BMP-2 mRNA Research Articles

Effects of imposed defocus of opposite sign on temporal gene expression patterns of BMP4 and BMP7 in chick RPE

This study investigated the effects of imposed optical defocus on the expression patterns of bone morphogenetic protein 4 and 7 (BMP4, BMP7) in chick retinal pigment epithelium (RPE), as indicators of roles in postnatal eye growth regulation. BMP4 and BMP7 gene and protein expression patterns were characterized for retina, RPE and choroid tissues of young normal White-Leghorn chickens. The effects of short-term (2 and 48 h) exposure to monocular +10 and −10 diopter (D) lenses on RPE gene expression of BMP4 and BMP7 were also examined. Tissues from both treated and fellow eyes as well as from eyes of age-matched untreated birds were included in the latter experiment. Of ocular tissues comprising the posterior wall of the chick eye, RPE showed the highest expression of BMP4 and BMP7 mRNA, compared to retina and choroid. Western blots and immunohistochemistry confirmed the expression of BMP4 and BMP7 protein in all layers – retina, RPE, choroid and sclera. With imposed defocus, both BMP4 and BMP7 showed bidirectional changes in expression in RPE, however, with different temporal patterns. With +10 D lenses, BMP4 gene expression was up-regulated after both 2 and 48 h of treatment, while BMP7 expression was up-regulated only after 48 h of lens wear. With −10 D lenses, both BMP4 and BMP7 showed down-regulation of gene expression for both 2 and 48 h treatment durations. With the −10 D lens treatment applied for 48 h, gene expression for both BMP4 and BMP7 was also down-regulated in contralateral fellows of treated eyes compared to eyes of untreated chicks. The rapid changes in gene expression in chick RPE observed for both BMP4 and BMP7, up or down according to the sign of imposed optical defocus, resemble similar trends reported for BMP2. Further studies are needed to confirm the roles of BMPs as ocular growth modulators, as suggested by these data. The data also suggest a role for the RPE as a conduit for relaying growth modulatory retinal signals.

Deletion of Fibroblast Growth Factor Receptor 2 from the Peri-Wolffian Duct Stroma Leads to Ureteric Induction Abnormalities and Vesicoureteral Reflux

PurposePax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity.MethodsWe conditionally deleted Fgfr2 in ST (Fgfr2ST−/−) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects.ResultsWe confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2ST−/− mice at embryonic day (E) 10.5. E11.5 Fgfr2ST−/− mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2ST−/− mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2ST−/− mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes.ConclusionMutations in FGFR2 could possibly cause VUR in humans.

Disruption of aldehyde dehydrogenase 2 gene results in altered cortical bone structure and increased cortical bone mineral density in the femoral diaphysis of mice

IntroductionAldehyde dehydrogenase 2 (ALDH2) degrades acetaldehyde produced by the metabolism of alcohol. The inactive ALDH2 phenotype is prevalent in East Asians, and an association between this ALDH2 polymorphism and osteoporosis has been reported. In our previous study, we found that alcohol consumption results in decreased trabecular bone volume in aldh2 knockout (aldh2−/−) mice compared with the volume in wild-type (aldh2+/+) mice. However, the effect of aldh2 gene on the skeletal phenotype in the absence of alcohol consumption remains unknown. The aim of this study was to clarify the effect of aldh2 disruption on femoral bone structure and dynamics in aldh2-disrupted mice in the absence of alcohol consumption. Materials and methodsWe examined aldh2−/− and aldh2+/+ mice at the ages of 4, 8 and 12weeks. The femoral bone length and bone mineral density (BMD) were measured using peripheral quantitative computed tomography. The mechanical strength was assessed by the three-point bending test at 12weeks, and cortical bone histomorphometry at the femur diaphysis was performed at all three time points. Osteogenic activities in aldh2−/− and aldh2+/+ mice were assessed by osteoblast culture from calvariae of the neonatal mice. Bilateral femoral and tibial bones containing no bone marrow cells of 8-week-old mice were used for analysis of mRNA expression. In addition, mRNA expression in aldh2−/− and aldh2+/+ mice after tail suspension or climbing exercise for 7days from 8weeks was analyzed to clarify the response to mechanical loading. ResultsAt 12weeks, there were no significant differences in femoral bone length, trabecular BMD in the distal metaphyses of the femurs, or mechanical strength between aldh2−/− and aldh2+/+ mice, whereas cortical BMD and cortical thickness were significantly increased and cross-sectional area and bone marrow area were significantly decreased in the femoral diaphysis of aldh2−/− mice relative to the corresponding values in aldh2+/+ mice. At 8weeks, bone formation rate and mineral apposition rate on the periosteal and endocortical surfaces were significantly increased in aldh2−/− mice relative to the rates in aldh+/+ mice. Calvarial osteoblast culture study revealed that the percentage of alkaline phosphatase stained cells was significantly higher in aldh2−/− mice compared to that in aldh+/+ mice. Quantitative real-time RT-PCR revealed a significant increase in the expressions of bmp2, osterix, runx2, and col1a1 mRNA in aldh2−/− mice, along with an increase in the expression of wnt5a mRNA and the lrp5/sost mRNA ratio. The mRNA expressions of bmp2, osterix, runx2 and pthr in aldh2−/− mice were significantly decreased after climbing exercise compared to those in the control, although the mRNA expressions of bmp2, osterix, runx2 were not significantly decreased and pthr mRNA expression was increased in aldh+/+ mice after climbing exercise. ConclusionOur results show that disruption of aldh2 gene resulted in altered cortical bone structure and dynamics in mice. Cross-sectional area was decreased. Cortical BMD was increased owing to the promotion of cortical bone formation on the periosteal and endocortical surfaces of the femoral diaphysis. The possible mechanisms underlying altered cortical bone structure in aldh2−/− mice were gene-related higher osteogenic activity of osteoblasts and weakened osteogenice response to mechanical loading in growth period.

Clinical immunology Comparison of phenotype, chondrogenic and osteogenic potential of rheumatoid mesenchymal stem cells derived from articular and subcutaneous adipose tissue - the role of adipocytokines

A study was carried out to compare the phenotype, chondrogenic and osteogenic potential of adipose mesenchymal stem cells (ASCs) derived from articular adipose tissue (AAT) and subcutaneous adipose tissue (ScAT) of rheumatoid arthritis (RA) patients. We also aimed to evaluate the role of leptin (LEP), low molecular weight adiponectin isoform (LMW), high molecular weight adiponectin isoform (HMW) and tumor necrosis factor (TNF) in ASCs differentiation. AAT and ScAT were obtained from RA patients undergoing total knee joint replacement surgery. ASCs were isolated and expanded in vitro. Cells phenotype was assesed by flow cytometry; leptin and adiponectin receptors mRNA expression by RT-PCR. Differentiation was performed in chondrogenic and osteogenic medium with or without LEP, LMW, HMW and TNF. After differentiation, expression of mRNA for Sox9, aggrecan (Agr), collagen 2a (Col2a) for chondrogenesis, and BMP-2, Runx-2 and osteopontin (OPN) for osteogenesis was evaluated by RT-PCR. Dickkopf-1 (DKK-1) and osteoprotegerin (OPG) proteins concentrations were measured in supernatants from osteogenic cultures using ELISA. AAT and ScAT-ASCs have the same CD105+CD90+CD73+CD45–CD34+/–CD19–CD14– phenotype. AAT-ASCs seem to be more susceptible to chondrogenesis and osteogenesis in vitro. Adipocytokines do not exert strong effect on ASCs differentiation, however HMW slightly increased expression of chondrogenesis markers mRNA in AATASCs. TNF diminished expression of all chondrogenesis markers and OPN, but enhanced expression of BMP-2 and Runx-2 mRNA.

Lithium Chloride Enhances Cathepsin H Expression and BMP-4 Degradation in C3H10T1/2 Cells

The effect of canonical Wnt/β-catenin signaling on chondrogenic differentiation induced by transfection of BMP4 expressing plasmid was analyzed. Lithium chloride (LiCl) which mimics canonical Wnt/β-catenin signaling was added to cells transfected with BMP4 expressing plasmid. Although BMP4 mRNA expression was not affected by LiCl, LiCl decreased BMP4 protein accumulation. Gene expression analysis exhibited upregulation of cathepsin H by LiCl treatment. Gene silencing of cathepsin H enhanced BMP4 protein accumulation from BMP4 expressing cells. These results suggested that cathepsin H is regulated by Wnt/β-catenin signaling and plays an important role in the regulation of BMP4 biological activity.

Osteogenic potential of adult stem cells from human maxillary sinus membrane by Simvastatinin vitro: preliminary report

ObjectivesThe objective of this study is to determine the adequate concentration and to evaluate the osteogenic potential of simvastatin in human maxillary sinus membrane-derived stem cells (hSMSC).Materials and MethodsMesenchymal stem cells derived from the human maxillary sinus membrane were treated with various concentrations of simvastatin. The adequate concentration of simvastatin for osteogenic induction was determined using bone morphogenetic protein (BMP-2). The efficacy of osteogenic differentiation of simavastatin was verified using osteocalcin mRNA, and the mineralization efficacy of hSMSCs and simvastatin treatment was compared with alkaline phosphatase and von Kossa staining.ResultsExpression of BMP-2 mRNA and protein was observed after three days and was dependent on the concentration of simvastatin. Expression of osteocalcin mRNA was observed after three days in the 1.0 µM simvastatin-treated group. Mineralization was observed after three days in the simvastatin-treated group.ConclusionThese results suggest that simvastatin induces the osteogenic potential of mesenchymal stem cells derived from the human maxillary sinus membrane mucosa.

Lin28 regulates BMP4 and functions with Oct4 to affect ovarian tumor microenvironment

Emerging evidence suggests that the tumor microenvironment plays a critical role in regulating cancer stem cells (CSCs) and tumor progression through both autocrine and paracrine signaling. Elevated production of bone morphogenetic proteins (BMPs) from human ovarian cancer cells and stroma has been shown to increase CSC proliferation and tumor growth. Here, we report that Lin28, a stem cell factor, binds to BMP4 mRNA in epithelial ovarian carcinoma cells, thereby promoting BMP4 expression at the post-transcriptional level. As co-expression of Lin28 and Oct4 (another stem cell factor) has been implicated in ovarian cancer CSCs, we also determined that high levels of Lin28 are associated with an unfavorable prognosis when co-expressed with high levels of Oct4. Together, these findings uncover a new level of regulation of BMP4 expression and imply a novel Lin28/Oct4/BMP4-mediated mechanism of regulating ovarian tumor cell growth, thus holding potential for the development of new strategies for the diagnosis and treatment of ovarian cancer.

Roles of Bmp4 during tooth morphogenesis and sequential tooth formation

Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas all tooth germs were arrested at the bud stage in Msx1(-/-) mice, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4(f/f);Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We found that expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, was significantly upregulated in the molar tooth mesenchyme in Bmp4(f/f);Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity partially rescued mandibular first molar morphogenesis in Bmp4(f/f);Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1(-/-)Osr2(-/-) mice, Osr2(-/-)Bmp4(f/f);Wnt1Cre compound mutant mice exhibited formation and subsequent arrest of supernumerary tooth germs that correlated with downregulation of Msx1 expression in the tooth mesenchyme. In addition, we found that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wild-type embryos and that Dkk2 expression was significantly upregulated in the molar mesenchyme in Bmp4(f/f);Wnt1Cre embryos, which correlated with the dramatic differences in maxillary and mandibular molar phenotypes in Bmp4(f/f);Wnt1Cre mice. Together, these data indicate that Bmp4 signaling suppresses tooth developmental inhibitors in the tooth mesenchyme, including Dkk2 and Osr2, and synergizes with Msx1 to activate mesenchymal odontogenic potential for tooth morphogenesis and sequential tooth formation.

Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells

The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (P<0.01). There was a significant difference in the expression level of BMP2 mRNA between group A and C (P<0.05) and this was markedly higher than that in groups B, D and E (P<0.01). The protein expression level of HIF1α in group A and B was markedly higher than that in groups C, D and E (P<0.01). The protein expression level of BMP2 in group A and C was markedly higher than that in groups B, D and E (P<0.01). The human BMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

BMP system expression in GCs from polycystic ovary syndrome women and the in vitro effects of BMP4, BMP6, and BMP7 on GC steroidogenesis

The bone morphogenetic proteins (BMPs) are growth factors involved in the folliculogenesis. Alteration in their expression may compromise the reproductive process in disease such as the polycystic ovary syndrome (PCOS). This study investigated the expression and role of granulosa cell (GC) BMP from normal cycling and PCOS women. This prospective study was performed in GCs obtained from 14 patients undergoing IVF: i) six women with normal ovulatory cycles and tubal or male infertility and ii) eight women with PCOS. BMP2, BMP4, BMP5, BMP6, BMP7, and BMP8A and their receptors BMPR1A, BMPR1B, and BMPR2 were identified by RT-PCR in GCs from normally cycling and PCOS women. BMP4, BMP6, and BMP7 expressions were confirmed by immunohistochemistry. Quantitative transcript analysis showed the predominant expression of BMP6. In GCs from PCOS women, an overexpression of BMP6 (P<0.01) and BMPR1A mRNA (P<0.05) was observed. GC culture experiments demonstrated that basal estradiol (E₂) production was threefold higher but FSH-induced E₂ increment was twofold lower in PCOS compared with controls. In PCOS, BMP6 and BMP7 exerted a stimulatory effect on basal E₂ production while BMP4 and BMP6 inhibited FSH-induced E₂ production. FSH receptor and aromatase expression were not different between both groups. The BMP system is expressed in human GCs from normal cycling and PCOS women. The BMP may be involved in reproductive abnormalities found in PCOS.

Phosphoinositide 3-kinase/Akt signaling is essential for prostaglandin E2-induced osteogenic differentiation of rat tendon stem cells

Tissue calcification is a typical histopathological feature of tendinopathy. The osteogenic differentiation of tendon stem cells (TSCs) induced by inflammatory mediators is believed to play a key role in this process. Previous studies showed that the major inflammatory mediator prostaglandin E2 (PGE2) induced osteogenic differentiation of TSCs via bone morphogenetic protein (BMP)-2 production. Using a rat TSC culture model, we showed that PGE2 induced BMP-2 production through up-regulation of BMP-2 mRNA expression. PGE2 activated Akt, but not extracellular-signal-regulated kinase, in TSCs. Increased BMP-2 mRNA expression mediated by PGE2 was prevented by phosphoinositide 3-kinase (PI3K) and Akt inhibitors, but not by a MEK inhibitor. Furthermore, in the presence of exogenous BMP-2, PI3K and Akt inhibitors blocked Runx2 and osteocalcin expression, although BMP-2 did not activate Akt. BMP-2-induced alkaline phosphatase activity and mineralization were also inhibited by PI3K and Akt inhibitors. However, these inhibitors did not block activation of Smad, implying that Akt was involved downstream of Smad. Taken together, these results indicate that the PI3K-Akt signaling cascade is essential for PGE2-induced BMP-2 production and BMP-2-mediated osteogenic differentiation, suggesting that PI3-kinase-Akt signaling contributes to the formation of calcified tissues in tendinopathy.

Prognostic significance of BMP7 as an oncogene in hepatocellular carcinoma

This study aims to evaluate the association between BMP7 tissue expression and patient prognosis in hepatocellular carcinoma (HCC). The expression of BMP7 mRNA in HCC was characterized using real-time PCR and 30 pairs of fresh frozen HCC tissues and corresponding noncancerous tissues. BMP7 protein expression in HCC was confirmed using immunohistochemistry on a tissue microarray chip. Finally, BMP7 expression was correlated with conventional clinicopathological features of HCC and patient outcome. The expression of BMP7 mRNA and protein in HCC cells was much higher than in normal hepatic cells. Our results showed that the high expression of BMP7 in HCC was related to tumor size (p < 0.001), histological differentiation (p = 0.041), serum AFP (p = 0.007), and tumor stage (p < 0.001). Kaplan-Meier survival analysis showed that a high-expression level of BMP7 resulted in a significantly poor prognosis of HCC patients. Multivariate analysis revealed that BMP7 expression level was an independent prognostic parameter for the overall survival rate of HCC patients. These findings provide evidence that a high-expression level of BMP7 serves as a biomarker for poor prognosis for HCC. Thus, we speculate that BMP7 may be a potential target of antiangiogenic therapy for HCC.

α2-Adrenoceptor agonist dexmedetomidine protects septic acute kidney injury through increasing BMP-7 and inhibiting HDAC2 and HDAC5

Bone morphogenetic protein (BMP)-7 protects sepsis-induced acute kidney injury (AKI). Dexmedetomidine (DEX), an α(2)-adrenoceptor (α(2)-AR) agonist, has anti-inflammatory effects. We investigated the protective effects of DEX on sepsis-induced AKI and the expression of BMP-7 and histone deacetylases (HDACs). In vitro, the effects of DEX or trichostatin A (TSA, an HDAC inhibitor) on TNF-α, monocyte chemotactic protein (MCP-1), BMP-7, and HDAC mRNA expression in LPS-stimulated rat renal tubular epithelial NRK52E cells, was determined using real-time PCR. In vivo, mice were intraperitoneally injected with DEX (25 μg/kg) or saline immediately and 12 h after cecal ligation and puncture (CLP) surgery. Twenty-four hours after CLP, we examined kidney injury and renal TNF-α, MCP-1, BMP-7, and HDAC expression. Survival was monitored for 120 h. LPS increased HDAC2, HDAC5, TNF-α, and MCP-1 expression, but decreased BMP-7 expression in NRK52E cells. DEX treatment decreased the HDAC2, HDAC5, TNF-α, and MCP-1 expression, but increased BMP-7 and acetyl histone H3 expression, whose effects were blocked by yohimbine, an α(2)-AR antagonist. With DEX treatment, the LPS-induced TNF-α expression and cell death were attenuated in scRNAi-NRK52E but not BMP-7 RNAi-NRK52E cells. In CLP mice, DEX treatment increased survival and attenuated AKI. The expression of HDAC2, HDAC5, TNF-α, and MCP-1 mRNA in the kidneys of CLP mice was increased, but BMP-7 was decreased. However, DEX treatment reduced those changes. DEX reduces sepsis-induced AKI by decreasing TNF-α and MCP-1 and increasing BMP-7, which is associated with decreasing HDAC2 and HDAC5, as well as increasing acetyl histone H3.

Stage-specific expression and effect of bone morphogenetic protein 2 on bovine granulosa cell estradiol production: regulation by cocaine and amphetamine regulated transcript

Members of the bone morphogenetic protein (BMP) family regulate follicular development and granulosa cell function. However, changes in expression of BMP2 and its receptors during follicular waves in cattle and ability of BMP2 to modulate bovine granulosa cell estradiol production are not well understood. The objectives of this study were to determine temporal regulation of mRNA for BMP2 and its type I and II receptors (BMPR1A and BMPR2) in bovine follicles collected at specific stages of a follicular wave (predeviation, early dominance, mid dominance, preovulatory), ability of BMP2 to modulate bovine granulosa cell steroidogenesis, and whether effects of BMP2 on granulosa cell estradiol production are influenced by cotreatment with cocaine- and amphetamine-regulated transcript (CART), an intrafollicular regulatory peptide shown to inhibit estradiol production in response to other trophic hormones (FSH and IGF1). Relative abundance of mRNAs for Bmp2 and Bmpr2 was elevated at the mid dominance stage relative to earlier stages of the follicular wave and further increased at the preovulatory stage. Abundance of mRNA for Bmpr1a was lowest at early dominance stage and highest at preovulatory stage relative to other stages of the follicular wave examined. Treatment of bovine granulosa cells in vitro with BMP2 increased estradiol but decreased progesterone concentrations. Co-incubation with CART reduced the BMP2-stimulated increase in granulosa cell estradiol production. Results suggest that BMP2 may play a regulatory role in development of bovine follicles to the preovulatory stage and that CART can inhibit granulosa cell estradiol production in response to multiple hormones/growth factors, including BMP2.

PARM-1 promotes cardiomyogenic differentiation through regulating the BMP/Smad signaling pathway

PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.

Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Polymicrogyria is a malformation of the developing cerebral cortex caused by abnormal organization and characterized by many small gyri and fusion of the outer molecular layer. We have identified autosomal-recessive mutations in RTTN, encoding Rotatin, in individuals with bilateral diffuse polymicrogyria from two separate families. Rotatin determines early embryonic axial rotation, as well as anteroposterior and dorsoventral patterning in the mouse. Human Rotatin has recently been identified as a centrosome-associated protein. The Drosophila melanogaster homolog of Rotatin, Ana3, is needed for structural integrity of centrioles and basal bodies and maintenance of sensory neurons. We show that Rotatin colocalizes with the basal bodies at the primary cilium. Cultured fibroblasts from affected individuals have structural abnormalities of the cilia and exhibit downregulation of BMP4, WNT5A, and WNT2B, which are key regulators of cortical patterning and are expressed at the cortical hem, the cortex-organizing center that gives rise to Cajal-Retzius (CR) neurons. Interestingly, we have shown that in mouse embryos, Rotatin colocalizes with CR neurons at the subpial marginal zone. Knockdown experiments in human fibroblasts and neural stem cells confirm a role for RTTN in cilia structure and function. RTTN mutations therefore link aberrant ciliary function to abnormal development and organization of the cortex in human individuals.

L51P — A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice

In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

Effect of iron overload and iron deficiency on liver hemojuvelin protein.

IntroductionHemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency.MethodsC57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv−/− mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit.ResultsLiver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv−/− mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA.DiscussionSince no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.