BMP-2 mRNA Expression Research Articles

Effects of 50Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts

Electromagnetic fields (EMFs) have been used clinically to slow down osteoporosis and promote fracture healing for many years. However, the underlying action mechanisms and optimal parameters of the EMF applications are unclear. In this study, we investigated the effects of treatment for different durations with 50 Hz sinusoidal electromagnetic fields (SEMFs) at different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts. Osteoblasts isolated from neonatal rats were treated with SEMFs (50 Hz at 0.9 mT-4.8 mT, 0.3 mT interval, 30 min/day up to 15 days). Compared to untreated control, SEMFs inhibited osteoblast proliferation (after 3 days' treatment) but increased alkaline phosphatase (ALP) activity (after treatment for 9 days) from 0.9 mT to 1.8 mT, declined from 1.8 mT until 3.0 mT, and then increased again from 3.0 mT to 3.6 mT and decreased once again from 3.6 mT to 4.8 mT. Numbers of colonies stained positive for ALP after 8 days and mineralized nodules stained by Alizarin red after 10 days showed the same bimodal tendency as with the ALP activity, with two peaks at 1.8 mT and 3.6 mT. SEMFs also bimodally increased Runx-2, Col1α2 and Bmp-2 mRNA expression levels in osteoblasts at 12, 24 and 96 h after exposure. The results indicated that while exposure to 50 Hz SEMFs inhibits the osteoblast proliferation, it significantly promotes differentiation and mineralization potentials of osteoblasts in an intensity-dependent manner with peak activity at 1.8 mT and 3.6 mT.

Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice

The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. Although liver iron content is independently positively correlated with hepatic Bmp6 messenger RNA (mRNA) expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice. Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, whereas the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.

Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure–activity relationship studies using harmine-related β-carboline alkaloids revealed that the C3–C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2 pathways. Our findings suggest that harmine has bone anabolic effects and may be useful for the treatment of bone-decreasing diseases and bone regeneration as a lead compound.

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects. MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins. Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation. Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.

Histamine and lipopolysaccharide induce endothelial cell bone morphogenetic protein 2 (60.1)

Abstract Vascular calcification is one of the complications of chronic inflammatory diseases. It is a complex and multifaceted disease and is recognized as a pathobiological process sharing many features of bone remodeling. BMP2 is a powerful osteogenic morphogen which belongs to TGFβ superfamily and plays a central role in bone remodeling and calcification of atherosclerotic plaques. We have previously shown that histamine (HIS) and LPS independently and synergistically induce endothelial cell inflammatory responses and contribute to vascular inflammation. Our hypothesis is that HIS and LPS stimulate BMP2 expression in endothelial cells. Human vascular endothelial cells were incubated with HIS (10µM), LPS (100 ng/ml) or HIS+LPS for different time periods and the mRNA and protein expressions were determined by qRT-PCR and Western Blot analyses. HIS and LPS induced BMP2 mRNA expression by 4-fold and 3-fold respectively. HIS-induced BMP2 expression was completely abrogated by cetirizine, a histamine receptor 1 (H1R) antagonist, indicating that histamine effect is mediated through H1R. Western blot analysis showed upregulation of BMP2 by HIS, LPS or HIS+LPS as early as 2 hrs post treatment which sustained upto 24 h. These results suggest that HIS and LPS play a role in the regulation BMP2 expression in chronic inflammation and infection.

Osr2 acts downstream of Pax9 and interacts with both Msx1 and Pax9 to pattern the tooth developmental field

Mammalian tooth development depends on activation of odontogenic potential in the presumptive dental mesenchyme by the Msx1 and Pax9 transcription factors. We recently reported that the zinc finger transcription factor Osr2 was expressed in a lingual-to-buccal gradient pattern surrounding the developing mouse molar tooth germs and mice lacking Osr2 developed supernumerary teeth lingual to their molars. We report here generation of a gene-targeted mouse strain that allows conditional inactivation of Pax9 and subsequent activation of expression of Osr2 in the developing tooth mesenchyme from the Pax9 locus. Expression of Osr2 from one copy of the Pax9 gene did not disrupt normal tooth development but was sufficient to suppress supernumerary tooth formation in the Osr2−/− mutant mice. We found that endogenous Osr2 mRNA expression was significantly downregulated in the developing tooth mesenchyme in Pax9del/del mice. Mice lacking both Osr2 and Pax9 exhibited early tooth developmental arrest with significantly reduced Bmp4 and Msx1 mRNA expression in the developing tooth mesenchyme, similar to that in Pax9del/del mutants but in contrast to the rescue of tooth morphogenesis in Msx1−/−Osr2−/− double mutant mice. Furthermore, we found that Osr2 formed stable protein complexes with the Msx1 protein and interacted weakly with the Pax9 protein in co-transfected cells. These data indicate that Osr2 acts downstream of Pax9 and patterns the mesenchymal odontogenic field through protein–protein interactions with Msx1 and Pax9 during early tooth development.

Chondrogenic and osteogenic potential of adipose derived stem cells from RA and OA patients

Background and objectivesAdipose derived stem cells (ADSC) have a potential to differentiate into cells of mesodermal origin (chondrocytes, osteoblasts, adipocytes) and recently emerged as an attractive source of mesenchymal stem...

BMP-2 Gene Modified Canine bMSCs Promote Ectopic Bone Formation Mediated by a Nonviral PEI Derivative

The study was to explore the effects of BMP-2 gene modified canine bone marrow stromal cells (bMSCs) mediated by a nonviral PEI derivative (GenEscort™ II) in promoting bone formation in vitro and in vivo. Canine bMSCs were cultured and transfected with plasmids containing bone morphogenetic protein-2 gene (pBMP-2) or enhanced green fluorescent protein gene (pEGFP). Gene transfection conditions were initially optimized by varying GenEscort™ II/plasmid ratios. Osteogenic differentiation of gene modified bMSCs was investigated via alkaline phosphatase (ALP) activity analysis and real-time quantitative PCR (RT-qPCR) analysis in vitro. The bone formation ability of pBMP-2 transfected bMSCs combined with apatite-coated silk scaffolds (mSS) was explored and compared with pEGFP transfected bMSCs/mSS or untreated bMSCs/mSS at 8, 12weeks after operation. Results showed that gene transfection efficiency reached up to 36.67±4.12% as demonstrated by EGFP expression. ALP staining and activity assay were stronger with pBMP-2 gene transfection, and the mRNA expression of BMP-2, bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx-2), and osteopontin (OPN) up-regulated in bMSCs 3, 6, 9days in pBMP-2 group. Besides, the tissue-engineered bone complex with pBMP-2 modified bMSCs achieved significantly increased de novo bone formation compared with control groups (p<0.01). We conclude that pBMP-2 transfection mediated by GenEscort™ II could enhance the osteogenic differentiation of canine bMSCs and promote the ectopic new bone formation in nude mice. GenEscort™ II mediated pBMP-2 gene transfer appears to be a safe and effective nonviral method for gene enhanced bone tissue engineering.

Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro

There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied.

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

Reactive oxygen species derived from Nox4 mediateBMP2gene transcription and osteoblast differentiation

BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.

187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.

Iron overload induces BMP6 expression in the liver but not in the duodenum

The bone morphogenetic protein BMP6 regulates hepcidin production by the liver. However, it is not yet known whether BMP6 derives from the liver itself or from other sources such as the small intestine, as has been recently suggested. This study was aimed at investigating the source of BMP6 further. We used three different strains of mice (C57BL/6, DBA/2, and 129/Sv) with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene. We examined Bmp6 expression at both the mRNA (by quantitative PCR) and protein (by immunohistochemistry and Western blotting analyses) levels. We showed that iron overload induces Bmp6 mRNA expression in the liver but not in the duodenum of these mice. Bmp6 is also detected by immunohistochemistry in liver tissue sections of mice with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene, but not in liver tissue sections from iron-loaded Bmp6-deficient mice. Bmp6 in the duodenum was below immunodetection threshold, thus confirming quantitative PCR data. Lack of specificity of available antibodies together with slight heterogeneity between 129 substrains may account for the differences with previously published data. Our data strongly support the importance of liver BMP6 for regulation of iron metabolism. Indeed, they demonstrate that intestinal Bmp6 expression is modulated by iron neither at the mRNA nor at the protein level.

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α(v)β(3) is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α(v)β(3) receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α(v)β(3) integrin/integrin-linked kinase/ERK signaling pathway.

The role of β-catenin signaling pathway on proliferation of rats neural stem cells after hyperbaric oxygen therapy in vitro.

β-catenin, a protein that functions in both cell adhesion and Wnt signaling, plays a key role in mammalian neural development. To investigate the role of β-catenin in hyperbaric oxygen therapy (HBO)-induced neurogenesis after hypoxic ischemic brain damage (HIBD), we transfected β-catenin siRNA and negative control siRNA into neural stem cells (NSCs) after HIBD. We found that HBO promoted NSCs differentiate into neurons or oligodendrocytes, and inhibited NSCs differentiate into astrocytes; HIBD brain tissue extract conditioned cultures promoted NSCs differentiate into neurons; β-Catenin siRNA decreased the NSE-positive neurons and increased GFAP-positive astrocytes in the NSCs in vitro. Furthermore, the expression of Ngn1 protein and mRNA in NSCs was increased when HBO promoted NSCs differentiate into neurons after HIBD, and the expression of BMP-4 protein and mRNA was decreased when HBO depressed NSCs differentiate into astrocytes after HIBD. These results showed that β-catenin-mediated transcriptional activation functions in the decision of NSCs to proliferate neurogenesis during HBO-induced after HIBD, and suggested that HBO therapy promotes the proliferation of neural stem cells in vitro, an effect that may be correlated with β-catenin protein and HBO therapy could promote neurogenesis by β-catenin-induced activated Ngn1 gene and repress astrocytogenesis by β-catenin-induced down-regulated BMP-4 gene.

P38 Mitogen-Activated Protein Kinase Controls a Switch Between Cardiomyocyte and Neuronal Commitment of Murine Embryonic Stem Cells by Activating Myocyte Enhancer Factor 2C-Dependent Bone Morphogenetic Protein 2 Transcription

Many studies have shown that it is possible to use culture conditions to direct the differentiation of murine embryonic stem (ES) cells into a variety of cell types, including cardiomyocytes and neurons. However, the molecular mechanisms that control lineage commitment decisions by ES cells remain poorly understood. In this study, we investigated the role of the 3 major mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38) in ES cell lineage commitment and showed that the p38 MAPK-specific inhibitor SB203580 blocks the spontaneous differentiation of ES cells into cardiomyocytes and instead induces the differentiation of these ES cells into neurons. Robust p38 MAPK activity between embryoid body culture days 3 and 4 is crucial for cardiomyogenesis of ES cells, and specific inhibition of p38 MAPK activity at this time results in ES cell differentiation into neurons rather than cardiomyocytes. At the molecular level, inhibition of p38 MAPK activity suppresses the expression of bmp-2 mRNA, whereas treatment of ES cells with bone morphogenetic protein 2 (BMP-2) inhibits the neurogenesis induced by SB203580. Further, luciferase reporter assays and chromatin immunoprecipitation experiments showed that BMP-2 expression in ES cells is regulated directly by the transcription factor myocyte enhancer factor 2C, a well-known substrate of p38 MAPK. Our findings reveal the molecular mechanism by which p38 MAPK activity in ES cells drives their commitment to differentiate preferentially into cardiomyocytes, and the conditions under which these same cells might develop into neurons.

Deficiency of vitamin A delays bone healing process in association with reduced BMP2 expression after drill-hole injury in mice

Although it is predicted that vitamin A and its active form, retinoic acid, regulate osteoblast lineage, this has not been elucidated in growing mammalians. To clarify the direct effect of retinoic acid on bone, we observed the process of filling up newly generating bone into a drill-hole of the bone, which is understood as membranous ossification, in vitamin A-deficient mice. Mice were assigned to three groups: a vitamin A-deficient group (VAD), which was fed a diet without vitamin A from the 10th day of gestation to the end of the experiments; a vitamin A-deficient-sufficient group (VADS), which was fed a diet without vitamin A from the 10th day of gestation to 4 weeks of age; and a vitamin A-sufficient group (VAS), which was fed a standard diet to the end of the experiment. In mice at 10 weeks of age (day 0), a drill-hole injury was made with a diameter of 1 mm at the anterior portion of the diaphysis of the bilateral femurs. In VAD, retardation in repairing the drill-hole was demonstrated by in vivo micro-CT and histomorphometry from day7 and after surgery. During repair of the bone defect, increases of bmp2, dlx5, msx2, col1a1, and osteocalcin mRNA expression were suppressed, and runx2-p2 mRNA expression was accelerated in VAD. Implantation of BMP2 in the bone defect of VAD normalized the delayed bone healing and mRNA expressions. We concluded that vitamin A regulates bmp2 mRNA expression and plays a crucial role in osteoblastogenesis and bone formation.

Osteogenic differentiation of intact human amniotic membrane

Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. In our approach, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials. For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM viability was evaluated. Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4 ± 6.8 mg/dl d28), increased AP activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3 ± 19.0% d14; 62.9 ± 22.3% d28) compared to CM (42.2 ± 15.2% d14; 35.1 ± 8.6% d28). By this strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment.

Adiponectin increases BMP‐2 expression in osteoblasts via AdipoR receptor signaling pathway

Adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. However, the effects of adiponectin on BMPs expression in cultured osteoblasts are largely unknown. Here we found that adiponectin increased mRNA expression of BMP-2 but not other BMPs in cultured osteoblastic cells. Stimulation of osteoblasts with adiponectin also increased protein levels of BMP-2 by Western blot and ELISA assay. Adiponectin-mediated BMP-2 expression was attenuated by 5'-AMP-activated protein kinase (AMPK) small interference RNA and AMPK inhibitor (araA and compound C). Activations of p38 and NF-kappaB pathways after adiponectin treatment were demonstrated, and adiponectin-induced expression of BMP-2 was inhibited by the specific inhibitor and mutant of p38 and NF-kappaB cascades. Taken together, our results provide evidence that adiponectin enhances BMP-2 expression in osteoblastic cells, and AdipoR1 receptor, AMPK, p38 and NF-kappaB signaling pathways may be involved in increasing BMP-2 expression by adiponectin.

Induced expression of bone morphogenetic protein-6 and Smads signaling in human monocytes derived dendritic cells during sickle-cell pathology with orthopedic complications

BMP-SMAD (bone morphogenetic protein) signaling pathways in association with APT play paramount roles in osteoblastic differentiation, bone formation and embryonic development of human and animals. However, the implications of potent components (BMP6, Smad1, Smad2 and APT) of this pathway in SCD (sickle cell disease) pathology with orthopedic complications (Ortho + SS) are poorly elucidated and substantially unknown. Here, we address the role of BMP6, Smad1, Smad2 and APT mRNA and protein expression in hMDDCs obtained from Ortho + SS patients, employing RT-PCR, qRT-PCR and immunoblotting. Interestingly, we observed that SCD pathology exhibited significantly up-regulated expression of those signaling components at the level of mRNA and protein. Furthermore, exogenous BMP6 induced apoptosis was observed to be significantly associated in Ortho + SS complication and markedly increased the percentage of cells undergoing apoptosis as compared to healthy group. Interestingly, the non-stimulated cells have shown higher apoptotic nuclei percentage than the stimulated cells in pathological condition. Thus, expression of BMP-SMAD signaling components augments apoptosis and up regulates the transcription of these genes and it suggests that induction is due to transcriptional regulation. Taken together, our findings provide evidence that BMP-SMAD signaling components along with APT were over expressed, mediates apoptosis and may play an important role in the SCD pathology with orthopedic complications.