin expression in the liver. Recently, the TGF-ß/BMP superfamily member activin B was implicated in hepcidin induction by inflammation via noncanonical SMAD1/5/8 signaling, but its mechanism of action and functional significance in vivo remain uncertain. Here, we show that low concentrations of activin B, but not activin A, stimulate prolonged SMAD1/5/8 signaling and hepcidin expression in liver cells to a similar degree as canonical SMAD2/3 signaling, and with similar or modestly reduced potency compared with BMP6. Activin B stimulates hepcidin via classical activin type II receptors ACVR2A and ACVR2B, noncanonical BMP type I receptors activin receptor-like kinase 2 and activin receptor-like kinase 3, and SMAD5. The coreceptor hemojuvelin binds to activin B and facilitates activin B-SMAD1/5/8 signaling. Activin B-SMAD1/5/8 signaling has some selectivity for hepatocyte-derived cells and is not enabled by hemojuvelin in other cell types. Liver activin B mRNA expression is up-regulated in multiple mouse models of inflammation associated with increased hepcidin and hypoferremia, including lipopolysaccharide, turpentine, and heat-killed Brucella abortus models. Finally, the activin inhibitor follistatin-315 blunts hepcidin induction by lipopolysaccharide or B. abortus in mice. Our data elucidate a novel mechanism for noncanonical SMAD activation and support a likely functional role for activin B in hepcidin stimulation during inflammation in vivo.

Recombinant biglycan promotes bone morphogenetic protein-induced osteogenesis.

The aim of this study was to determine the effects of glutathione-S-transferase-fused recombinant biglycan (GST-BGN) on craniofacial bone regeneration. We recently demonstrated a positive effect of tissue-derived BGN on bone morphogenetic protein 2 (BMP-2) function, which is exerted likely via the BGN core protein. Here, we investigated the effects of GST-BGN lacking any posttranslational modifications on BMP-2 function in vitro and in vivo. In the C2C12 cell culture system, BMP-2-induced Smad 1/5/8 phosphorylation and alkaline phosphatase activity were both enhanced by the addition of GST-BGN. For the in vivo effect, we employed a Sprague-Dawley rat mandible defect model utilizing 1 µg (optimal) or 0.1 µg (suboptimal) of BMP-2 combined with 0, 2, 4, or 8 µg of GST-BGN. At 2 weeks post-surgery, newly formed bone was evaluated by microcomputed tomography and histologic analyses. The results revealed that the greatest amounts of bone within the defect were formed in the groups of suboptimal BMP-2 combined with 4 or 8 µg of GST-BGN. Also, bone was well organized versus that formed by the optimal dose of BMP. These results indicate that recombinant BGN is an efficient substrate to promote low-dose BMP-induced osteogenesis.

Bone morphogenetic protein 6 stimulates mineralization in human dental follicle cells without dexamethasone.

OBJECTIVE: The aim of this study is to investigate the osteogenic differentiation human dental follicle cells (hDFCs) cultured with in osteogenic induction medium (OIM) without dexamethasone (DEX), and to analyze the gene expression profile during osteogenic differentiation. METHODS: hDFCs, which isolated from dental follicle tissue from impacted third molar teeth, were cultured with OIM with or without DEX. Osteogenic differentiation of hDFCs was examined using Alkaline phosphatase activity and Arizarin red staining. Gene expression analysis was performed by Microarray and real time-PCR. RESULTS: We showed that hDFCs have the capacity to differentiate into osteogenic lineages in osteogenic induction medium lacking DEX. We also analyzed gene expression profiling of hDFCs during osteogenic differentiation. BMP6 is up-regulated in both the presence and absence of DEX. In addition, BMP6 enhances gene expression levels of DLX-5, Runx2, and Osterix, which are transcription factors associated with osteogenic differentiation. BMP6 also stimulates phosphorylation of Smad1/5/8 which are transcription factors associated with BMP signalling at protein levels. Additionally BMP6 stimulates mineralization of hDFCs monolayers examined by Arizarin red S staining. CONCLUSION: These findings suggest that hDFCs can differentiate to osteogenic lineage cells osteogenic induction medium without DEX, and BMP6 is a key gene in the osteogenic differentiation of hDFCs, and has therapeutic utility for bone regeneration and bone research.

Effects of recombinant dentin sialoprotein in dental pulp cells.

Dentin sialophosphoprotein (DSPP) is critical for dentin mineralization. However, the function of dentin sialoprotein (DSP), the cleaved product of DSPP, remains unclear. This study aimed to investigate the signal transduction pathways and effects of recombinant human DSP (rh-DSP) on proliferation, migration, and odontoblastic differentiation in human dental pulp cells (HDPCs). The exogenous addition of rh-DSP enhanced the proliferation and migration of HDPCs in dose- and time-dependent manners. rh-DSP markedly increased ALP activity, calcium nodule formation, and levels of odontoblastic marker mRNA. rh-DSP increased BMP-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist, noggin. Furthermore, rh-DSP phosphorylated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), Akt, and IκB-α, and induced the nuclear translocation of the NF-κB p65 subunit. Analysis of these data demonstrates a novel signaling function of rh-DSP for the promotion of growth, migration, and differentiation in HDPCS via the BMP/Smad, JNK, ERK, MAPK, and NF-κB signaling pathways, suggesting that rh-DSP may have therapeutic utility in dentin regeneration or dental pulp tissue engineering.