Analysis of furin (FURIN) gene expression in the U-87 MG cell line as a potential target for gene inhibiting therapies in (auto-) immune diseases

Karolina Zapletal-Pudełko; Grzegorz Machnik; Katarzyna Stec-Grosman; Łukasz Bułdak; Bogusław Okopień

doi:10.18778/1730-2366.18.16

Authors

Karolina Zapletal-Pudełko Medical University of Silesia, Faculty of Medical Science in Katowice, Department of Internal Medicine and Clinical Pharmacology, Poland https://orcid.org/0009-0003-2211-1764
Grzegorz Machnik Medical University of Silesia, Faculty of Medical Science in Katowice, Department of Internal Medicine and Clinical Pharmacology, Poland https://orcid.org/0000-0002-9081-0984
Katarzyna Stec-Grosman Medical Laboratory Synevo, Poland
Łukasz Bułdak Medical University of Silesia, Faculty of Medical Science in Katowice, Department of Internal Medicine and Clinical Pharmacology, Poland
Bogusław Okopień Medical University of Silesia, Faculty of Medical Science in Katowice, Department of Internal Medicine and Clinical Pharmacology, Poland

DOI:

https://doi.org/10.18778/1730-2366.18.16

Keywords:

neuroinflammation, FURIN, HERVs, neurodegenerative diseases, gene therapies

Abstract

Purpose: Furin is a proprotein convertase commonly found in the human body. The enzymatic activity of furin is necessary for the activation of numerous substrates including e.g. hormones and growth factors. Nevertheless, furin is involved in various pathological conditions caused by, among others, chronic inflammation. Therefore furin is considered as a potential target in autoimmune diseases therapy.

We performed an experiment in which the expression of FURIN gene in U-87 MG astrocytoma cells was investigated. Additionally, this cell line contains some sequences coding human endogenous retroviruses (HERVs), including ERVW-1 and its receptor- SLC1A5. Deregulation of HERV expression has been observed in some neurodegenerative diseases as well as in inflammatory process.

Material and Methods: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) techniques were utilized for analysis. Phorbol 12-myristate 13-acetate (PMA) were used for cell stimulation. Short interfering RNAs (siRNA) were used for gene expression inhibition in U-87 MG cells in vitro.

Results: Cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p<0.01). Moreover addition of PMA significantly stimulates the autocatalytic action of cellular furin itself resulting in the dissociation of its propeptide that was clearly visible in a time-dependent manner. SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p<0.01).

Conclusion: Our results showed that stimulation by PMA and inhibition expression by siRNA affects the expression of FURIN in U-87 MG astrocytoma cells. Moreover, furin shows a complex relationship on the expression of ERVW-1 and SLC1A5 genes, as well as on the form (precursor or mature) and the amount of the final translation products of the transcripts. The regulation of FURIN may pose a potential therapeutic strategy in the treatment of neurodegenerative diseases caused by autoimmunity.

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References

Amos, S., Martin, P.M., Polar, G.A., Parsons, S.J., Hussaini, I.M. 2005. Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells. The Journal of Biological Chemistry, 280, 7729–7738.
Google Scholar

Antony, J.M., Izad, M., Bar-Or, A., Warren, K.G., Vodjgani, M., Mallet, F., Power, C. 2006. Quantitative analysis of human endogenous retrovirus-W env in neuroinflammatory diseases. AIDS Research and Human Retroviruses, 22, 1253–1259.
Google Scholar

Bennett, B.D., Denis, P., Haniu, M., Teplow, D.B., Kahn, S., Louis, J.C., Citron, M., Vassar, R. 2000. A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer’s β-secretase. The Journal of Biological Chemistry 275, 37712–37717.
Google Scholar

Chomczynski, P., Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochememistry, 162, 156–159.
Google Scholar

Dolei, A. 2018. The aliens inside us: HERV-W endogenous retroviruses and multiple sclerosis. Multiple Sclerosis (Houndmills, Basingstoke, England) 24, 42–47.
Google Scholar

Dolei, A., Ibba, G., Piu, C., Serra, C. 2019. Expression of HERV Genes as Possible Biomarker and Target in Neurodegenerative Diseases. International Journal of Molecular Sciences, 20.
Google Scholar

Dolei, A., Uleri, E., Ibba, G., Caocci, M., Piu, C., Serra, C. 2015. The aliens inside human DNA: HERV-W/MSRV/syncytin-1 endogenous retroviruses and neurodegeneration. Journal of Infection in Developing Countries, 9, 577–587.
Google Scholar

Abu El-Asrar, A.M., Nawaz, M.I., Ahmad, A., Siddiquei, M.M., Allegaert, E., Gikandi, P.W., De Hertogh, G., Opdenakker, G. 2022. Proprotein convertase furin is a driver and potential therapeutic target in proliferative diabetic retinopathy. Clinical & Experimental Ophthalmology, 50, 632–652.
Google Scholar

Gawlik, K., Shiryaev, S.A., Zhu, W., Motamedchaboki, K., Desjardins, R., Day, R., Remacle, A.G., Stec, B., Strongin, A.Y. 2009. Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme’s N-terminus from the active site. PloS One, 4, e5031.
Google Scholar

Machnik, G., Bułdak, Ł., Zapletal-Pudełko, K., Grabarek, B.O., Staszkiewicz, R., Sobański, D., Okopień, B. 2024. The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells. Advances in Medical Sciences, 69, 113–124.
Google Scholar

Ni, J., Dai, W., Liu, C., Ling, Y., Mou, H. 2023. A pan-cancer analysis of SLC1A5 in human cancers. Heliyon, 9, e17598.
Google Scholar

Noorali, S., Rotar, I.C., Lewis, C., Pestaner, J.P., Pace, D.G., Sison, A., Bagasra, O. 2009. Role of HERV-W syncytin-1 in placentation and maintenance of human pregnancy. Applied Immunohistochemistry & Molecular Morphology, 17, 319–328.
Google Scholar

Oliveira, A.C.P. de, Candelario-Jalil, E., Fiebich, B.L., Santos, M. da S., Palotás, A., Reis, H.J. dos 2015. Neuroinflammation and Neurodegeneration: Pinpointing Pathological and Pharmacological Targets. BioMed Research International, 2015, 487241.
Google Scholar

Poma, P. 2020. NF-κB and Disease. International Journal of Molecular Sciences, 21.
Google Scholar

Roe, K. 2021. An inflammation classification system using cytokine parameters. Scandinavian Journal of Immunology 93, e12970.
Google Scholar

Schmittgen, T.D., Livak, K.J. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3, 1101–1108.
Google Scholar

Schneider, C.A., Rasband, W.S., Eliceiri, K.W. 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675.
Google Scholar

Singh, S., Borkar, M.R., Bhatt, L.K. 2024. Transposable Elements: Emerging Therapeutic Targets in Neurodegenerative Diseases. Neurotoxicity Research 42, 9.
Google Scholar

Valli, A., Ranta, N., Grönholm, A., Silvennoinen, O., Pesu, M., Isomäki, P. 2019. Increased expression of the proprotein convertase enzyme FURIN in rheumatoid arthritis. Scandinavian Journal of Rheumatology 48, 173–177.
Google Scholar

Vardi, G., Merrick, J. 2007. Neurological Disorders. Public Health Challenges. Journal of Policy and Practice in Intellectual Disabilities 4.
Google Scholar

Wang, X., Huang, J., Zhu, F. 2018a. Human Endogenous Retroviral Envelope Protein Syncytin-1 and Inflammatory Abnormalities in Neuropsychological Diseases. Frontiers in Psychiatry, 9, 422.
Google Scholar

Wang, X., Liu, Z., Wang, P., Li, S., Zeng, J., Tu, X., Yan, Q., Xiao, Z., Pan, M., Zhu, F. 2018b. Syncytin-1, an endogenous retroviral protein, triggers the activation of CRP via TLR3 signal cascade in glial cells. Brain, Behavior, and Immunity, 67, 324–334.
Google Scholar

Zhang, Y., Gao, X., Bai, X., Yao, S., Chang, Y.-Z., Gao, G. 2022. The emerging role of furin in neurodegenerative and neuropsychiatric diseases. Translational Neurodegeneration, 11, 39.
Google Scholar