Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers

Our plain-English summary of Receptor role of the annexin A2 in the mesothelial endocytosis of crocidolite fibers, by K. Yamashita, H. Nagai, S. Toyokuni (Laboratory Investigation; a Journal of Technical Methods and Pathology, 2015).

The study

• Authors: K. Yamashita, H. Nagai, S. Toyokuni
• Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology
• Published: 2015;95(7):749-64
• Cited by: 8 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Asbestos-induced mesothelioma is a worldwide problem. Parietal mesothelial cells internalize asbestos fibers that traverse the entire lung parenchyma, an action that is linked to mesothelial carcinogenesis. Thus far, vitronectin purified from serum reportedly enhances the internalization of crocidolite by mesothelial cells via integrin αvβ5. To reveal another mechanism by which mesothelial cells endocytose (phagocytose) asbestos, we first evaluated the effects of serum on asbestos uptake, which proved to be nonessential. Thereafter, we undertook a study to identify proteins on the surface of mesothelial cells (MeT5A) that act as receptors for asbestos uptake based on the assumption that receptors bind to asbestos with physical affinity. To this end, we incubated the membrane fraction of MeT5A cells with crocidolite or chrysotile and evaluated the adsorbed proteins using sodium dodecyl sulfate polyacrylamide gel analysis. Next, we extensively identified the proteins using an in-solution or in-gel digestion coupled with mass spectrometry. Among the identified proteins, annexin A2 (ANXA2) and transferrin receptor protein 1 (TFRC) were distinguished because of their high score and presence at the cell surface. Crocidolite uptake by MeT5A cells was significantly decreased by shRNA (short hairpin RNA)-induced knockdown of ANXA2 and direct blockade of cell surface ANXA2 using anti-ANXA2 antibody. In addition, abundant ANXA2 protein was present on the cell membrane of mesothelial cells, particularly facing the somatic cavity. These findings demonstrate that ANXA2 has a role in the mesothelial phagocytosis of crocidolite and may serve as its receptor.

In plain English

This laboratory study used a human mesothelial cell line, MeT5A, plus rat tissue from the authors' earlier work. The authors looked for cell-surface proteins that might help these cells take in asbestos fibers (UICC chrysotile, crocidolite and amosite). With uncoated crocidolite in serum-free medium, cells took up significantly more fiber than in three groups using serum in the medium, serum-coated fibers, or both. For chrysotile, no significant difference was found. The authors believed that differences in how fast crocidolite fibers sank influenced this result. Using mass spectrometry, they identified membrane proteins that stuck to the fibers, focusing on annexin A2 (ANXA2) and transferrin receptor protein 1 (TFRC). Reducing ANXA2 in the cells significantly lowered crocidolite uptake, while reducing TFRC did not. Blocking ANXA2 with one antibody significantly lowered crocidolite uptake, but a second antibody showed no significant difference. For chrysotile, reducing either protein produced no significant difference. ANXA2 was found on cell membranes, including the peritoneal-cavity side of epithelioid rat mesothelioma cells. The authors suggested ANXA2 is involved in crocidolite uptake and could be its putative 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: Laboratory (cell culture) study of how mesothelial cells take in asbestos fibers, using protein identification by mass spectrometry, gene knockdown and antibody blocking, plus staining of rat tissues
• Who was studied: The human mesothelial cell line MeT5A, plus rat peritoneal mesothelium and rat peritoneal malignant mesothelioma tissues obtained in the authors' previous work
• Exposure: UICC chrysotile A, crocidolite and amosite fibers, uncoated or coated with fetal bovine serum, added to cells at several doses for 2 or 24 hours
• Main results:
  • Crocidolite uptake by MeT5A cells was significantly higher in Group A (serum-free medium with uncoated fibers) than in the other three groups (Group B: medium with 10% serum and uncoated fibers; Group C: serum-free medium and serum-coated fibers; Group D: medium with 10% serum and serum-coated fibers). There were nine tests.
  • For chrysotile, no significant difference in uptake was found among the four serum and coating groups (six tests)
  • The authors believed that differences in how fast crocidolite fibers sank to the cells influenced the serum results
  • ANXA2 and TFRC were chosen as candidate proteins: ANXA2 had the highest score among cell-surface and transmembrane proteins adsorbed to crocidolite, and TFRC is a known receptor for endocytosis
  • In MeT5A cells, knockdown of ANXA2 significantly reduced crocidolite uptake, while knockdown of TFRC gave no significant difference (P = 0.219)
  • Blocking cell-surface ANXA2 with one antibody (ab41803) significantly lowered crocidolite uptake compared with control antibody (nine tests)
  • A second anti-ANXA2 antibody (sc-9061) showed no significant difference in crocidolite uptake (P = 0.440)
  • For chrysotile, neither ANXA2 knockdown (P = 0.593) nor TFRC knockdown (P = 0.267) significantly changed uptake (six experiments each)
  • Chrysotile adsorbed more TFRC and less ANXA2 than crocidolite and amosite
  • In epithelioid rat mesothelioma cells, ANXA2 was found on the cell membrane on the side facing the peritoneal cavity
• The authors' conclusion: The authors concluded that serum was not essential for asbestos uptake, and suggested that ANXA2 is involved in crocidolite uptake by mesothelial cells and could be its putative receptor
• Limitations the authors state:
  • The uptake measurements cannot be compared across fiber types because the fibers differ in shape and properties
  • The flow cytometry signal may increase slightly over time, so timing differences between samples matter
  • The effect of serum coating, which made fibers clump together, may have persisted despite sonication
  • The chrysotile fiber-sinking experiment did not give consistent data because of technical difficulties
  • The authors did not rule out the importance of the vitronectin-coating uptake mechanism in living bodies

Topics

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

Medical terms in this study, explained in our Lexicon: Crocidolite, Mesothelioma, Mesothelial Cells, Parenchyma, Carcinogenesis, Vitronectin, Chrysotile, Phagocytosis.

Related studies

• Role of alphavbeta5 integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells — P. Pande, T.A. Mosleh, et al. (2006)
• Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells — K. Maruyama, H. Haniu, et al. (2015)
• Increased expression of epidermal growth factor receptor in rat pleural mesothelial cells correlates with carcinogenicity of mineral fibres — S.P. Faux, C.E. Houghton, et al. (2000)
• DNA damage in bronchial epithelial and mesothelial cells with and without associated crocidolite asbestos fibers — J. Nygren, S. Suhonen, et al. (2004)
• Analysis of cell cycle disruptions in cultures of rat pleural mesothelial cells exposed to asbestos fibers — V. Levresse, A. Renier, et al. (1997)
• Asbestos upregulates expression of the urokinase-type plasminogen activator receptor on mesothelial cells — R.C. Perkins, V.C. Broaddus, et al. (1999)

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