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Cell Biology and Toxicology

, Volume 35, Issue 1, pp 67–80 | Cite as

Aflatoxin-induced upregulation of protein arginine methyltransferase 5 is mediated by protein kinase C and extracellular signal-regulated kinase

  • Md. Sajid Ghufran
  • Priyanka Soni
  • Santosh R. KanadeEmail author
Original Article
  • 99 Downloads

Abstract

Aflatoxins are fungal metabolites classified into four major groups such as B1, B2, G1, and G2. These natural aflatoxins are designated as group I carcinogen by the International Agency for Research on Cancer. Among these, the aflatoxin B1 is more potent. Protein arginine methyltransferase 5, an epigenetic modulator, emerged as an oncoprotein, is overexpressed in diverse forms of cancers. The present study aims to explore the AFB1-mediated overexpression of PRMT5. The AFB1 at nanomolar concentrations increased the cell viability, as well as the expression of PRMT5 and its binding partner methylosome protein 50 level significantly in L-132 and HaCaT cells. The knockdown of PRMT5 by its siRNA is attenuated by AFB1, thus substantiating AFB1-mediated PRMT5 overexpression. The PKC isoform-specific inhibitor study revealed direct relation with PKCα and an inverse relation with PKCδ. The analysis of mitogen-activated protein kinase pathway revealed reduced p38 phosphorylation with increased phosphorylation of ERK1/2 upon exposure to AFB1. The combination of MEK and PKC inhibitors with AFB1 treatment revealed that PKCα activates downstream kinase ERK which leads to overexpression of PRMT5. In summary, we propose that PKCα and extracellular signal-regulated kinases are conjointly involved in the induction of PRMT5 upon AFB1 exposure.

Keywords

Aflatoxin B1 Arginine methylation Aspergillus ERK PKC PRMT5 

Abbreviations

AFB1

Aflatoxin B1

ERK

Extracellular signal-related kinases

MAPK

Mitogen-activated protein kinase

MEP50

Methylosome protein 50

PKC

Protein kinase C

PRMT5

Protein arginine methyltransferase 5

TPA

Tetradecanoylphorbol-13-acetate

Notes

Funding information

The authors are grateful to Kerala State Council for Science, Technology, and Environment (KSCSTE), Govt. of Kerala, for the financial support.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

10565_2018_9439_Fig7_ESM.png (717 kb)
Figure S1

Determination of number of viable cells using trypan blue exclusion assay in HaCaT cells. The cells were counted using trypan blue exclusion assay. Cells grown in 0.1% DMSO were used as control and considered as 100% viable. The data are presented as mean ± SD of three individual experiments that gave similar results. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control. (PNG 717 kb).

10565_2018_9439_MOESM1_ESM.tif (2.1 mb)
High resolution image (TIF 2191 kb).
10565_2018_9439_Fig8_ESM.png (3.4 mb)
Figure S2

Knockdown of PRMT5 on treatment with PRMT5-siRNA in human L-132 and HaCaT cells. The cells were transfected with either 1 μg of control or PRMT5-siRNA for 4 hours followed by treatment with indicated concentration of AFB1 in (A) L-132 cells (B) HaCaT cells. RNA was isolated and assessed for PRMT5 mRNA level by qRT-PCR analysis (C) 20 μg of total protein was electrophoresed on a 10% SDS gel to assess PRMT5 protein level. β-actin was used as loading control. (D, E) The relative intensities of bands with respect to control are plotted. (F) The cells were pre-incubated with PRMT5 inhibitor (0.2 and 0.4 μM, EPZ015666) for 6 hour followed by treatment with 1000 nM AFB1. The cellular lysates were prepared for the detection of PRMT5, H4R3me2 and SDMA protein level. β-actin was used as loading control. (G) The relative intensities of bands with respect to control are plotted The data are presented as mean ± SD of three individual experiments that gave similar results. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control. #p < 0.05 and ##p < 0.01 versus 1000 nM AFB1. (PNG 3475 kb).

10565_2018_9439_MOESM2_ESM.tif (6.5 mb)
High resolution image (TIF 6623 kb).
10565_2018_9439_Fig9_ESM.png (2.2 mb)
Figure S3

Knockdown of PKCδ on treatment with PKCδ-siRNA. (A, B) Dose dependent expression of PKCδ mRNA and protein. (C, D) PKCδ mRNA and protein abundance in presence of PKCδ-siRNA. (E) The cells were transfected with 1 μg of control-, PKCδ-siRNA for 4 hours followed by treatment with indicated concentration of AFB1. RNA was isolated and assessed for PRMT5 mRNA level by qRT-PCR analysis (F) 20 μg of total protein was electrophoresed on a 10% SDS gel to assess PRMT5 protein level. β-actin was used as loading control. The relative intensities of bands with respect to control are plotted. The data are presented as mean ± SD of three individual experiments that gave similar results. *p < 0.05, **p < 0.01 and ***p < 0.001 versus control. (PNG 2205 kb).

10565_2018_9439_MOESM3_ESM.tif (5.2 mb)
High resolution image (TIF 5296 kb).

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Md. Sajid Ghufran
    • 1
  • Priyanka Soni
    • 1
  • Santosh R. Kanade
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, School of Biological Sciences, Riverside transit CampusCentral University of KeralaKasargod DistrictIndia

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