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Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation

Andrea U. Steinbicker, Chetana Sachidanandan, Ashley J. Vonner, Rushdia Z. Yusuf, Donna Y. Deng, Carol S. Lai, Kristen M. Rauwerdink, Julia C. Winn, Borja Saez, Colleen M. Cook, Brian A. Szekely, Cindy N. Roy, Jasbir S. Seehra, Gregory D. Cuny, David T. Scadden, Randall T. Peterson, Kenneth D. Bloch and Paul B. Yu

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Impact of BMP signaling on IL-6–mediated regulation of hepcidin. HepG2 cells were pretreated with varying concentrations of LDN-193189 for 30 minutes and (A) stimulated with IL-6 (100 ng/mL) or (B) vehicle for 90 minutes. Hepcidin mRNA levels were measured by qRT-PCR (values are mean ± SEM, n ≥ 3, 1-way ANOVA P < .05, *P < .05 vs untreated control, #P < .05 vs IL-6–treated control). (C) HepG2 cells were pretreated with and without LDN-193189 (100nM) for 30 minutes and stimulated with or without IL-6 (100 ng/mL). Hepcidin mRNA levels were measured by qRT-PCR (1-way ANOVA P = .01, *P = .01 vs control, #P < .05 vs control, and †P < .02 vs IL-6–stimulated cells). (D) HepG2 cells were pretreated with noggin (1 μg/mL), ALK3-Fc (1 μg/mL), or vehicle for 30 minutes and incubated with IL-6 (100 ng/mL) or vehicle for 90 minutes, and hepcidin mRNA levels were measured by qRT-PCR (n = 3, 1-way ANOVA P < .0001, *P < .04 vs vehicle-treated controls, #P < .05 vs IL-6–treated controls). (E) HepG2 cells were treated with either vehicle, BMP6 (2.5 ng/mL), IL-6 (100 ng/mL), or BMP6 and IL-6, and hepcidin mRNA levels were measured by qRT-PCR (n = 3, 1-way ANOVA P = .003, *P < .05 vs untreated control, #P < .05 vs IL-6–treated control). (F) Protein extracts were prepared from HepG2 cells treated either with vehicle, BMP6 (10 ng/mL), IL-6 (100 ng/mL), or BMP6 and IL-6 for 30 minutes. Phosphorylated SMAD1/5/8 and total SMAD1 were detected by immunoblot techniques.

  • Figure 2

    LDN-193189 inhibits the induction of hepcidin by inflammation in zebrafish. (A) Human IL-6 protein was detected in homogenates of double transgenic (cmlc-IL-6) but not in those of WT zebrafish hearts (arrows). β-actin was used to control for protein loading. (B) Hepcidin mRNA levels were measured by qRT-PCR in the livers of 7-day-old WT and cmlc-IL-6 zebrafish treated with vehicle or LDN-193189 (6μM) for 16 hours. (n = 3, *,†P < .05 vs WT, #P < .05 vs cmlc-IL-6).

  • Figure 3

    LDN-193189 inhibits short-term IL-6– and turpentine-induced hypoferremia and hepcidin expression. (A) IL-6 (16 μg) or vehicle were injected IV into 10-week-old mice pretreated with or without LDN-193189 (3 mg/kg IP) or vehicle. After 2 hours, livers were harvested, and hepcidin mRNA levels were measured using qRT-PCR (n ≥ 3 each group, 1-way ANOVA P < .005, *P < .05 vs control, †P < .05 vs saline, #P < .05 vs IL-6–stimulated mice). (B-C) Mice were pretreated with LDN-193189 (3 mg/kg IP repeated each 12 hours) or drug vehicle, followed by a single intrascapular injection with turpentine (5 mL/kg) or saline. Twenty-four hours after the turpentine injection, hepcidin mRNA levels were measured by qRT-PCR (B), and serum iron levels were assayed (C; n ≥ 4, *P ≤ .01 vs untreated controls, #P < .05 vs turpentine). (D) Mice were pretreated with ALK3-Fc (2 mg/kg IP) or vehicle, followed by intrascapular injection with turpentine or saline, and serum iron levels measured (n ≥ 5, *P < .000 01 vs untreated control, #P < .05 vs turpentine). Serum IL-6 levels (E) were determined by ELISA before and 6, 12, 24, 48, and 96 hours after turpentine injection with or without LDN-193189 (3 mg/kg) administered every 12 hours (n = 5 mice per time point and treatment). Hepcidin gene expression in the livers of treated mice was measured by qRT-PCR at 0, 24 and 96 hours (F; n = 5, 1-way ANOVA P = .003, *P < .05 vs 0 hours, #P < .01 vs vehicle at 24 hours, †P = .01 vs vehicle at 96 hours). Levels of phosphorylated-SMAD1/5/8 (p-SMAD) and SMAD1 proteins (G) in the livers of mice treated with vehicle, turpentine, or LDN-193189 were measured by immunoblot (n = 4 mice each).

  • Figure 4

    LDN-193189 prevents the development of turpentine-induced anemia. C57BL/6 mice were injected once a week with turpentine (5 mL/kg intrascapularly) or saline for 3 weeks, while receiving daily injections of LDN-193189 (3 mg/kg IP) or vehicle. Blood Hb levels (A) were reduced by repeated turpentine injection, not affected by LDN-193189 treatment alone, and were increased to levels of untreated mice in turpentine-injected mice treated with LDN-193189 (n = 7 mice each, *,#P < .0001 turpentine-injected vs saline-injected controls, and turpentine-injected vs turpentine-injected treated with LDN-193189). (B) Mean corpuscular volume (MCV) was decreased as a result of repeated turpentine injection, and normalized when LDN-193189 was administered in combination with turpentine (*P < .0001 turpentine-injected vs saline-injected controls, #P = .01 turpentine-injected vs turpentine-injected treated with LDN-193189). Serum iron levels (C) were similarly decreased as a result of turpentine-injection, and partially normalized by the concurrent administration of LDN-193189 (*P < .05 turpentine-injected vs saline-injected animals). Data shown are representative of 5 independent experiments.

  • Figure 5

    LDN-193189 increases Hb levels in established turpentine-induced anemia. (A-B) Mice were injected with turpentine (5 mL/kg intrascapularly) or saline weekly for 3 weeks resulting in microcytic anemia in turpentine-injected mice (left panels; n = 5 per group, *P < .05 turpentine-injected vs saline-injected controls). After 3 weeks, turpentine-treated mice were treated daily with LDN-193189 (3 mg/kg IP) or vehicle, while intrascapular turpentine or saline injections continued for an additional 3 weeks. At 6 weeks, Hb levels (A) in turpentine-injected mice were less than those in saline-injected mice, while administration of LDN-193189 by itself had no significant effect, and administration of LDN-193189 in turpentine-injected mice increased Hb levels to a significant degree (right panel; n = 5 per group, 1-way ANOVA P = .005, #P < .05 turpentine-injected vs saline-injected controls, †P < .05 turpentine-injected vs turpentine-injected mice treated with LDN-193189). MCV (B) was persistently decreased as a result of turpentine injections after 6 weeks, and tended to improve with LDN-193189 treatment (right panel; n = 5 per group, 1-way ANOVA P = .0002, #P < .01 turpentine-injected vs saline-injected controls, †P = .1 turpentine-injected vs turpentine-injected treated with LDN-193189). Data shown are representative of 3 independent experiments.