Serum Iron Overload Activates the SMAD Pathway and Hepcidin Expression of Hepatocytes via SMURF1.

Background and Aims: Liver iron overload can induce hepatic expression of bone morphogenic protein (BMP) 6 and activate the BMP/SMAD pathway. However, serum iron overload can also activate SMAD but does not induce BMP6 expression. Therefore, the mechanisms through which serum iron overload activates the BMP/SMAD pathway remain unclear. This study aimed to clarify the role of SMURF1 in serum iron overload and the BMP/SMAD pathway. Methods: A cell model of serum iron overload was established by treating hepatocytes with 2 mg/mL of holo-transferrin (Holo-Tf). A serum iron overload mouse model and a liver iron overload mouse model were established by intraperitoneally injecting 10 mg of Holo-Tf into C57BL/6 mice and administering a high-iron diet for 1 week followed by a low-iron diet for 2 days. Western blotting and real-time PCR were performed to evaluate the activation of the BMP/SMAD pathway and the expression of hepcidin. Results: Holo-Tf augmented the sensitivity and responsiveness of hepatocytes to BMP6. The E3 ubiquitin-protein ligase SMURF1 mediated Holo-Tf-induced SMAD1/5 activation and hepcidin expression; specifically, SMURF1 expression dramatically decreased when the serum iron concentration was increased. Additionally, the expression of SMURF1 substrates, which are important molecules involved in the transduction of BMP/SMAD signaling, was significantly upregulated. Furthermore, in vivo analyses confirmed that SMURF1 specifically regulated the BMP/SMAD pathway during serum iron overload. Conclusions: SMURF1 can specifically regulate the BMP/SMAD pathway by augmenting the responsiveness of hepatocytes to BMPs during serum iron overload.

A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo.

The Activin A Receptor type I (ALK2) is a critical component of BMP-SMAD signaling that, in the presence of ligands, phosphorylates cytosolic SMAD1/5/8 and modulates important biological processes, including bone formation and iron metabolism. In hepatocytes, the BMP-SMAD pathway controls the expression of hepcidin, the liver peptide hormone that regulates body iron homeostasis via the BMP receptors ALK2 and ALK3, and the hemochromatosis proteins. The main negative regulator of the pathway in the liver is transmembrane serine protease 6 (TMPRSS6), which downregulates hepcidin by cleaving the BMP coreceptor hemojuvelin. ALK2 function is inhibited also by the immunophilin FKBP12, which maintains the receptor in an inactive conformation. FKBP12 sequestration by tacrolimus or its silencing upregulates hepcidin in primary hepatocytes and in vivo in acute but not chronic settings. Interestingly, gain-of-function mutations in ALK2 that impair FKBP12 binding to the receptor and activate the pathway cause a bone phenotype in patients affected by Fibrodysplasia Ossificans Progressiva but not hepcidin and iron metabolism dysfunction. This observation suggests that additional mechanisms are active in the liver to compensate for the increased BMP-SMAD signaling. Here we demonstrate that Fkbp12 downregulation in hepatocytes by antisense oligonucleotide treatment upregulates the expression of the main hepcidin inhibitor Tmprss6, thus counteracting the ALK2-mediated activation of the pathway. Combined downregulation of both Fkbp12 and Tmprss6 blocks this compensatory mechanism. Our findings reveal a previously unrecognized functional cross talk between FKBP12 and TMPRSS6, the main BMP-SMAD pathway inhibitors, in the control of hepcidin transcription.NEW & NOTEWORTHY This study uncovers a previously unrecognized mechanism of hepcidin and BMP-SMAD pathway regulation in hepatocytes mediated by the immunophilin FKBP12 and the transmembrane serine protease TMPRSS6.

Osteoprotective Effect and Mechanisms of Taxillus chinensis from Different Hosts on Ovariectomized Osteoporotic Rats / 中国实验方剂学杂志

ObjectiveTo compare the effects of Taxillus chinensis from different hosts with different meridian affinity on bone microstructure and bone metabolism in ovariectomized osteoporotic rats， and investigate its mechanism of action. MethodEighty-eight specific-pathogen-free （SPF）-grade female Sprague-Dawley （SD） rats were selected and randomly divided into 11 groups： sham-operated group， model group， low-， medium- and high-dose groups of T. chinensis from Morus alba （2.5， 5， and 10 g·kg-1）， low-， medium- and high-dose groups of T. chinensis from Cinnamomum cassia （2.5， 5， and 10 g·kg-1）， and low-， medium- and high-dose groups of T. chinensis from C. burmannii （2.5， 5， and 10 g·kg-1）. After 12 weeks of drug intervention， the rats were examined for proximal femur bone density and bone microstructure using dual-energy X-ray absorptiometry （DXA） and micro-computed tomography （Micro-CT）. Histopathological changes in rat femur were observed by the hematoxylin-eosin staining （HE）. Contents of serum estradiol （E2）， bone Gla protein （BGP）， bone alkaline phosphatase （BALP）， tartrate-resistant acid phosphatase 5b （TRACP-5b） and pre-collagen type Ⅰ amino-terminal protopeptide （PINP） were measured by the enzyme-linked immunosorbent assay （ELISA）. Real-time quantitative polymerase chain reaction （Real-time PCR） was employed to detect the messenger ribonucleic acid （mRNA） expressions of bone morphogenetic protein-2 （BMP-2）， Smad1， Smad9 and recombinant runt-related transcription factor 2 （Runx2） in rat humerus. Western blot was used to detect the protein expressions of BMP-2， p-Smad1/5/9 and Runx2 in rat humerus. ResultCompared with that in the sham-operated group， the femur microstructure of rats in the model group was significantly disrupted， with significant decreases in bone mineral density （BMD） value， bone volume fraction （BV/TV）， trabecular number （Tb.N）， and trabecular thickness （Tb.Th） （P<0.01）， and significant increases in trabecular separation （Tb.Sp） and structure model index （SMI） （P<0.01）. The serum levels of BGP， BALP， TRACP-5b and PINP were significantly increased （P<0.05， P<0.01）， and E2 levels were significantly decreased （P<0.01）. The mRNA expressions of BMP-2， Smad1， Smad9， and Runx2 were significantly decreased in rat humerus （P<0.01）， and the protein expressions of BMP-2， p-Smad1/5/9， and Runx2 were significantly reduced （P<0.01）. Compared with the model group， the administration groups of T. chinensis from different hosts all elevated the BMD， BV/TV， Tb.N， Tb.Th， Tb.Sp， and SMI levels in the femur， improved bone microstructure， increased serum E2 levels （P<0.05， P<0.01）， lowered the levels of serum BGP， BALP， TRACP-5b， and PINP， upregulated the mRNA expression of BMP-2， Smad1， and Runx2 and upregulated the mRNA expression levels of Smad9 （P<0.05， P<0.01）， and upregulated the protein expressions levels of BMP-2， p-Smad1/5/9， and Runx2 （P<0.01）. The best effect was observed in the group of T. chinensis from C. cassia. ConclusionT. chinensis from different hosts improved osteoporosis in ovariectomized rats， with the group of T. chinensis from C. cassia being the most potent among the administered groups， and its treatment of osteoporosis may regulate the balance of bone conversion by regulating BMP/Smad signaling pathway.

Exploring the boost of steaming with wine on Ligustri Lucidi Fructus in treating postmenopausal osteoporosis based on superior "multi-component structure" and iron/bone metabolism coregulation.

BACKGROUND: Clinical studies indicated that postmenopausal osteoporosis (PMOP) often accompanied by iron overload risk factor, which exacerbated bone metabolism disorders and accelerated PMOP. Previous research found that multicomponent in Ligustri Lucidi Fructus (FLL) or wine-steamed FLL (WFLL) acted on the common targets of iron overload and PMOP simultaneously, which indicated that FLL and WFLL probably regulated iron/bone metabolism dually. Additionally, WFLL had more superior effect according to the theory of Chinese medicine for thousands of years. PURPOSE: To reveal the "superior multi-component structure (SMCS)" and its molecular mechanisms in parallelly down-regulating iron overload and rescuing bone metabolism by WFLL. DESIGNS AND METHODS: HPLC fingerprinting was established to compare the chemical profiles of FLL and WFLL; Then, the chemical compositions and quality markers of FLL and WFLL were analyzed by UPLC-Orbitrap-MS/MS coupled with OPLS-DA; the dynamic contents of quality markers and the multi-component structure at different wine steaming times (WST) were simultaneously determined by HPLC-DAD. Meanwhile, the dynamic efficacy of FLL at different WST were hunt by systematic zebrafish model. Subsequently, potential mechanism of WFLL in treating PMOP accompanied with iron overload was obtained from network pharmacology (NP) and molecular docking (MD). Finally, zebrafish and ovariectomy rat model were carried out to validate this potential mechanism. RESULTS: HPLC fingerprints similarity of 15 batches in FLL and WFLL were among 0.9-1.0. 126 compositions were identified, including 58 iridoids, 25 terpenes, 30 phenylethanoids, 7 flavonoids and 6 others. 20 quality markers associated with WFLL was revealed, and the ratio of phenylethanols: Iridoids: Triterpenes (P/I/T) was converted from 1: 15: 4.5 to 1: 0.8: 0.9 during steaming (0 - 24 h) calculated by the quantification of 11 quality markers; the bone mineralization and motor performance of zebrafish larvae indicated that the optimum efficacy of WFLL at 12 h (p < 0.05) in which the SMCS of P/I/T was converted to 1: 4: 1.8. NP discovered that BMP-Smad pathway is one of the potential mechanisms of FLL in anti PMOP and then regulated bone formation and iron overload simultaneously. MD revealed that 17 active ingredients and 10 core targets genes could spontaneously bind with appropriate affinity. Rats model verified that FLL and WFLL significantly reversed PMOP, based on the improvement in bone formation indexes (ALP, OPG, OGN), iron metabolism indicators (hepcidin, ferritin), bone microstructure (BMD, BV/TV, Tb. Th, Tb. N); Moreover, WFLL significant enhanced reversal effect in anti-PMOP compared to FLL (p < 0.05). FLL and WFLL increased genes and proteins expression (Hep, BMP-6, p-Smad1/5, Smad4) related to BMP-Smad pathway compared with model group, and WFLL was more superior than FLL (p< 0.05). CONCLUSION: The SMCS of FLL was optimized by wine-steam, WFLL represented a dual effect in downregulating iron overload and promoting bone formation, and the BMP-Smad pathway is one of the potential molecular mechanisms.

Ataluren prevented bone loss induced by ovariectomy and aging in mice through the BMP-SMAD signaling pathway.

Both estrogen deficiency and aging may lead to osteoporosis. Developing novel drugs for treating osteoporosis is a popular research direction. We screened several potential therapeutic agents through a new deep learning-based efficacy prediction system (DLEPS) using transcriptional profiles for osteoporosis. DLEPS screening led to a potential novel drug examinee, ataluren, for treating osteoporosis. Ataluren significantly reversed bone loss in ovariectomized mice. Next, ataluren significantly increased human bone marrow-derived mesenchymal stem cell (hBMMSC) osteogenic differentiation without cytotoxicity, indicated by the high expression index of osteogenic differentiation genes (OCN , BGLAP, ALP, COL1A, BMP2, RUNX2). Mechanistically, ataluren exerted its function through the BMP-SMAD pathway. Furthermore, it activated SMAD phosphorylation but osteogenic differentiation was attenuated by BMP2-SMAD inhibitors or small interfering RNA of BMP2. Finally, ataluren significantly reversed bone loss in aged mice. In summary, our findings suggest that the DLEPS-screened ataluren may be a therapeutic agent against osteoporosis by aiding hBMMSC osteogenic differentiation.

Curculigo orchioides polysaccharide COP70-1 stimulates osteogenic differentiation of MC3T3-E1 cells by activating the BMP and Wnt signaling pathways.

The crude polysaccharide CO70 isolated from Curculigo orchioides could alleviate ovariectomy-induced osteoporosis in rats. To clarify the bioactive components, a new heteropolysaccharide (COP70-1) was purified from CO70 in this study, which was consisted of ß-D-Manp-(1→, →4)-α-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →3,4)-ß-D-Manp-(1→, →4,6)-ß-D-Manp-(1→, and →4,6)-α-D-Galp-(1→. COP70-1 significantly promoted the osteoblastic differentiation of MC3T3-E1 cells through improving alkaline phosphatase activity, the deposition of calcium as well as up-regulating the expression of osteogenic markers (RUNX2, OSX, BSP, OCN, and OPN). Furthermore, COP70-1 stimulated the expression of critical transcription factors of the BMP and Wnt pathways, including BMP2, p-SMAD1, active-ß-catenin, p-GSK-3ß, and LEF-1. In addition, LDN (BMP pathway inhibitor) and DKK-1 (Wnt pathway inhibitor) suppressed the COP70-1-induced osteogenic differentiation of MC3T3-E1 cells. Therefore, COP70-1 was one of the bioactive constituents of C. orchioides for targeting osteoblasts to treat osteoporosis by triggering BMP/Smad and Wnt/ß-catenin pathways.

Functional mechanism of miR-92b-3p in osteogenic differentiation of fibroblasts in patients with ankylosing spondylitis via the TOB1/BMP/Smad pathway.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory arthritis. Upregulation of microRNA (miR)-92b-3p is associated with enhanced osteoblastic differentiation. The current study sought to investigate the functional mechanism of miR-92b-3p in osteogenic differentiation of AS fibroblasts. METHODS: First, fibroblasts were isolated from AS and non-AS patients and cultured. Next, cell morphology was observed, cell proliferation was assessed and the vimentin expression pattern was determined. Alkaline phosphatase (ALP) activity and levels of osteogenic markers RUNX2, OPN, OSX, and COL I were additionally measured, followed by determination of miR-92b-3p and TOB1 levels. The binding site of miR-92b-3p and TOB1 was predicted, and their target relationship was validated. Lastly, miR-92b-3p inhibitor, si-TOB1, and the BMP/Smad signaling pathway inhibitor LDN193189 were delivered into AS fibroblasts to evaluate the osteogenic differentiation of AS fibroblasts and the activation of the BMP/Smad pathway. RESULTS: miR-92b-3p was highly expressed in AS fibroblasts. AS fibroblasts showed enhanced osteogenic differentiation and proliferation, while inhibition of miR-92b-3p suppressed osteogenic differentiation and proliferation of AS fibroblasts. miR-92b-3p targeted TOB1, and TOB1 was poorly expressed in AS fibroblasts. The concurrent downregulation of TOB1 and inhibition of miR-92b-3p elevated the levels of RUNX2, OPN, OSX, and COL I and ALP activity and further enhanced the proliferation of AS fibroblasts. The BMP/Smad pathway was activated in AS fibroblasts. Silencing miR-92b-3p could inhibit the activation of the BMP/Smad pathway by upregulating TOB1. Inhibition of the BMP/Smad pathway reduced the number of calcified nodules and hindered the osteogenic differentiation and proliferation of AS fibroblasts. CONCLUSION: Our findings highlighted that silencing miR-92b-3p inhibited the osteogenic differentiation and proliferation of AS fibroblasts by upregulation of TOB1 and inhibition of the BMP/Smad pathway.

Progress on the effect of FecB mutation on BMPR1B activity and BMP/SMAD pathway in sheep.

BMPR1B is the first major gene of litter size identified in sheep. However, the molecular mechanism of the FecB mutation that increases the ovulation rate in sheep is still unclear. In recent years, it has been demonstrated that BMPR1B activity is regulated by the small molecule repressor protein FKBP1A, which acts as a key activity switch of the BMPR1B in the BMP/SMAD pathway. The FecB mutation is located close to the binding site of FKBP1A and BMPR1B. In this review, we summarize the structure of BMPR1B and FKBP1A proteins, and clarify the spatial interactive domains of the two proteins with respect to the location of the FecB mutation. Then the relationship between the FecB mutation and the degree of affinity of the two proteins are predicted. Finally, the hypothesis that FecB mutation causes change of activity in BMP/SMAD pathway by affecting the intensity of the interactions between BMPR1B and FKBP1A is proposed. This hypothesis provides a new clue to investigate the molecular mechanism of FecB mutation affecting ovulation rate and litter size in sheep.

Histone methyltransferase SETDB1 promotes osteogenic differentiation in osteoporosis by activating OTX2-mediated BMP-Smad and Wnt/ß-catenin pathways.

Osteogenic differentiation plays important roles in the pathogenesis of osteoporosis. In this study, we explored the regulatory mechanism of histone methyltransferase SET domain bifurcated 1 (SETDB1) underlying the osteogenic differentiation in osteoporosis. The common osteoporosis-related genes were retrieved from the GeneCards, CTD, and Phenolyzer databases. The enrichment analysis was conducted on the candidate osteoporosis-related genes using the PANTHER software, and the binding site between transcription factors and target genes predicted by hTFtarget. The bioinformatics analyses suggested 6 osteoporosis-related chromatin/chromatin binding protein or regulatory proteins (HDAC4, SIRT1, SETDB1, MECP2, CHD7, and DKC1). Normal and osteoporosis tissues were collected from osteoporosis patients to examine the expression of SETDB1. It was found that SETDB1 was poorly expressed in osteoporotic femoral tissues, indicating that SETDB1 might be involved in the development of osteoporosis. We induced SETDB1 overexpression/knockdown, orthodenticle homeobox 2 (OTX2) overexpression, activation of Wnt/ß-catenin or BMP-Smad pathways alone or in combination in osteoblasts or ovariectomized mice. The data indicated that SETDB1 methylation regulated H3K9me3 in the OTX2 promoter region and inhibited the expression of OTX2. Besides, the BMP-Smad and Wnt/ß-catenin pathways were inhibited by OTX2, thereby resulting in inhibited osteogenic differentiation. Animal experiments showed that overexpressed SETDB1 could promote the increase of calcium level and differentiation of femoral tissues. In conclusion, upregulation of SETDB1 promotes osteogenic differentiation by inhibiting OTX2 and activating the BMP-Smad and Wnt/ß-catenin pathways in osteoporosis.

Efficacy of total flavonoids of Rhizoma drynariae on the blood vessels and the bone graft in the induced membrane.

BACKGROUND: Total flavonoids of Rhizoma drynariae (TFRD), a Chinese medicine, is widely used in the treatment of orthopedic diseases. However, there are few basic and clinical studies on the effect of TFRD on induced membrane technique (Masquelet technique). PURPOSE: This trial is to explore effects of TFRD on vascularization of the induced membrane, and mineralization of the bone graft in rats with femoral bone defects. STUDY DESIGN AND METHODS: Forty-eight Sprague-Dawley rats were randomly divided into high dose group (H-TFRD), medium dose group (M-TFRD), low dose group (L-TFRD) and control group (control). The segmental bone defects were established with 12 rats in per group. The polymethyl methacrylate (PMMA) spacer was implanted into the femoral bone defect of rats in the first-stage surgery. About 4 weeks after first-stage surgery, induced membranes of 6 rats in each group were selected. The blood vessels and angiogenesis-related factors in the induced membrane were analyzed by hematoxylin-eosin (HE) and masson staining, western blot, qPCR and immunohistostaining. The remaining rats in per group underwent second-stage surgery (bone grafting). Twelve weeks after the bone grafting, the bone tissues was examined by X-ray, micro-computed tomography (Micro-CT), HE staining and enzyme-linked immunosorbent assay (ELISA) to evaluate the growth of the bone graft. Meanwhile, the TFRD-containing serum was collected from rats to culture osteoblasts in vitro. Cell Counting Kit-8 (CCK-8) method, Alizarin Red S (ARS) staining, western blot and immunofluorescence were used to detect effects of TFRD on the osteoblasts' proliferation and BMP-SMAD signaling pathway. RESULTS: Compared with the L-TFRD and control groups, the number of blood vessels and the expression of angiogenesis-related factors (VEGF, TGF-ß1, BMP-2, PDGF-BB and CD31) were higher in the H-TFRD and M-TFRD groups. The Lane-Sandhu X-ray score, bone mass and growth rate of the bone graft in the H-TFRD and M-TFRD groups were significantly better than those in the L-TFRD and control groups. In addition, medium and high doses of TFRD significantly increased the expression of BMP-SMAD pathway proteins (BMP-2, SMAD1, SMAD4, SMAD5 and RUNX2) in rat serum and bone graft. In vitro, after osteoblasts were intervened with TFRD-containing serum from the H-TFRD and M-TFRD groups, the cell viability, the number of mineralized nodules and the phosphorylation of BMP-SMAD pathway proteins were markedly increased. CONCLUSION: TFRD could promote the formation of blood vessels and the expression of angiogenesis-related factors during the formation of the induced membrane. During the growing period of bone graft, it could facilitate the growth and mineralization of bone graft in a dose-dependent manner, which is partly related to the activation and phosphorylation of BMP-SMAD signaling pathway.

Low-level laser irradiation enhances the proliferation and osteogenic differentiation of PDLSCs via BMP signaling.

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs). We designed one control group, without irradiation and four testing groups, treated with LLLT (Nd:YAG;1064 nm) at 2, 4, 6, and 8 J/cm2 for human PDLSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining, and the gene levels of runt-related transcription factor 2 (Runx2), ALP, osteocalcin, and bone morphogenetic protein 2 (BMP2). The effects of LLLT on secretion of TNF-α and IL-1ß in PDLSCs were measured by enzyme-linked immunosorbent assay. BMP/Smad pathway was measured through the expression of Smad1/5/8 phosphorylation (P-Smad1/5/8). LDN-193189, an inhibitor of the BMP/Smad pathway, was used to explore the underlying effects of BMP/Smad signaling on the process of LLLT regulating the proliferation and osteogenesis of PDLSCs. Our results demonstrated LLLT could promote the proliferation and osteogenesis of PDLSCs at 2-6 J/cm2 and LLLT at 8 J/cm2 significantly suppress osteogenic differentiation of PDLSCs. Moreover, LLLT stimulated the secretion of TNFα and IL-ß1. Finally, we found the irradiation positively modulates the P-Smad1/5/8 level. When the cells were treated with LDN-193189, the proliferation and osteogenic effects of LLLT on PDLSCs were attenuated. In conclusion, LLLT may upregulate the proliferation and bone formation ability of PDLSCs via the BMP/Smad signaling.

CircRFWD2 Promotes Osteogenic Differentiation of human Dental Pulp Stem Cells by Targeting miR-6817-5p Through BMP-Smad and p38 MAPK Pathway.

Dental pulp stem cells (DPSCs) are one promising cell source of mesenchymal stem cells in bone tissue engineering. However, it remains unknown that the molecules and signaling pathways involved in osteogenesis of DPSCs. Hence, this study investigated the functional roles and underlying mechanisms of circRFWD2 during osteogenesis of DPSCs. Knockdown of circRFWD2 suppressed osteogenesis of DPSCs significantly. Mechanistically, circRFWD2 could crosstalk with miR-6817-5p, which was an inhibitor of DPSCs osteogenesis. MiR-6817-5p functioned as a sponge of BMPR2, which regulated the phosphorylation of Smad5 and p38 to impact osteogenesis activity of DPSCs. Collectively, circRFWD2/miR-6817-5p/BMPR2 axis could regulate DPSCs osteogenesis via BMP-Smad and p38 MAPK pathway, which are novel mechanisms in the osteogenic differentiation of DPSCs and suggest potential therapeutic methods for bone defects regeneration.

[Corrigendum] Combination treatment of adipose­derived stem cells and adiponectin attenuates pulmonary arterial hypertension in rats by inhibiting pulmonary arterial smooth muscle cell proliferation and regulating the AMPK/BMP/Smad pathway.

Following the publication of the above article, an interested reader drew the authors' attention that the data featured in Fig. 1B (for adipogenic differentiation of adipose­derived stem cells) and Fig. 1F (for expression of green fluorescent protein of adipose­derived stem cells) of the above article appeared to have already been published as Fig. 1A (for adipogenic differentiation of adipose­derived stem cells) and Fig. 2D (for expression of green fluorescent protein of adipose­derived stem cells) in the following article: Luo L, Lin T, Zheng S, Xie Z, Chen M, Lian G, Xu C, Wang H and Xie L: Adipose­derived stem cells attenuate pulmonary arterial hypertension and ameliorate pulmonary arterial remodeling in monocrotaline­induced pulmonary hypertensive rats. Clin Exp Hypertens 37: 241­248, 2015. The authors consulted their original data and were able to determine that the duplication of these figure parts had arisen inadvertently during the process of compiling the figure. The revised version of Fig. 1, featuring the corrected data panels for the above­mentioned experiments in Fig. 1B and F, is shown on the next page. The authors confirm that the errors associated with this figure did not have any significant impact on either the results or the conclusions reported in this study, and are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum. Furthermore, they apologize to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 51­60, 2018; DOI: 10.3892/ijmm.2017.3226].

Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe-/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis.

The interplay between TGF-ß/SMAD and BMP/SMAD signaling pathways in the epithelial mesenchymal transition of A549 cells induced by silica.

The epithelial-mesenchymal transition (EMT) is a phenotype transdifferentiation of epithelial into mesenchymal cells and contributes to pulmonary fibrotic disease. SMAD-dependent pathway has been reported to play a key role in the multiple fibrotic diseases. We hypothesized that TGF-ß/SMAD signaling could cross-interact with BMP/SMAD signaling pathways in silica-induced EMT in A549 cells. We investigated that the ability of silica-induced EMT in A549 cells, and this process was significantly inhibited by SB431542 through up-regulation of Vimentin, α-SMA and collagen type I expression and down-regulation of E-cadherin expression. Whereas BMP/SMAD inhibition using LDN193189 enhanced EMT. In addition, we also demonstrated that SB431542 could enhance BMP/SMAD signaling pathways in silica-induced EMT and vice versa. Therefore, our study provides evidence that the TGF-ß/SMAD pathway was a crucial regulator in silica-induced EMT and that SB431542 could prevent the EMT. More importantly, we have identified that the interplay of TGF-ß/SMAD and BMP/SMAD pathways in silica-induced EMT in A549 cells.

Effect of BMP-7 on osteoblastic differentiation of rat periodontal ligament cells / 대한치주과학회지

Periodontal therapy has dealt primarily with attempts at arresting progression of disease, however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein (BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-1, BSP, and OCN was increased by BMP-7 in concentration of 10 ng/ml~100 ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.