Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes.

Hepcidin is a peptide hormone that targets the iron exporter ferroportin, thereby limiting iron entry into the bloodstream. It is generated in hepatocytes mainly in response to increased body iron stores or inflammatory cues. Iron stimulates expression of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells, which in turn binds to BMP receptors on hepatocytes and induces the SMAD signaling cascade for transcriptional activation of the hepcidin-encoding HAMP mRNA. SMAD signaling is also essential for inflammatory HAMP mRNA induction by the IL-6/STAT3 pathway. Herein, we utilized human Huh7 hepatoma cells and primary murine hepatocytes to assess the effects of iron perturbations on signaling to hepcidin. Iron chelation appeared to slightly impair signaling to hepcidin. Subsequent iron supplementation not only failed to reverse these effects, but drastically reduced basal HAMP mRNA and inhibited HAMP mRNA induction by BMP6 and/or IL-6. Thus, treatment of cells with excess iron inhibited basal and BMP6-mediated SMAD5 phosphorylation and induction of HAMP, ID1 and SMAD7 mRNAs in a dose-dependent manner. Iron also inhibited IL-6-mediated STAT3 phosphorylation and induction of HAMP and SOCS3 mRNAs. These responses were accompanied by induction of GCLC and HMOX1 mRNAs, known markers of oxidative stress. We conclude that hepatocellular iron overload suppresses hepcidin by inhibiting the SMAD and STAT3 signaling pathways downstream of their respective ligands.

Suppression of osteoblast-related genes during osteogenic differentiation of adipose tissue derived stromal cells.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.

Screening identifies the Chinese medicinal plant Caulis Spatholobi as an effective HAMP expression inhibitor.

Hepcidin, the pivotal regulator of iron metabolism, plays a critical role in multiple diseases including anemia of chronic disease and hemochromatosis. Recent studies have focused on identifying antagonists of hepcidin. We hypothesized that bioactive extracts from Chinese medicinal plants may be efficacious in the inhibition of expression of the hepcidin-encoding gene (HAMP) product, hepcidin. To test this, we measured the level of hepcidin expression in cultured cells treated with 16 different medicinal plant extracts, all of which are used to treat anemia-related disorders in traditional Chinese medicine. Among the extracts tested, that of Caulis Spatholobi (CS; also called Jixueteng, the stem of Spatholobus suberectus Dunn) showed the most potent inhibitory effect on HAMP expression in the Huh7 cell line and was therefore selected for further mechanistic study. In cells treated with 400 µg/mL of extract, phosphorylated mothers against decapentaplegic homolog proteins 1/5/8 levels were 80% less than those of controls (P < 0.001), and the inhibitory effect on interleukin-6-induced HAMP expression (65% inhibition) was weaker than the strong inhibition on bone morphogenetic protein 6-induced HAMP expression (97% inhibition). Seven-week-old C57BL/6 female mice were fed an AIN-76A diet containing 10.8% dried CS and then analyzed on d 0, 5, 10, or 15. On d 5, there was a 60% decrease in hepatic HAMP expression (P < 0.05), an 18% decrease in hepatic iron concentration, and a 100% increase in serum iron concentration (P < 0.05) compared with the d 0 group. In conclusion, we identify the extract of CS as a novel, potent HAMP expression inhibitor, which may be further modified and optimized to become a dietary supplement or a therapeutic option for the amelioration of hepcidin-overexpression-related diseases, including iron deficiency anemia.

Tailoring adipose stem cell trophic factor production with differentiation medium components to regenerate chondral defects.

Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 µg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-ß1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-ß1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-ß2, TGF-ß3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-ß2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-ß2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-ß1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-ß2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.

Bone morphogenetic protein-6 (BMP-6): mRNA expression and effect on steroidogenesis during in vitro maturation of porcine cumulus oocyte complexes.

Oocyte secreted factors (OSFs) have emerged as important factors for follicular development. The present study investigated the effect of the potential OSF bone morphogenic protein (BMP)-6 on steroidogenesis in porcine cumulus oocyte complexes during in vitro maturation. Cumulus oocyte complexes (COCs), cumulus complexes (CCs) without oocytes and CCs with supplemented BMP-6 were cultured for 0, 5, 26 or 46 h. BMP-6 transcripts were detected in oocytes and cumulus cells at all time points. In both cell types the mRNA expression was most intense after 5h, and decreased during further maturation. After 26 and 46 h of culture, CCs secreted significantly less 17ß-estradiol than COCs. This effect was reversed by adding BMP-6 to CCs cultures. In addition, a down-regulation of Cyp19A1, the rate-limiting enzyme of 17ß-estradiol synthesis, was detected in CC cultures after 5h. As seen for 17ß-estradiol secretion, the addition of BMP-6 caused a significant increase in Cyp19A1 mRNA levels after 5, 26 and 46 h of culture. Progesterone secretion and transcripts of steroidogenic marker proteins StAR and 3ß-HSD were not affected considerably by oocyte removal or addition of BMP-6. Furthermore, BMP-6 did not affect the activity of the mitogen-activated protein kinase. The results indicated that BMP-6 is a potential OSF and is involved in the prevention of premature luteinisation in cumulus cells via enhancing 17ß-estradiol synthesis.

Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid, and ß-glycerophosphate may not enhance osteogenic differentiation of human periodontal ligament cells.

This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6 and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and ß-glycerophosphate] on osteogenic differentiation of periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay, alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6+AA increased ALP activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.