Hepcidin promoter activity was measured by the promoter-driven firefly luciferase activity normalized to constitutive Renilla luciferase activity (Promega E1910)

Hepcidin promoter activity was measured by the promoter-driven firefly luciferase activity normalized to constitutive Renilla luciferase activity (Promega E1910). in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound 58Fe, the amount of 58Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to BMP-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. Conclusion: Testosterone inhibits hepcidin transcription through its interaction with BMP-Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells. Introduction Testosterone is an important regulator of erythropoiesis in men and women (Bhasin em et al. /em , 2010; Mirand EA, 1965). Circulating testosterone levels have been associated with hemoglobin levels in community-dwelling middle-aged and older men (Ferrucci L, 2006; Yeap em et al. /em , 2009) and in men with prostate cancer who are receiving androgen-deprivation therapy (Hara N, 2010). Before the advent of erythropoietin (EPO), androgens were used widely to treat anemia-associated with chronic disease, end stage renal disease, and aplastic anemia (Shahidi NT, 1959; Watkinson G, Lanopepden 1947). Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially in older men. However, the mechanisms by which testosterone stimulates erythropoiesis remain poorly understood. An understanding of these mechanisms has Lanopepden important implications for the therapeutic applications of androgens for the treatment of androgen deficiency, anemia, and sarcopenia associated with aging or chronic disease. Several hypotheses have been proposed to explain the mechanisms by which testosterone increases hemoglobin and hematocrit, including stimulation of EPO, direct effects on erythroid progenitors, ferrokinetics, and red cell survival (Bachman E, 2010; Beran M, 1982; Moriyama Y, 1975); however, the evidence supporting these hypotheses is inconclusive (Coviello AD, 2008; Mirand EA, 1971). We show here that testosterone regulates hepcidin expression, in association with decreased splenic iron retention and increased iron PIK3R1 incorporation into red cells. Additionally, we investigated the mechanisms by which testosterone regulates hepcidin expression; we provide multiple lines of evidence that testosterone, through activation of its nuclear receptor, interferes with bone morphogenetic protein (BMP)/Smad signaling to reduce hepcidin transcription. We also tested whether testosterone regulates hepcidin by stimulating EPO or by activating hypoxia-sensing mechanisms. Results 1. The effects of testosterone on red cell indices In adult female C57BL/6 mice, testosterone administration by subcutaneous implants was associated with a gradual increase in hematocrit after a lag of two days (Fig. 1A). Two weeks after initiation of testosterone administration, mice assigned to testosterone group had significantly higher levels of hemoglobin and hematocrit, and a trend towards higher mean corpuscular hemoglobin than those in the control group (Fig. 1B). Mean corpuscular volume did not differ between groups. Serum iron and transferrin saturation were significantly higher and total iron binding capacity significantly Lanopepden lower in testosterone-treated mice than in placebo-treated mice (Fig. 1C). Reticulocyte count and reticulocyte Lanopepden hemoglobin ratio (CHr), a sensitive marker of iron available for hemoglobin synthesis (Fishbane S, 1997), were also significantly higher in testosterone-treated mice than in placebo-treated mice (Fig. 1D). Open in a separate window Figure 1 Effects of Testosterone on Red Cell Indices, Reticulocyte Count, Reticulocyte Hemoglobin Ratio, and Circulating Iron IndicesA: Time course of hematocrit change after subcutaneous insertion of empty or testosterone implants in female mice (*p=0.0002, **p 0.0001, ***p=0.0002 for between-group comparison at each time). The slope of hematocrit change over time was 0.65 for testosterone group (p 0.0001) and 0.02 for control group (p=0.6121). B: The mice treated with testosterone implants for 2-weeks had significantly higher hemoglobin (Hgb), hematocrit (Hct), and a trend towards higher mean corpuscular hemoglobin Lanopepden (CHm) than mice that received empty implants. C: Testosterone-treated mice had significantly higher serum iron, lower total iron binding capacity (TIBC), and higher transferrin (Tf) saturation than controls 2 weeks after insertion of either empty (C) or testosterone implants (T). D. Testosterone-treated mice (T) had significantly higher reticulocyte count (Retic-C) and higher reticulocyte hemoglobin ratio (CHr) than controls (C) after 2 weeks of treatment. The data are meanSEM, n=10-20 mice for each measurement. Similar data were obtained in castrated male mice (supplementary.