Fig 1.
HepG2 cells transiently transfected with heparanase showed a reduction of hepcidin mRNA.
HepG2 cells were transfected with pcDNA3.1-HPA plasmid (HPA) or empty pcDNA3.1 as control (MOCK) and harvested 48 h after the transfection. (A) Relative level of HPA mRNA was measured by qRT-PCR (B) Western blot of SDS-PAGE with anti-HPA antibodies show the levels of its latent (65 kDa) and active (50 kDa) form. Densitometry quantification of the two protein forms was performed in relation to Actin. (C) The level of hepcidin mRNA and (D) Id1 mRNA was analyzed by qPCR and normalized for Hprt1. (E) The phosphorylated (pSMAD5) and total SMAD5 were analyzed by western blot and pSMAD5 densitometry was normalized to actin. In (A) the values are expressed as–dCt for HPA mRNA, in C and D as fold change over the control (MOCK) for hepcidin and Id1 mRNA., respectively
Fig 2.
HepG2 clones overexpressing heparanase showed a reduction of hepcidin expression and indices of iron loading.
Two stable clones of HepG2 cells transfected with pcDNA3.1-HPA (HPA3 and HPA6) were analyzed for hepcidin expression, BMP/SMAD signaling and indices of iron status. (A) qPCR was assessed to analyze the level of HPA mRNA. (B) Western blot for HPA shows the level of the latent (65 kDa) and active (50 kDa) forms. Densitometry quantification of the two forms was performed in relation to actin as calibrator. (C) qPCR was performed to analyze the level of hepcidin mRNA, (D) and the level of Id1 mRNA in relation to Hprt1. (E) WB of phosphorylated SMAD5 and their densitometry quantification referred to actin and WB of total SMAD5, (F) WB of transferrin Receptor 1, Tfr1, and its densitometry, (G) WB of ferritin light chain, FTL and its densitometry; (H) WB of Ferroportin (FPN) and its densitometry. The values are expressed as–dCt (for HPA mRNA) or as fold change over the control (MOCK) (for hepcidin and Id1 mRNA). The images are representative from three different analyses
Fig 3.
Treatment with heparin and IL6 of HepG2 clones overexpressing heparanase.
(A) The two HepG2 clones overexpressing HPA (HPA3 and HPA6) and control (MOCK) cells were treated with 0.12 μg/mL of RO-82 heparin for 6 h and hepcidin mRNA evaluated. (B) The clones and control cells were treated with 50 ng/mL IL6 for 3 h and analyzed for mRNA level of Socs3 and (C) hepcidin in relation to Hprt1. The values are expressed as fold change of their respective untreated controls (-). (D) Western blot of pSMAD5, total SMAD5, pSTAT3, total STAT3 and of ACTIN as housekeeping.
Fig 4.
Transgenic mice overexpressing heparanase showed reduced liver hepcidin mRNA and serum protein.
(A) Liver hepcidin mRNA levels of wild type (WT) and transgenic HPA mice (TG-HPA) normalized to Hprt1. The values are expressed as fold change of wild type mice. (B) Quantification of serum hepcidin by SELDI-TOF in the same mice.
Fig 5.
Transgenic mice overexpressing heparanase showed altered iron homeostasis.
(A) Levels of serum iron in the WT and TG-HPA mice, (B) non-heme liver iron and (C) non-heme spleen iron levels, measured by a spectrophotometric assay. (D) Perl’s stain of spleen sections that showed a lower number of iron granules in TG-HPA compared to WT mice. The sections were counterstained with hematoxylin and eosin
Fig 6.
Transgenic mice overexpressing heparanase have increased ferritin-iron and ferritin protein content in the liver.
(A and B) Western blot of liver extracts from WT and TG-HPA mice (A) for ferritin L-chain (FTL) subunits in SDS-PAGE with GAPDH as calibrator and (B) for assembled ferritin in non-denaturing PAGE. (C) Prussian blue stain of non-denaturing PAGE loaded with 50 ug protein, before (upper) and after enhancing with DAB and H2O2 (lower). rFTL is control purified recombinant mouse FTL. (D) Western blot of Ferroportin (FPN) and (E) of Transferrin Receptor1 (TfR1) and their respective GAPDH as calibrator. Densitometry data were obtained from 3 independent experiments.
Fig 7.
Transgenic mice overexpressing Heparanase showed increased BMP6/SMAD signaling.
(A) evaluation of BMP6 mRNA in the liver of WT and TG-HPA mice (B) evaluation of Id1 mRNA, in relation to Hprt1. (C) Western blot of pSMAD5, of total SMAD5 and of GAPDH as housekeeping for normalization in densitometry quantification. The values are expressed as fold change of wild type mice.
Fig 8.
Transgenic mice overexpressing Heparanase are more sensitive to inflammatory stimuli.
WT and TG-HPA mice were treated with a single dose of LPS (1 mg/Kg) IP and sacrified after 6 h. (A) Analysis of Socs3 mRNA, (B) CRP mRNA and (C) HAMP1 mRNA in the liver of WT and TG-HPA mice in relation to Hprt1, before and after the treatment. The values are expressed as fold change of wild type mice. (D) Quantification of serum Hepcidin by SELDI-TOF. (E) Western blot of pSMAD5, total SMAD5, pSTAT3, total STAT3 and of GAPDH as housekeeping.