Functional Characterization of the Hephaestin Variant D568H Provides Novel Mechanistic Insights on Iron-Dependent Asbestos-Induced Carcinogenesis

Our plain-English summary of Functional Characterization of the Hephaestin Variant D568H Provides Novel Mechanistic Insights on Iron-Dependent Asbestos-Induced Carcinogenesis, by P. Zacchi, F. Longo, A. Marconato, et al (International Journal of Molecular Sciences, 2025).

The study

• Authors: P. Zacchi, F. Longo, A. Marconato, M. Amadei, M.C. Bonaccorsi di Patti, E. Avolio, P. Li, H. Fan, T.D. Tetley, G. Zabucchi, V. Borelli
• Journal: International Journal of Molecular Sciences
• Published: 2025;26(6)
• Read it: PubMed · Publisher · Free full text

Abstract

A local disruption of iron homeostasis leading to oxidative stress is considered one of the main mechanisms of asbestos-related genotoxicity. Another aspect contributing to the risk of developing pathological consequences upon asbestos exposure is individual genetic factors. In a previous study, we identified a coding SNP in the hephaestin gene (HEPH) that protects against developing asbestos-related thoracic cancer. Heph is a ferroxidase that promotes iron export in concert with the permease ferroportin (Fpn1). Here, we performed an in-depth functional characterization of the HephD568H variant to gain insights into the molecular basis of its protective activity. We showed that HephD568H forms a complex with Fpn1 and possesses full ferroxidase activity. Although HephD568H is more efficiently recruited to the plasma membrane, it is impaired in binding iron-deficient Tfn, whose interaction with wild-type (WT) ferroxidase emerged as a novel mechanism to perceive brain iron needs. Heph is expressed in the human lung by pericytes and fibroblasts, and lung pericytes were shown to respond to iron demand by upregulating the iron exporter pair. These results extend the paradigm of local iron regulation discovered at the blood-brain barrier to the pulmonary vasculature. Furthermore, they establish a mechanistic link between changes in iron sensing and the risk of developing asbestos-related malignancies.

In plain English

This laboratory study examined the hephaestin (Heph) variant D568H, which the authors had earlier identified as protective against asbestos-related thoracic cancer; the experiments used cultured cells and purified protein, not asbestos fibers. In transfected HEK293T cells, HephD568H distributed like wild-type (WT) Heph. After protein synthesis was blocked, HephD568H levels stayed elevated with or without the iron exporter ferroportin (Fpn1), while HephWT halved after 16 h unless Fpn1 was co-expressed. Both variants bound Fpn1, but HephD568H/Fpn1 complexes were more enriched at the cell surface. Purified HephD568H was enzymatically active: Vmax was unchanged and the Km decrease was not significant (WT Km 7.71, Vmax 10.60 micromolar per minute; D568H Km 5.38, Vmax 10.30). In proximity ligation and pull-down assays, HephD568H recruited iron-depleted transferrin (apo-Tfn) poorly compared with WT. Among the cultured human lung and pleural cell types tested, surface biotinylation detected Heph only in lung pericytes and fibroblasts. The authors noted they lacked data on where Heph is located in healthy human lung. Cultured pericytes raised Heph levels after apo-Tfn treatment without raising the transferrin receptor.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: in vitro laboratory study using transfected cell lines, recombinant protein biochemistry and cultured human lung cells
• Who was studied: Cultured cells: HEK293T and Expi293F cells expressing Heph variants, plus immortalized human lung pericytes, primary human lung fibroblasts, human pulmonary microvascular endothelial cells, Huvec, TT1 alveolar type 1-like cells, MeT-5A and primary human mesothelial cells
• Exposure: Not an asbestos exposure study. The comparison was the hephaestin variant D568H (SNP rs3747359) versus wild-type Heph. Cells were also treated with 0.25 micromolar apo-transferrin or holo-transferrin, and with cycloheximide.
• Main results:
  • After cycloheximide treatment, HephD568H protein stayed elevated at all time points, independently of Fpn1 expression. HephWT halved after 16 h, but co-expressed Fpn1 stabilized it and prolonged its half-life.
  • HephD568H bound Fpn1 like WT. Its complexes were pulled down comparably with membrane-permeable and membrane-impermeable cross-linkers, indicating surface enrichment relative to HephWT.
  • Ferroxidase kinetics: HephWT Km 7.71 (SEM 1.55), Vmax 10.60 micromolar per minute (SEM 0.80); HephD568H Km 5.38 (SEM 0.97), Vmax 10.30 (SEM 0.60). The authors reported no impact on Vmax and a non-significant decrease in Km.
  • In the proximity ligation assay, cells expressing HephD568H showed very few apo-Tfn/Heph puncta, while HephWT showed a strong signal.
  • In co-immunoprecipitation of full-length Heph, HephD568H recruited apo-Tfn poorly compared with HephWT (n = 5, p less than 0.01).
  • Among the cultured cell types tested, Heph at the pulmonary barrier was detected only in lung pericytes and fibroblasts. Pulmonary microvascular endothelial cells were negative for Heph and ceruloplasmin.
  • Cultured lung pericytes treated for 72 h with apo-Tfn upregulated Heph without upregulating the transferrin receptor.
• The authors' conclusion: The authors concluded that HephD568H is impaired in apo-Tfn recruitment and therefore poorly responsive to iron demand. They proposed that this may underlie the variant's protective effect against asbestos-dependent cancers.
• Limitations the authors state:
  • The authors state that they lack data on where Heph is located in healthy human lung tissue.
  • The authors note that the relative contribution of pericytes versus fibroblasts to regulating iron release remains to be studied.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer.

Medical terms in this study, explained in our Lexicon: Carcinogenesis, Oxidative Stress, Genotoxicity, Asbestos Exposure, Fibroblast.

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