Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1

Sudha Priya Soundara Pandi; Michael J Shattock; Bruce M Hendry; Claire C Sharpe

doi:10.1186/s12882-022-02844-1

Stimulated phosphorylation of ERK in mouse kidney mesangial cells is dependent upon expression of Cav3.1

BMC Nephrol. 2022 Jun 16;23(1):211. doi: 10.1186/s12882-022-02844-1.

Authors

Sudha Priya Soundara Pandi^{1

2}, Michael J Shattock³, Bruce M Hendry¹, Claire C Sharpe⁴

Affiliations

¹ Department of Inflammation Biology, King's College London, Denmark Hill Campus, James Black Centre, London, SE5 9NU, UK.
² Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK.
³ School of Cardiovascular Medicine and Sciences, King's College London, London, UK.
⁴ Department of Inflammation Biology, King's College London, Denmark Hill Campus, James Black Centre, London, SE5 9NU, UK. Claire.sharpe@kcl.ac.uk.

Abstract

Background: T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases. METHODS: In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms Ca_V3.1 and Ca_V3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-β1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177.

Results: We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-β1) in Ca_V3.1 SKO clone, whereas the Ca_V3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death.

Conclusion: This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the Ca_V3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca _V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.

Keywords: CRISPR-cas9; Calcium channels; ERK1/2; Glomerulonephritis; Mesangial cell.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Calcium Channels, T-Type*
Humans
Mesangial Cells* / metabolism
Mibefradil / metabolism
Mibefradil / pharmacology
Mice
Phosphorylation
Rats
Transforming Growth Factor beta1 / metabolism

Substances

Calcium Channels, T-Type
Transforming Growth Factor beta1
Mibefradil