Iron-deficiency anemia from matriptase-2 inactivation is dependent on the presence of functional Bmp6

Abstract

Hepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv) whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6^−/− mice (characterized by increased hepcidin levels and anemia) and Bmp6^−/− mice (exhibiting severe iron overload due to hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels, increased hepatic iron and, importantly, corrected hematological abnormalities in Tmprss6^−/− mice. This suggests that elevated hepcidin levels in patients with familial iron-refractory iron deficiency anemia are due to excess signaling through the Bmp6/Hjv pathway.

Introduction

Body iron supply is provided both by iron recycling from senescent erythrocytes within the reticuloendothelial system and dietary iron absorption by duodenal enterocytes[1]. The liver-iron regulatory hormone, hepcidin, controls these two iron-delivery pathways via its targeted degradation of the cell surface iron exporter, ferroportin. As a consequence, iron availability in the circulation is decreased, leading to hypoferraemia[2,3]. Neither the lack of hepcidin nor its excess can be compensated for by the body, the results of which ultimately manifest in either iron overload or iron deficiency anemia, respectively.

Hepcidin gene expression is tightly regulated by body iron status and is dependent upon bone morphogenetic protein 6 (Bmp6) and hemojuvelin (Hjv). Binding of the iron-regulated Bmp6 ligand[4] to its receptors activates a signaling cascade leading to hepcidin transcription via phosphorylation of son of mother against decapentaplegic (Smad) 1/5/8 effectors[5]. Hjv, a GPI-linked membrane protein synthesized by the hepatocytes, is a Bmp6 coreceptor[5]. The critical role of the Bmp6/Hjv/Smad pathway in iron homeostasis is supported by the loss of hepcidin expression and massive parenchymal iron overload observed in Bmp6^−/− and Hjv^−/− mice as well as in mice with targeted liver deletion of Smad4[6–9].

Recently, the serine protease matriptase-2 (also known as TMPRSS6) has been connected to this iron-pathway[10–12] due to its proteolysis of Hjv[13]. Matriptase-2 is a type II serine protease that is predominately expressed in the liver (for review[14]). Matriptase-2 deficient mice[10,12] have very high levels of hepcidin, which lead to the inhibition of dietary iron absorption and cause a severe iron deficiency anemia phenotype. Matriptase-2 was thus characterized as a negative regulator of hepcidin gene expression. Accordingly, Du et al. demonstrated that overexpression of normal matriptase-2 protein in hepatoma cells suppresses the activation of hepcidin expression[10]. The anemic phenotype of matriptase-2 deficient mice is mirrored in patients with matriptase-2 mutations who present with iron-refractory iron deficiency anemia (IRIDA)[11]. Indeed, IRIDA patients show inappropriately high hepcidin levels[11,14,15], which explain lack of dietary iron absorption and partial response to parenteral iron treatment[16].

The goal of this study was to characterize the in vivo relationship between matriptase-2 and the iron-regulated ligand of Hjv, Bmp6, by analyzing the role of Bmp6 in the setting of anemia in mice deficient for matriptase-2. Towards this purpose, we intercrossed matriptase-2 and Bmp6-deficient mice and compared the iron status of the double mutant mice with that of wild-type controls or single mutant mice.

Material and methods

Tmprss6^tm1Otin mice on a mixed 129/Ola x C57BL/6 background[12] were mated to Bmp6^tm1Rob mice on an outbred CD1 background[6]. F1 mice, heterozygous for both the Tmprss6^tm1Otin (hereafter referred to as Tmprss6^+/−) and the Bmp6^tm1Rob alleles (referred to as Bmp6^+/−) were then intercrossed and the F2 progeny genotyped as previously described[6,12]. As expected, the nine possible genotypic combinations were observed among the F2 mice.

Mice were cared for in accordance with the «European convention for the protection of laboratory animals». Animals were given free access to tap water and standard laboratory mouse chow diet (AO3, iron content 280 mg/kg, UAR, France). Mice used in this study were 8 to 13-week-old females and had a mixed 129/Ola × C57BL/6 × CD1 background. Hematological parameters as well as plasma and liver iron were obtained as previously described[17].

RNA extraction and real-time quantification of the hepcidin and β actin transcripts were performed as reported in[18]. Standardized genetic nomenclature for mouse hepcidin is Hamp and Atcb for β actin.

Student t tests were used to compare quantitative traits between mouse groups. P values less than .05 were considered as statistically significant.

Results and discussion

To investigate the role of Bmp6 in the pathogenesis of iron-refractory iron deficiency anemia, we intercrossed matriptase-2 and Bmp6-deficient mice and analyzed iron metabolism in their F2 progeny.

As recently published[6,7], liver hepcidin expression was repressed in Bmp6^−/− mice compared with Bmp6^+/+ controls (Fig. 1A), leading to increased liver and plasma iron levels (Fig. 1B & C). Id1, a marker of activation of the Bmp/Smad signaling pathway, had an expression pattern similar to that of hepcidin in these Bmp6^−/− mice (Fig. 1D). However, their expression of Tmprss6 was not significantly different from the wild-type controls (data not shown). Furthermore, as shown in Table 1, hematological parameters of Bmp6^−/− mice were similar to those of Bmp6^+/+ controls.

Figure 1. Phenotypic analysis of female mice according to their Bmp6/Tmprss6 genotypes.

The phenotypes analyzed include hepcidin mRNA expression relative to β-actin (A), non-heme iron concentration of liver (B), plasmatic iron (C) and Id1 mRNA expression relative to β-actin (D). Data are presented as mean ± SD.

a, p<0.05 when compared with Bmp6^+/+Tmprss6^+/+controls.

b, p<0.05 when compared with Bmp6^−/−Tmprss6^+/+ mice.

c, p<0.05 when compared with Bmp6^+/+Tmprss6^−/− mice.

Table 1.

Hematological parameters of female mice according to their Bmp6/Tmprss6 genotypes.

Genotype	RBC (1012/L)	Hb (g/dL)	Hct (%)	MCV (fL)
Bmp6+/+ Tmprss6+/+	9,77 ± 0,35c	16,22 ± 0,63c	48,12 ± 2,36c	49,28 ± 1,86c
Bmp6+/− Tmprss6+/+	9,59 ± 0,49c	16,01 ± 0,62c	47,96 ± 2,46c	50,04 ± 1,62c
Bmp6−/− Tmprss6+/+	9,52 ± 0,48c	16,40 ± 0,66c	48,61 ± 2,01c	51,13 ± 2,12c
Bmp6+/+ Tmprss6+/−	9,54 ± 0,37c	15,60 ± 0,45abc	46,27 ± 1,44bc	48,52 ± 1,86bc
Bmp6+/− Tmprss6+/−	9,53 ± 0,35c	16,08 ± 0,59c	47,67 ± 2,69c	50,10 ± 2,26c
Bmp6−/− Tmprss6+/−	9,45 ± 0,45c	16,51 ± 0,72c	48,98 ± 2,57c	51,82 ± 1,62ac
Bmp6+/+ Tmprss6−/−	6,99 ± 1,1ab	11,97 ± 1,44ab	32,06 ± 4,86ab	45,94 ± 1,02ab
Bmp6+/− Tmprss6−/−	8,45 ± 0,63abc	13,48 ± 0,85abc	38,40 ± 2,54abc	45,47 ± 0,63ab
Bmp6−/− Tmprss6−/−	9,28 ± 0,35ac	15,92 ± 1,13c	46,88 ± 4,08c	50,42 ± 2,61c

RBC indicates red cell count; Hb, hemoglobin; Hct, hematocrit; MCV, mean corpuscular volume. Data are presented as mean ± SD.

^a,p<0.05 when compared with Bmp6^+/+Tmprss6^+/+controls.

^bp<0.05 when compared with Bmp6^−/−Tmprss6^+/+ mice.

^cp<0.05 when compared with Bmp6^+/+Tmprss6^−/− mice.

Conversely, hepcidin and Id1 gene expressions were significantly up-regulated in Tmprss6^−/− mice (Fig. 1A & D), and as expected[12], these mice had reduced hepatic and plasma iron indices, compared with Tmprss6^+/+ controls (Fig. 1B & C). In addition, Tmprss6^−/− mice were anemic and presented with significantly decreased Hb levels, as well as RBC, Hct and MCV (Table 1). In addition, as previously reported[19], they exhibited lower Bmp6 gene expression than wild-type controls (data not shown).

Interestingly, in double mutant Bmp6^−/− Tmprss6^−/− mice, hepcidin expression was repressed to the same extent as in Bmp6^−/− mice (Fig. 1A). A similar pattern of expression was observed for Id1, although the comparison did not reach statistical significance, due to a slightly higher variability of gene expression levels between mice for Id1 than for hepcidin (Fig. 1D). In addition, phosphorylation of Smad1/5/8 appeared similarly decreased in Bmp6^−/− Tmprss6^+/+ mice and in Bmp6^−/− Tmprss6^−/− mice, compared with wild-type controls (data not shown), which is concordant with the similar reduction we observed in their levels of hepcidin expression. However, although liver and plasmatic iron levels were higher in Bmp6^−/− Tmprss6^−/− mice than in Bmp6^+/+ mice, these levels remained significantly lower than in Bmp6^−/− mice (Fig. 1B & C). Lastly, and most importantly, iron deficiency anemia observed in the Tmprss6^−/− mice was completely rescued by Bmp6 deficiency. As shown in Table 1, Hb, Hct and MCV values observed in Bmp6^−/− Tmprss6^−/− mice were comparable to control values. Furthermore, heterozygous loss of Bmp6 in Tmprss6^−/− mice was able to partially correct systemic iron homeostasis by decreasing hepcidin gene expression and increasing plasma and liver iron (Fig. 1).

Although hematological parameters were found to be normal in mice deficient for both Hjv and matriptase-2[19,20], supporting the role of matriptase-2 as a regulator of Hjv expression at the hepatocyte membrane, the role of Bmp6 in this process was not clearly defined. The data obtained in this study indicate that hepcidin overexpression which results from matriptase-2 inactivation requires the presence of Bmp6. Indeed, neither the activation of the Hjv/Smad signaling pathway nor the establishment of the anemic phenotype were observed in double knockout mice.

However, in contrast to Hjv^−/− Tmprss6^−/− mice[19,20] which had a phenotype very similar to Hjv^−/− mice, we found that loss of matriptase-2 in Bmp6^−/− Tmprss6^−/− mice attenuates the effects of Bmp6 deficiency on hepatic and plasma iron accumulation. It could be speculated that, due to the lack of matriptase-2, Hjv is stabilized at the hepatocyte plasma membrane and can serve as a coreceptor for ligands other than Bmp6. However, neither hepcidin nor Id1 gene expression were found up-regulated in the double mutant female mice compared with Bmp6^−/− mice. Alternatively, matriptase-2 could, in absence of Bmp6, regulate other iron-related proteins or initiate a signaling pathway involved in maintaining hepatic iron balance and/or systemic iron regulation, independently of hepcidin.

In conclusion, the present data further support that Bmp6 is the physiological ligand of Hjv and demonstrate that the regulation of Hjv membrane expression by matriptase-2 serves to tightly control the signaling pathway induced by Bmp6.