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Objective: The bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix., Design: We generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts., Results: The first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype., Conclusions: These data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

As a member of transforming growth factor β (TGF-β) family, bone morphogenetic proteins (BMPs) are multi-functional factors and play critical roles in heart, cartilage, neural development and postnatal bone formation. It has been demonstrated that among the family, BMP2/4 have been reported to be especially important for the developmental and maturation of central nervous system (CNS). It has different, even opposite functions, in certain given circumstances, which could be a potential risk for BMPs' clinical use.

Bmp2 and Bmp4 genes were ablated in adult mice (KO) using a conditional gene knockout technology. Bones were evaluated by microcomputed tomography (μCT), bone strength tester, histomorphometry and serum biochemical markers of bone turnover. Drill-hole was made at femur metaphysis and bone regeneration in the hole site was measured by calcein binding and μCT. Mice were either sham operated (ovary intact) or ovariectomized (OVX), and treated with human parathyroid hormone (PTH), 17β-estradiol (E2) or vehicle. KO mice displayed trabecular bone loss, diminished osteoid formation and reduced biomechanical strength compared with control (expressing Bmp2 and Bmp4). Both osteoblast and osteoclast functions were impaired in KO mice. Bone histomorphomtery and serum parameters established a low turnover bone loss in KO mice. Bone regeneration at the drill-hole site in KO mice was lower than control. However, deletion of Bmp2 gene alone had no effect on skeleton, an outcome similar to that reported previously for deletion of Bmp4 gene. Both PTH and E2 resulted in skeletal preservation in control-OVX, whereas in KO-OVX, E2 but not PTH was effective which suggested that the skeletal action of PTH required Bmp ligands but E2 did not. To determine cellular effects of Bmp2 and Bmp4, we used bone marrow stromal cells in which PTH but not E2 stimulated both Bmp2 and Bmp4 synthesis leading to increased Smad1/5 phosphorylation. Taken together, we conclude that Bmp2 and Bmp4 are essential for maintaining adult skeletal homeostasis and mediating the anabolic action of PTH., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

During vertebrate embryogenesis the interdigital mesenchyme is removed by programmed cell death (PCD), except in species with webbed limbs. Although bone morphogenetic proteins (BMPs) have long been known to be players in this process, it is unclear if they play a direct role in the interdigital mesenchyme or if they only act indirectly, by affecting fibroblast growth factor (FGF) signaling. A series of genetic studies have shown that BMPs act indirectly by regulating the withdrawal of FGF activity from the apical ectodermal ridge (AER); this FGF activity acts as a cell survival factor for the underlying mesenchyme. Other studies using exogenous factors to inhibit BMP activity in explanted mouse limbs suggest that BMPs do not act directly in the mesenchyme. To address the question of whether BMPs act directly, we used an interdigit-specific Cre line to inactivate several genes that encode components of the BMP signaling pathway, without perturbing the normal downregulation of AER-FGF activity. Of three Bmps expressed in the interdigital mesenchyme, Bmp7 is necessary for PCD, but Bmp2 and Bmp4 both have redundant roles, with Bmp2 being the more prominent player. Removing BMP signals to the interdigit by deleting the receptor gene, Bmpr1a, causes a loss of PCD and syndactyly, thereby unequivocally proving that BMPs are direct triggers of PCD in this tissue. We present a model in which two events must occur for normal interdigital PCD: the presence of a BMP death trigger and the absence of an FGF survival activity. We demonstrate that neither event is required for formation of the interdigital vasculature, which is necessary for PCD. However, both events converge on the production of reactive oxygen species that activate PCD., (Copyright © 2016. Published by Elsevier Inc.)

The objective was to investigate the expression of bone morphogenetic protein (BMP) family members in the mouse uterus during the estrous cycle by real-time polymerase chain reaction (PCR) and immunohistochemistry. Uterine samples from Swiss ICR mice were collected and dissected free of surrounding tissue. One uterine horn was snap frozen in liquid nitrogen immediately after collection and stored at -80 °C for RNA extraction, and the other was fixed in 40 mg/ml paraformaldehyde at room temperature for immunolocalization of BMP2 protein. Real-time PCR analysis showed that the expression level of Bmp2 was significantly higher at proestrus than at estrus and metestrus (P<0.05). The relative abundance of Bmp4 exhibited significant fluctuations, but there were no statistically significant differences between the expression levels of Bmp2 and Bmp4 (P>0.05). The expression levels of Bmpr1a and Bmpr2 remained unchanged during estrous cycles. However, the level of Bmpr1b mRNA decreased significantly at estrus (P<0.05), increasing subsequently at metestrus. Furthermore, the level of Bmpr1b mRNA was significantly lower than those of Bmpr1a and Bmpr2 mRNA at the corresponding stages (P<0.05). All three receptor-regulated Smads (R-Smads) detected were differentially expressed in the mouse uterus and the expression levels of Smad1 and Smad5 were significantly higher than that of Smad8 (P<0.05). In addition, the expression level of Smad4 did not change substantially throughout the estrous cycle. Immunohistochemical experiments revealed that BMP2 protein was differentially expressed and localized mainly in the uterine luminal and glandular epithelial cells throughout the estrous cycle. In conclusion, our results provide information about the variation in the mRNA levels of Bmp2 and Bmp4 and related components of the BMP signaling pathway. The data provide quantitative and useful information about the roles of endometrial BMP proposed and demonstrated by others, such as the degradation and remodeling of the endometrium.

Rare retinal stem cells (RSCs) within the ciliary epithelium at the retinal margin of the adult mouse and human eyes can divide in vitro in the absence of growth factors to generate clonal, self-renewing spheres which can generate all the retinal cell types. Since no regenerative properties are seen in situ in the adult mammalian eye, we sought to determine the factors that are involved in the repression of endogenous RSCs. We discovered that factors secreted by the adult lens and cornea block the proliferation of adult RSCs in vitro. Bone morphogenetic protein (BMP)2, BMP4, and secreted frizzled related protein 2 were identified as principal effectors of the anti-proliferative effects on RSCs. As a similar induced quiescence was observed in vitro on both mouse and human RSCs, targeting these molecules in vivo may reactivate RSCs directly in situ in the eyes of the blind., (© AlphaMed Press.)

Background: Hyperoxia or clinical oxygen (O2) therapy is known to result in increased oxidative burden. Therefore, understanding susceptibility to hyperoxia exposure is clinically important. Bone morphogenetic proteins (BMPs) 2 and 4 are involved in cardiac development and may influence responses to hyperoxia., Methods: Bmp2(+/)(-). Bmp4(+/)(-) and wild-type mice were exposed to hyperoxia (100% O2) for 24 hrs. Electrocardiograms (ECG) were recorded before and during exposure by radio-telemetry., Results: At baseline, a significantly higher low frequency (LF) and total power (TP) heart rate variability (HRV) were found in Bmp2(+/)(-) mice only (p < 0.05). Twenty-four hours hyperoxia-induced strain-independent reductions in heart rate, QTcB and ST-interval and increases in QRS, LF HRV and standard deviation of RR-intervals were observed. In Bmp4(+/)(-) mice only, increased PR-interval (PR-I) (24 hrs), P-wave duration (P-d; 18 and 21-24 hrs), PR-I minus P-d (PR - Pd; 24 hrs) and root of the mean squared differences of successive RR-intervals (24 hrs) were found during hyperoxia (p < 0.05)., Discussion: Elevated baseline LF and TP HRV in Bmp2(+/)(-) mice suggests an altered autonomic nervous system regulation of cardiac function in these mice. However, this was not related to strain specific differences in responses to 24 hrs hyperoxia. During hyperoxia, Bmp4(+/-) mice were the most susceptible in terms of atrioventricular conduction changes and risk of atrial fibrillation, which may have important implications for patients treated with O2 who also harbor Bmp4 mutations. This study demonstrates significant ECG and HRV responses to 24 hrs hyperoxia in mice, which highlights the need to further work on the genetic mechanisms associated with cardiac susceptibility to hyperoxia.

Purpose of this study was to investigate whether human β-defensins (hBDs) affect maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were stimulated with hBD-1, -2, and -3 under control conditions and with hBD-2 during experimental inflammation (induced by interleukin-1β, tumor necrosis factor-α, toll-like receptor-2 and -4 agonists). Expression of different osteogenic markers and hBDs were analyzed by real-time PCR, immunohistochemistry, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were monitored. All tested hBDs were expressed on mRNA and protein level in MG63 cells. Only stimulation with hBD-2 elevated the proliferation rate. hBD-2 and hBD-3 positively affected the differentiation of osteoblast-like cells provided by increased transcript levels of osteogenic markers, up-regulated ALP enzyme activity and enhanced mineralized nodule formation. All pro-inflammatory stimuli enhanced interleukin-6 and hBD-2 expression and down-regulated markers of osteoblastic differentiation. In accordance, inflammation increased transcript level of Notch-1 (an inhibitor of osteoblastic differentiation). hBD-2 was not able to revert effects of inflammation on differentiation. In bone cells human β-defensins exhibit further functions than antimicrobial peptide activity. These include stimulation of proliferation and differentiation. Differentiation arrest due to inflammation could not be overcome by hBD-2 alone., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for BMP2 and 4, an innate inhibitor noggin (NOG), and BMP2/4 receptors (BMPRII, ACVR1, BMPR1A, BMPR1B), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression., Methods: RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated., Results: Transcripts for BMP2 and 4 were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for NOG were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced IFNT mRNA abundance but had no effect on CSH1 mRNA abundance in CT1 cells., Conclusions: The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.

A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Both of bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4) are two closely related members of the transforming growth factor beta superfamily and play diverse roles in normal and pathological processes. However, detail understandings of mechanisms through which Bmp2 and Bmp4 exert their effects remain elusive due to their functional compensations each other. To study roles of Bmp2/Bmp4 in osteoblast differentiation and extracellular matrix (ECM) remodeling, calvarial osteoblasts from Bmp2/4 conditional mice with Cre recombinase recognition site (loxP) were isolated and transfected with simian virus 40 large T antigen to generate immortalized BMP2C/C4C/C (iBMP2 C/C/4C/C) osteoblast lines. The BMP2/4 genes in the iBMP2 C/C/4C/C cells were double knocked out by Ad-Cre recombinase infection. Differentiation and mineralization of iBMP2C/C/4C/C knock-out (iBmp2C/C/4C/C KO) cells were detected by alkaline phosphatase (ALP) and alizarin (ALZ) red S staining analyses. ECM remodeling was also observed in fluorescent microscope. Cell differentiation was dramatically decreased in the iBMP2C/C/4C/C KO cells compared to that of the iBMP2C/C/4C/C osteoblasts. Mineralization was also reduced in these KO cells by ALZ staining. Furthermore, Bmp2/4 double knock-out cells have major defects in remodeling the ECM as reflected by changes in collagen type I processing. Here we for the first time demonstrate the establishment of iBmp2C/C/4C/C KO osteoblasts. Cell differentiation and mineralization in the iBmp2C/C/4C/C KO cells were decreased. Furthermore, ECM processing in these KO cells was impaired. This indicates that BMP2/4 play important roles in osteoblast differentiation and ECM remodeling.