Figure 2.
HJV overexpression in control animals induced BMP signaling and increased hepcidin expression. (A) Relative hepatic hepcidin (Hamp) mRNA levels were determined by qRT-PCR. Transcripts were normalized to 18S ribosomal RNA as internal controls, and the average of respective Alk2fl/fl and Alk3fl/fl control mice was set to 1 (control PBS, n = 13; control AAV-HJV, n = 12; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 6; Alk3fl/fl;Alb-Cre PBS, n = 11; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 5). (B) Serum hepcidin levels were decreased in hepatocyte-specific Alk3-deficient mice compared with control animals as determined by enzyme-linked immunosorbent assay (control PBS, n = 11; control AAV-HJV, n = 7; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 5; Alk3fl/fl;Alb-Cre PBS, n = 5; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 6). (C) HJV overexpression in control animals increased the level of pSMAD1/5/8 determined by immunoblotting. GAPDH was used as a loading control. (D) Hepatic Id1 mRNA levels were determined by qRT-PCR. Transcripts were normalized to 18S ribosomal RNA, and the average of control mice treated with PBS was set to 1 (control PBS, n = 8; control AAV-HJV, n =10; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 5; Alk3fl/fl;Alb-Cre PBS, n = 5; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 6). (E) Ferroportin staining (red) and DAPI (4′,6-diamidino-2-phenylindole) staining (white) of representative duodenal sections of control mice and Alk3fl/fl;Alb-Cre mice injected with PBS or AAV-HJV are shown. (F) In Alk3fl/fl;Alb-Cre mice the ferroportin signal is increased compared with control littermates. AAV-HJV injection in control mice decreased the ferroportin signal, albeit not significant. In Alk3fl/fl;Alb-Cre mice there is no difference in ferroportin signal detectable between AAV-HJV–injected mice and PBS-injected mice (control PBS, n = 5; control AAV-HJV, n = 3; Alk3fl/fl;Alb-Cre PBS, n = 6; and Alk2fl/fl;Alb-Cre AAV-HJV, n = 3). Data are presented as box plots with minimum to maximum whiskers and medians. Significances were presented relative to the indicated control with ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

HJV overexpression in control animals induced BMP signaling and increased hepcidin expression. (A) Relative hepatic hepcidin (Hamp) mRNA levels were determined by qRT-PCR. Transcripts were normalized to 18S ribosomal RNA as internal controls, and the average of respective Alk2fl/fl and Alk3fl/fl control mice was set to 1 (control PBS, n = 13; control AAV-HJV, n = 12; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 6; Alk3fl/fl;Alb-Cre PBS, n = 11; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 5). (B) Serum hepcidin levels were decreased in hepatocyte-specific Alk3-deficient mice compared with control animals as determined by enzyme-linked immunosorbent assay (control PBS, n = 11; control AAV-HJV, n = 7; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 5; Alk3fl/fl;Alb-Cre PBS, n = 5; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 6). (C) HJV overexpression in control animals increased the level of pSMAD1/5/8 determined by immunoblotting. GAPDH was used as a loading control. (D) Hepatic Id1 mRNA levels were determined by qRT-PCR. Transcripts were normalized to 18S ribosomal RNA, and the average of control mice treated with PBS was set to 1 (control PBS, n = 8; control AAV-HJV, n =10; Alk2fl/fl;Alb-Cre PBS, n = 6; Alk2fl/fl;Alb-Cre AAV-HJV, n = 5; Alk3fl/fl;Alb-Cre PBS, n = 5; and Alk3fl/fl;Alb-Cre AAV-HJV, n = 6). (E) Ferroportin staining (red) and DAPI (4′,6-diamidino-2-phenylindole) staining (white) of representative duodenal sections of control mice and Alk3fl/fl;Alb-Cre mice injected with PBS or AAV-HJV are shown. (F) In Alk3fl/fl;Alb-Cre mice the ferroportin signal is increased compared with control littermates. AAV-HJV injection in control mice decreased the ferroportin signal, albeit not significant. In Alk3fl/fl;Alb-Cre mice there is no difference in ferroportin signal detectable between AAV-HJV–injected mice and PBS-injected mice (control PBS, n = 5; control AAV-HJV, n = 3; Alk3fl/fl;Alb-Cre PBS, n = 6; and Alk2fl/fl;Alb-Cre AAV-HJV, n = 3). Data are presented as box plots with minimum to maximum whiskers and medians. Significances were presented relative to the indicated control with ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

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