TNFα Amplifies DNaseI Expression in Renal Tubular Cells while IL-1β Promotes Nuclear DNaseI Translocation in an Endonuclease-Inactive Form.

Dhivya Thiyagarajan,Ole Petter Rekvig,Natalya Seredkina,Jean-Claude Dussaule

doi:10.1371/journal.pone.0129485

Dhivya Thiyagarajan, Ole Petter Rekvig + Show 2 more

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https://doi.org/10.1371/journal.pone.0129485

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Abstract

We have demonstrated that the renal endonuclease DNaseI is up-regulated in mesangial nephritis while down-regulated during progression of the disease. To determine the basis for these reciprocal DNaseI expression profiles we analyse processes accounting for an early increase in renal DNaseI expression. Main hypotheses were that i. the mesangial inflammation and secreted pro-inflammatory cytokines directly increase DNaseI protein expression in tubular cells, ii. the anti-apoptotic protein tumor necrosis factor receptor-associated protein 1 (Trap 1) is down-regulated by increased expression of DNaseI due to transcriptional interference, and iii. pro-inflammatory cytokines promote nuclear translocation of a variant of DNaseI. The latter hypothesis emerges from the fact that anti-DNaseI antibodies stained tubular cell nuclei in murine and human lupus nephritis. The present study was performed on human tubular epithelial cells stimulated with pro-inflammatory cytokines. Expression of the DNaseI and Trap 1 genes was determined by qPCR, confocal microscopy, gel zymography, western blot and by immune electron microscopy. Results from in vitro cell culture experiments were analysed for biological relevance in kidneys from (NZBxNZW)F1 mice and human patients with lupus nephritis. Central data indicate that stimulating the tubular cells with TNFα promoted increased DNaseI and reduced Trap 1 expression, while TNFα and IL-1β stimulation induced nuclear translocation of the DNaseI. TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI. IL-1β-stimulation solely induced nuclear DNaseI translocation. Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear translocation. This demonstrates that IL-1β promotes nuclear translocation of a cytoplasmic variant of DNaseI since translocation clearly was not dependent on DNaseI gene activation. Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

Highlights

Lupus nephritis is a prototype immune complex disease where antibodies to dsDNA play a central role [1,2,3]
Increased renal DNaseI expression in mesangial nephritis and nuclear DNaseI translocation had its experimental analogy when showing the same phenomena in tubular cells stimulated with TNFα and IL-1β, but not by hypoxic stress, nor by IFNγ, IL-6 or IL-10 in renal proximal tubule epithelial cells (RPTEC)
The clinical consequence of renal DNaseI gene silencing is a predictable transformation of mild mesangial lupus nephritis into end-stage organ disease

Summary

Introduction

Lupus nephritis is a prototype immune complex disease where antibodies to dsDNA play a central role [1,2,3]. Deposition of chromatin fragment-anti-dsDNA antibody complexes has been shown to be one of the core factors that impose progressive renal inflammation [4,5,6,7,8,9]. DNaseI executes the initial degradation of whole chromatin in context of apoptosis and necrosis [16,17]. This is important to perceive, as the initial chromatin degradation is a prerequisite for other secondary endonucleases to digest the chromatin fragments into small oligonucleosomes This is important to perceive, as the initial chromatin degradation is a prerequisite for other secondary endonucleases to digest the chromatin fragments into small oligonucleosomes (see e.g. [16,17] for review)

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