Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells - PubMed (original) (raw)

. 2009 Jul 2;114(1):181-6.

doi: 10.1182/blood-2008-12-195503. Epub 2009 May 4.

Prashanth Porayette, Orapan Sripichai, Seung-Jae Noh, Colleen Byrnes, Ajoy Bhupatiraju, Y Terry Lee, Julia B Goodnough, Omid Harandi, Tomas Ganz, Robert F Paulson, Jeffery L Miller

Affiliations

Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells

Toshihiko Tanno et al. Blood. 2009.

Abstract

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.

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Figures

Figure 1

Figure 1

TWSG1 expression in human erythropoiesis. (A) Microarray confirmation by quantitative PCR using erythroblasts from 6 separate donors (y-axis, copy number/ng total RNA). Each line associated values from the same donor on separate days. D7, culture day 7; D14, culture day 14. *P < .05. Bars represent the mean values. Flow cytometry analysis for CD71 and GPA surface expression on culture day 7 (B) and day 14 (C). Flow cytometric gates denoted as 1, 2, 3 (panel B) and 4, 5, 6 (panel C) were sorted for quantitative PCR quantitation of TWSG1. (D) TWSG1 expression level in the sorted populations according to the sorted gates shown in panels B and C.

Figure 2

Figure 2

TWSG1 inhibit up-regulation of hepcidin expression by BMP2/4 in human hepatoma cell line. (A) Relative hepcidin expression as a dosed response to BMP2 (□) and BMP4 (■) are shown. Final BMP concentrations are on the x-axis, and relative hepcidin expression on the y-axis (0 ng/mL assigned a value of 1 for comparison). (B) TWSG1 dose response of relative hepcidin expression in human HuH-7 cells in cultures supplemented with 10 ng/mL BMP2 (—), 10 ng/mL BMP4 (---), or in the absence of added BMP (formula image). Bars represent mean values with asterisks signifying P < .05.

Figure 3

Figure 3

TWSG1 inhibits BMP2/4 up-regulation of hepcidin in primary human hepatocytes. Dose response of relative hepcidin expression levels to (A) BMP2 and (C) BMP4 are shown using primary hepatocytes from 4 separate donors (shaded bars). Final BMP concentrations are on the x-axis, and relative hepcidin expression on the y-axis (0 ng/mL assigned a value of 1 for comparison). In panels B and D, dosed response of relative hepcidin expression to TWSG1 in human primary hepatocytes cultured in 10 ng/mL BMP2 or BMP4, respectively. Lines represent results from 4 separate donors' cells from triplicate cultures. *P < .05.

Figure 4

Figure 4

TWSG1 inhibits BMP-mediated Smad phosphorylation. HuH-7 cells were cultured in the presence or absence of 1000 ng/mL TWSG1, 10 ng/mL BMP2, or both for 1 hour. Total cell lysates (20 μg/lane) were immunoblotted using a rabbit antiserum specific to human phosphorylated-Smad1/5/8 (p-Smad1/5/8). Anti–total Smad1 antibody and anti–β-actin antibody were used as internal controls.

Figure 5

Figure 5

Twsg1 dose response of hepcidin levels in primary murine hepatocytes. In primary hepatocytes from 4- to 6-week-old C57Bl/6 mice, dosed increased Twsg1 (x-axis) was added, and hepcidin (y-axis; hamp1) mRNA was quantified. *P < .05 compared with untreated cells.

Figure 6

Figure 6

Twsg1 mRNA expression in murine thalassemia. Murine Twsg1 mRNA in (A) spleen, (B) liver, and (C) bone marrow from wild-type mice (WT, n = 7), Hbbth3/+ β-thalassemia intermedia mice (th3/+, n = 13), and Hbbth3/th3 β-thalassemia major mice (th3/th3, n = 5) was determined by quantitative PCR using total RNA isolated from the tissues. Bars represent mean values; *P < .05 compared with wild-type. (D) Murine Twsg1 protein (Twsg1) expression in spleen from wild-type mice (WT), Hbbth3/+ β-thalassemia intermedia mice (th3/+), and Hbbth3/th3 β-thalassemia major mice (th3/th3) was detected by immunoblotting using a rabbit anti–serum-specific Twsg1. Anti–β-actin antibody were used as internal control. Splenic tissues from 2 mice were studied for comparison (#1, #2).

Figure 7

Figure 7

Model of hepcidin regulation by TWSG1 and GDF15.

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