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Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both in vitro and in vivo Specifically, enzymatic removal of heparan sulfate from primary human hepatocytes, CRISPR/Cas9 manipulation of heparan sulfate biosynthesis in human hepatoma cells, or pharmacological manipulation of heparan sulfate-protein interactions using sodium chlorate or surfen dramatically reduced baseline and BMP6/SMAD1/5/8-dependent hepcidin expression. Moreover inactivation of the heparan sulfate biosynthetic gene N-deacetylase and N-sulfotransferase 1 (Ndst1) in murine hepatocytes (Ndst1 f/f AlbCre +) reduced hepatic hepcidin expression and caused a redistribution of systemic iron, leading to iron accumulation in the liver and serum of mice. Manipulation of heparan sulfate had a similar effect on IL6-dependent hepcidin expression in vitro and suppressed IL6-mediated iron redistribution induced by lipopolysaccharide in vivo These results provide compelling evidence that hepatocyte heparan sulfate plays a key role in regulating hepcidin expression and iron homeostasis in mice and in human hepatocytes.

Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants. [ABSTRACT FROM AUTHOR]

Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional ‘type-2’ variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT–NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT–NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue. [ABSTRACT FROM AUTHOR]

Advances in genetic screening technologies have considerably accelerated the discovery of rare alpha-1-antitrypsin (AAT) variants. Expression in cellular models is an effective approach to evaluate the pathogenic potential of these new AAT variants, whose clinical significance would otherwise remain uncertain. Here we provide a detailed description of established methods for in vitro characterization of AAT coding variants expressed in HEK293T/17 cells. The protocols include determination of secretion efficiency, the tendency to form polymeric chains and  the anti-elastase inhibitory activity., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Hepcidin is considered the major regulator of systemic iron homeostasis in human and mice, and its expression in the liver is mainly regulated at a transcriptional level. Central to its regulation are the bone morphogenetic proteins, particularly BMP6, that are heparin binding proteins. Heparin was found to inhibit hepcidin expression and BMP6 activity in hepatic cell lines and in mice, suggesting that endogenous heparan sulfates are involved in the pathway of hepcidin expression. This was confirmed by the study of cells and mice overexpressing heparanase, the enzyme that hydrolyzes heparan sulfates, and by cellular models with altered heparan sulfates. The evidences supporting the role of heparan sulfate in hepcidin expression are summarized in this chapter and open the way for new understanding in hepcidin expression and its control in pathological condition., (© 2019 Elsevier Inc. All rights reserved.)