Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is induced by BMP6. (A) Hep3B cells were treated with 5, 25, and 50 ng/mL of human BMP6 for 16 hours and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 3 to 8 (depending of the dose) independent experiments is presented. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock. (B-C) Hep3B cells were transfected with siRNA control (5nM), siRNA TMPRSS6 (5nM), or TMPRSS6-FLAG (8 μg), and treated with 25 ng/mL of BMP6 for 48 hours. Cells were analyzed for matriptase-2 level relative to pan-cadherin protein by Western blot (B) followed by chemiluminescence quantification (C). (B) *A shorter exposure of lane 5 to better distinguish the 2 bands. (C) The mean of 3 experiments is presented, and results are reported as the mean ± SEM. (D) A total of 15 μg of protein from conditioned media of Hep3B cells transfected with siRNA control (5nM) and siRNA TMPRSS6 (5nM) and treated with BMP6 (25 ng/mL) for 48 hours were incubated with 666μM of N-(tert-butoxycarbonyl)-Gln-Ala-Arg-p-nitroanilide. Activity of matriptase-2 was assessed by measurement of the release of the dye p-nitroaniline during up to 20 minutes at a wavelength of 405 nm at 37°C using a spectrophotometer. The resulting activities (1 U corresponds to a release rate of 1 mmol of p-nitroaniline per minute) were measured in duplicate in 3 independent experiments. Results are reported as the mean ± SEM. (A,C-D) Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Incubation, Activity Assay, Spectrophotometry

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Regulation of TMPRSS6 mRNA expression in response to BMP6. Hep3B cells were treated with 25 ng/mL of BMP6 for several time points between 1 and 48 hours (A), and were analyzed for hepcidin and TMPRSS6 relative to RPL19 mRNA expression by quantitative real-time RT-PCR. (B-C) Hep3B cells received 10 μg/mL of cycloheximide (B) or 60nM of LDN-193189 (C) before BMP6 (25 ng/mL) treatment and were analyzed for gene expression relative to RPL19 mRNA expression by quantitative real-time RT-PCR. The mean of 6 (A-B) and 3 (C) independent experiments is presented. (B-C) Results are reported as the mean ± SEM for the fold change from mock (just before adding BMP6) and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; **P < .01; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is controlled by ID1 in response to BMP6. Hep3B cells transfected with 10nM of siRNA control, siRNA SMAD7 (A-B), or siRNA ID1 (C-D), and treated in the absence or presence of 25 ng/mL of BMP6 for 24 hours were analyzed for TMPRSS6 (B,D) SMAD7 (A), and ID1 (C) mRNA expression relative to RPL19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock, and significant changes represent the comparisons with mock (siRNA control alone). Significant changes are as follows: *P < .05.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: TMPRSS6 expression is up-regulated in vivo by BMP6 and chronic iron treatment. (A) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of BMP6 750 μg/kg animal weight (BMP6, black bars) or vehicle alone (mock, gray bars; n = 3 per group) for 6 and 12 hours. (B) Eight-week-old male C57BL/6 mice received an intraperitoneal injection of neutralizing BMP6 antibody 15 mg/kg once a day for 1 week (n = 5 per group). (C) Seven-week-old male C57BL/6 mice were killed at time zero (baseline) or after initiation of a 2% carbonyl iron diet for 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks (n = 6 per group). Tissues were analyzed for hepatic hepcidin and Tmprss6 relative to Rpl19 mRNA by quantitative real-time RT-PCR. Results are reported as the mean ± SEM for the fold change from mock and significant changes represent the comparisons with mock. Significant changes are as follows: *P < .05; and ***P < .005.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, In Vivo, Injection, Quantitative RT-PCR

Journal: Blood

Article Title: Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

doi: 10.1182/blood-2011-04-348698

Figure Lengend Snippet: Schematic representation showing proposed role of TMPRSS6 regulation by the BMP6-SMAD signaling pathway and iron via ID1. We propose that, in addition to being stimulated by several signals that inhibit hepcidin, such as iron deficiency, erythropoeitic drive, and hypoxia, TMPRSS6 expression is also stimulated indirectly by the hepcidin activators BMP6 and iron. Stimulation by BMP6 and/or iron induces an increase of the BMP6-HJV-SMAD pathway activity, possibly through a mechanism involving HFE and transferrin receptor 2 (TFR2), leading to binding of SMAD complexes to BMP-responsive elements (BMP-REs) on the hepcidin promoter and up-regulation of hepcidin transcription. In parallel, BMP6-SMAD pathway directly up-regulates SMAD7 and ID1 transcription. ID1 induction leads to the up-regulation of TMPRSS6 expression. TMPRSS6 then serves as a negative feedback inhibitor of BMP-SMAD pathway activity and hepcidin expression by cleaving the BMP coreceptor HJV. Inhibitory SMAD7 can also act as a negative feedback inhibitor by blocking SMAD activation. By acting as negative feedback inhibitors, TMPRSS6 and SMAD7 are important to prevent excessive hepcidin increases in response to BMP6 and iron, thereby maintaining tight control of iron homeostasis. Abbreviations: BMPR indicates BMP receptor; TF-Fe, holotransferrin; sHJV, soluble hemojuvelin; and BMP-RE, BMP responsive element.

Article Snippet: After 16 hours, cells were treated with recombinant human BMP6 (25 ng/mL; R&D Systems) and then harvested for total RNA extraction 1, 4, 9, 16, 24, 32, and 48 hours after treatment.

Techniques: Expressing, Activity Assay, Binding Assay, Blocking Assay, Activation Assay, Control

Journal: Experimental & Molecular Medicine

Article Title: Hepatocyte toll-like receptor 4 mediates lipopolysaccharide-induced hepcidin expression

doi: 10.1038/emm.2017.207

Figure Lengend Snippet: LPS induces hepcidin expression by hepatocytes. ( a ) AML12 cells were treated with different concentrations of LPS for 24 h, and the expression of mouse hepcidin (Hamp1) mRNA was measured by quantitative real time polymerase chain reaction (qRT-PCR). ( b ) AML12 cells were treated with LPS (1 μg ml −1 ) for the designated times. ( c ) Expression of mRNA encoding inducible nitric oxide synthase (iNOS) and Hamp1 in AML12 cells treated with 1 μg ml −1 LPS for 24 h. ( d ) AML12 cells were treated for 12 h with BMP6 (20 ng ml −1 ), IL-6 (20 ng ml −1 ) or LPS (1 μg ml −1 ), and the expression of Hamp1 mRNA was measured by qRT-PCR. ( e ) Hepcidin concentration in cell culture medium from LPS (1 μg ml −1 )-treated AML12 cells. ( f ) Iron concentration in AML12 cells treated with LPS (1 μg ml −1 ) for 24 h.

Article Snippet: LPS ( Escherichia coli 026:B6, L2654, Sigma Aldrich, St Louis, MO, USA), BMP6 (6325-BM, R&D Systems, Minneapolis, MN, USA) and IL-6 (CYT-213, PROSPEC, Ness-Ziona, Israel) were dissolved in manufacturer-recommended solvents.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Cell Culture