Post-transcriptional regulation of plasminogen activator inhibitor type-1 expression in human pleural mesothelial cells

Our plain-English summary of Post-transcriptional regulation of plasminogen activator inhibitor type-1 expression in human pleural mesothelial cells, by S. Shetty, T. Velusamy, R.S. Shetty, et al (American Journal of Respiratory Cell and Molecular Biology, 2010).

The study

• Authors: S. Shetty, T. Velusamy, R.S. Shetty, A.S. Marudamuthu, S.K. Shetty, G. Florova, T. Tucker, K. Koenig, P. Shetty, Y.P. Bhandary, S. Idell
• Journal: American Journal of Respiratory Cell and Molecular Biology
• Published: 2010;43(3):358-67
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The plasminogen activator inhibitor type-1 (PAI-1) effectively blocks the activities of free and receptor-bound urokinase-type plasminogen activator. Incubation of cultured human pleural mesothelial (Met5A) cells with TGF-beta increased PAI-1 protein. TGF-beta, phorbol myristate acetate, and the translation inhibitor cycloheximide induced PAI-1 mRNA and slowed its degradation, suggesting that PAI-1 mRNA could be regulated by interaction of a PAI-1 binding protein (PAI-1 mRNABp) with PAI-1 mRNA. We found that an approximately 60 kD cytoplasmic PAI-1 mRNABp is detectable in cytoplasmic extracts of MeT5A human pleural mesothelial and malignant mesothelioma cells. The PAI-1 mRNABp specifically binds to a 33-nt sequence in the 3' untranslated region of PAI-1 mRNA. Insertion of this 33-nt sequence destabilizes otherwise stable beta-globin mRNA, indicating that the binding sequence accelerates decay of endogenous PAI-1 mRNA. Competitive inhibition by overexpression of the 33-nt binding sequence in MeT5A cells reduced PAI-1 mRNA decay and increased PAI-1 protein and mRNA expression, indicating that the PAI-1 mRNABp destabilizes PAI-1 mRNA by its interaction with the endogenous 33-nt binding sequence. Incubation of Met5A cells with TGF-beta attenuated the interaction of the PAI-1 mRNABp with the 33-nt sequence. By conventional and affinity purification, we isolated the PAI-1 mRNABp and confirmed its identity as 6-phospho-d-gluconate-NADP oxidoreductase, which specifically interacts with the full-length and the 33-nt sequence of the PAI-1 mRNA 3' untranslated region. This newly recognized pathway could influence expression of PAI-1 by mesothelial or mesothelioma cells at the level of mRNA stability in the context of pleural inflammation or malignancy.

In plain English

This was a laboratory study in cultured cells. It did not study asbestos exposure or people. The authors examined how PAI-1, a protein that blocks the breakdown of fibrin, is controlled in human pleural mesothelial cells (the MeT5A line), in human malignant mesothelioma cell lines, and in primary rabbit pleural mesothelial cells. Treating MeT5A cells with the signaling proteins TGF-beta or TNF-alpha increased PAI-1. In MeT5A cells the PAI-1 messenger RNA was unstable, with a half-life of less than 3 hours. TGF-beta, PMA and cycloheximide lengthened this to about 8 to 12 hours. The authors found a cell protein of about 60 kD that bound a 33-nucleotide stretch of the PAI-1 messenger RNA and made it break down faster. TGF-beta and TNF-alpha treatment reduced this binding. The authors identified the protein as the enzyme 6-PGD. Adding extra 6-PGD reduced PAI-1 by more than 50% in rabbit mesothelial cells. The authors concluded that this interaction regulates PAI-1 messenger RNA stability. They said it could contribute to pleural disease if it operates in the living body, which they had not tested.

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 (in vitro) study of cultured cell lines and primary cells, using molecular biology methods (RNA binding assays, chimeric gene constructs, protein purification and overexpression)
• Who was studied: Cultured cells, not people. These were human pleural mesothelial cells (MeT5A); human pleural malignant mesothelioma cell lines MS-1, REN, M33K and M9K obtained from ATCC; primary rabbit pleural mesothelial cells; and H157 lung carcinoma cells, which were used to make recombinant 6-PGD.
• Exposure: No asbestos or fiber exposure was studied. Cells were treated with TGF-beta (2 ng/ml), TNF-alpha (10 ng/ml), phorbol myristate acetate (PMA, 150 ng/ml) and the translation inhibitor cycloheximide. Cells were also genetically altered to overexpress the 33-nt binding sequence or the enzyme 6-PGD.
• Main results:
  • TGF-beta and TNF-alpha increased PAI-1 protein in MeT5A and MS-1 cells. The increase was larger in MeT5A cells than in MS-1 mesothelioma cells.
  • In MeT5A cells, PAI-1 messenger RNA had a half-life of less than 3 hours. PMA, TGF-beta and cycloheximide lengthened the half-life to about 8 to 12 hours.
  • A protein of about 60 kD from MeT5A cells bound the 3' untranslated region of PAI-1 messenger RNA but not its coding region. Treating the cells with TGF-beta or TNF-alpha inhibited this binding.
  • The binding protein from non-malignant MeT5A cells bound PAI-1 messenger RNA to a greater extent than the binding protein from the M33K, M9K and MS-1 mesothelioma cell lines.
  • The protein bound a specific 33-nucleotide sequence (nucleotides 1690 to 1722, called B3a) in the PAI-1 messenger RNA.
  • Inserting this 33-nucleotide sequence into otherwise stable beta-globin messenger RNA made it unstable in MeT5A cells. A control sequence of similar size did not.
  • When MeT5A cells overexpressed the 33-nucleotide binding sequence, the cells' own PAI-1 messenger RNA broke down more slowly, and PAI-1 protein and messenger RNA increased. A non-binding control sequence did not have this effect.
  • Purification gave two protein bands, of about 45 and 55 kD, identified as citrate synthase and 6-PGD. Only 6-PGD bound the PAI-1 messenger RNA sequence.
  • In MeT5A cells overexpressing 6-PGD, TGF-beta-induced PAI-1 expression was at least 50% lower than in cells transfected with an empty vector.
  • In primary rabbit mesothelial cells, 6-PGD expressed from an adenovirus reduced PAI-1 expression by more than 50% compared with empty vector. It also blunted TGF-beta-induced PAI-1.
• The authors' conclusion: The authors concluded that the interaction of 6-PGD with the 33-nucleotide sequence of PAI-1 messenger RNA regulates the stability of that RNA. They also concluded that TGF-beta raises PAI-1 in cultured MeT5A cells by stabilizing PAI-1 messenger RNA. They suggested that, if this pathway operates in the living body, it could contribute to PAI-1 overexpression in inflammatory pleural diseases.
• Limitations the authors state:
  • The work was done in cultured cells. The authors said that whether the pathway operates in the living body remains a possibility, to be pursued in later work.
  • The authors noted an apparent inconsistency between PAI-1 expression and the strength of the binding interaction in MeT5A versus MS-1 cells. They suggested it may reflect changes in transcription or translation.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelial Cells, Malignant Mesothelioma, Mesothelioma, Inflammation.

Related studies

• Asbestos upregulates expression of the urokinase-type plasminogen activator receptor on mesothelial cells — R.C. Perkins, V.C. Broaddus, et al. (1999)
• Absence of post-transcriptional RNA modifications of BCL10 in human malignant mesothelioma and colorectal cancer — S. Apostolou, S.S. Murthy, et al. (2001)
• Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma — T. He, K. McColl, et al. (2018)
• Increased production of plasminogen activator inhibitor in vitro by pleural leukocytes from rats intratracheally instilled with crocidolite asbestos — X.Y. Li, G.M. Brown, et al. (1991)
• Aberrant expression of FoxP3 in a human T cell line possessing regulatory T cell‑like function and exposed continuously to asbestos fibers — M. Maeda, H. Matsuzaki, et al. (2018)
• Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers — C.R. Timblin, G.D. Guthrie, et al. (1998)

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