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

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.

Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

“…Interestingly, the localization of renal DNase I in humans with class IV lupus nephritis is mostly in the nuclei of tubular cells. Previously we have found that DNase I in nuclei of human renal proximal tubule epithelial cells is enzymatically inactive, and may possess another function besides the endonucleolytic role [28].…”

IL-1β Promotes a New Function of DNase I as a Transcription Factor for the Fas Receptor Gene

“…During a longitudinal study on expression profiles in (NZBxNZW)F1 (BW) mice, we observed a tendency for DNase I up-regulation during mesangial nephritis, before a subsequent and distinct down-regulation of the gene during progressive disease (Fenton et al, 2009 ; Seredkina and Rekvig, 2011 ). In addition, we have observed nuclear localization of renal DNase I in tubular cells in human lupus nephritis (Thiyagarajan et al, 2015 ). Studies on cultured human renal proximal tubular epithelial cells (RPTEC) have also shown translocation of DNase I into the nucleus under certain conditions (Thiyagarajan et al, 2015 ).…”

“…In addition, we have observed nuclear localization of renal DNase I in tubular cells in human lupus nephritis (Thiyagarajan et al, 2015 ). Studies on cultured human renal proximal tubular epithelial cells (RPTEC) have also shown translocation of DNase I into the nucleus under certain conditions (Thiyagarajan et al, 2015 ).…”

“…Two DNase I variants were determined to be products of the DNASE1 gene by mass spectrometry (MS) and were expressed in the cytoplasm; one enzymatically active with molecular weight (MW) of 40 kDa, and another enzymatically inactive with MW around 55 kDa. The DNase I variant that translocated into the nucleus was enzymatically inactive and differed from the other detected DNase I molecules, as it had a MW of 52 kDa (Thiyagarajan et al, 2015 ). Studies from Oliveri et al suggested that recombinant DNase I added to cell cultures could act as transcriptional factor for FAS and increase the expression of cell surface fas receptor (FasR) in human cells (Oliveri et al, 2004 ).…”

“…The present study aims to describe three phenomena linked to renal DNASE1 gene expression and subcellular compartmentation in response to different stimuli. First, since the DNASE1 gene tended to be upregulated in mesangial nephritis (Fenton et al, 2009 ; Thiyagarajan et al, 2015 ), we assumed that pro-inflammatory cytokines may be responsible for this phenomenon. Second, the same pro-inflammatory cytokines were expected to induce nuclear translocation of DNase I; and third, nuclear translocated DNase I presumably had another biological significance than to promote chromatin degradation.…”