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

Thiyagarajan D, Rekvig OP, Seredkina N - PLoS ONE (2015)

Bottom Line: 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.Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

View Article: PubMed Central - PubMed

Affiliation: RNA and Molecular Pathology Research Group, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.

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.

No MeSH data available.


Related in: MedlinePlus

Cytoplasmic and nuclear DNaseI and Trap 1 expression in tubular cells after stimulation with TNFα.Confocal microscopy of sham-stimulated human renal proximal tubule epithelial cells (RPTEC) (A, upper panels), and cells stimulated with 20ng/ml of TNFα for 48 hrs (A, lower panels) was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). No nuclear staining of DNaseI was observed in sham-stimulation of the cells (A, upper panels). After stimulation of RPTEC with TNFα, expression of DNaseI generally increased in staining intensity, and DNaseI translocated into the nucleus (A, lower panels). Nuclear location of DNaseI was confirmed by co-staining with DAPI (A, blue). The data demonstrate that DNaseI and DAPI were in confocus (violet in the merged picture, A, lower panel), thus confirming that DNaseI indeed was translocated to the nucleus. Correspondingly, by western blots both the 40 kDa and the 55 kDa bands increased in response to increasing TNFα stimulation (0, 10, 20ng/ml TNFα) (B). In the same cells, Trap 1 expression was reduced after TNFα stimulation, as determined by confocal microscopy (A, lower panels versus upper panels, green staining) and by western blot (B). Nuclear and cytoplasmic protein fractions were isolated from RPTEC after 48 hrs stimulation by TNFα (C). The 40 kDa and 55 kDa bands were detected in cytoplasmic fraction (C, lane 1), while weak 52 kDa band was observed in nuclear fraction of sham-stimulated RPTEC (C, lane 2). After 48 hrs stimulation with 20ng of TNFα, an increased DNaseI expression in cytoplasm (C, lane 3) and remarkably strong expression of the 52 kDa DNaseI in the nuclei was apparent (C, lane 4). Thus data from western blot analysis corresponded with data from confocal microscopy with respect to TNFα-induced nuclear translocation of DNaseI. Only nuclear fractions of RPTEC contained histone H1 protein as shown in D, confirming controlled separation of nuclear and cytoplasmic fractions in the cells. Actin was used as a loading control in all western blot analyses.
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pone.0129485.g005: Cytoplasmic and nuclear DNaseI and Trap 1 expression in tubular cells after stimulation with TNFα.Confocal microscopy of sham-stimulated human renal proximal tubule epithelial cells (RPTEC) (A, upper panels), and cells stimulated with 20ng/ml of TNFα for 48 hrs (A, lower panels) was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). No nuclear staining of DNaseI was observed in sham-stimulation of the cells (A, upper panels). After stimulation of RPTEC with TNFα, expression of DNaseI generally increased in staining intensity, and DNaseI translocated into the nucleus (A, lower panels). Nuclear location of DNaseI was confirmed by co-staining with DAPI (A, blue). The data demonstrate that DNaseI and DAPI were in confocus (violet in the merged picture, A, lower panel), thus confirming that DNaseI indeed was translocated to the nucleus. Correspondingly, by western blots both the 40 kDa and the 55 kDa bands increased in response to increasing TNFα stimulation (0, 10, 20ng/ml TNFα) (B). In the same cells, Trap 1 expression was reduced after TNFα stimulation, as determined by confocal microscopy (A, lower panels versus upper panels, green staining) and by western blot (B). Nuclear and cytoplasmic protein fractions were isolated from RPTEC after 48 hrs stimulation by TNFα (C). The 40 kDa and 55 kDa bands were detected in cytoplasmic fraction (C, lane 1), while weak 52 kDa band was observed in nuclear fraction of sham-stimulated RPTEC (C, lane 2). After 48 hrs stimulation with 20ng of TNFα, an increased DNaseI expression in cytoplasm (C, lane 3) and remarkably strong expression of the 52 kDa DNaseI in the nuclei was apparent (C, lane 4). Thus data from western blot analysis corresponded with data from confocal microscopy with respect to TNFα-induced nuclear translocation of DNaseI. Only nuclear fractions of RPTEC contained histone H1 protein as shown in D, confirming controlled separation of nuclear and cytoplasmic fractions in the cells. Actin was used as a loading control in all western blot analyses.

Mentions: RPTEC were stimulated for 24 hrs (S2A Fig) or for 48 hrs (Fig 5) by TNFα. In sham-stimulated RPTEC the anti-DNaseI antibody (Abcam) predominantly stained cytoplasm but not the nucleus (S2A Fig for 24h and Fig 5A for 48 hrs respectively, upper panels). In cells stimulated with 20ng/ml TNFα the anti-DNaseI antibody stained more strongly in cytoplasm and distinctly stronger in the nucleus (S2A Fig for 24 hrs and Fig 5A for 48 hrs respectively, lower panels). This nuclear translocation was consistent with an increased expression, not only of the 40 kDa protein, but also the approximately 55 kDa protein as demonstrated by western blot at 48 hrs of stimulation (Fig 5B). In agreement with data in Fig 1, Trap 1 expression was reduced concomitant with increased DNaseI expression (Fig 5B, see below). In order to characterize the sub-cellular origin of these proteins, we performed western blot analysis with anti-DNaseI antibodies on nuclear and cytoplasmic fractions of RPTEC (Fig 5C). The data demonstrated presence of a very weak 52 kDa band in the nuclear fraction of un-stimulated RPTEC (Fig 5C) but a very strong nuclear 52 kDa band after TNFα stimulation (Fig 5C). As is demonstrated, the 52 kDa protein was distinctly different from the 55 kDa protein in size.


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

Thiyagarajan D, Rekvig OP, Seredkina N - PLoS ONE (2015)

Cytoplasmic and nuclear DNaseI and Trap 1 expression in tubular cells after stimulation with TNFα.Confocal microscopy of sham-stimulated human renal proximal tubule epithelial cells (RPTEC) (A, upper panels), and cells stimulated with 20ng/ml of TNFα for 48 hrs (A, lower panels) was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). No nuclear staining of DNaseI was observed in sham-stimulation of the cells (A, upper panels). After stimulation of RPTEC with TNFα, expression of DNaseI generally increased in staining intensity, and DNaseI translocated into the nucleus (A, lower panels). Nuclear location of DNaseI was confirmed by co-staining with DAPI (A, blue). The data demonstrate that DNaseI and DAPI were in confocus (violet in the merged picture, A, lower panel), thus confirming that DNaseI indeed was translocated to the nucleus. Correspondingly, by western blots both the 40 kDa and the 55 kDa bands increased in response to increasing TNFα stimulation (0, 10, 20ng/ml TNFα) (B). In the same cells, Trap 1 expression was reduced after TNFα stimulation, as determined by confocal microscopy (A, lower panels versus upper panels, green staining) and by western blot (B). Nuclear and cytoplasmic protein fractions were isolated from RPTEC after 48 hrs stimulation by TNFα (C). The 40 kDa and 55 kDa bands were detected in cytoplasmic fraction (C, lane 1), while weak 52 kDa band was observed in nuclear fraction of sham-stimulated RPTEC (C, lane 2). After 48 hrs stimulation with 20ng of TNFα, an increased DNaseI expression in cytoplasm (C, lane 3) and remarkably strong expression of the 52 kDa DNaseI in the nuclei was apparent (C, lane 4). Thus data from western blot analysis corresponded with data from confocal microscopy with respect to TNFα-induced nuclear translocation of DNaseI. Only nuclear fractions of RPTEC contained histone H1 protein as shown in D, confirming controlled separation of nuclear and cytoplasmic fractions in the cells. Actin was used as a loading control in all western blot analyses.
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pone.0129485.g005: Cytoplasmic and nuclear DNaseI and Trap 1 expression in tubular cells after stimulation with TNFα.Confocal microscopy of sham-stimulated human renal proximal tubule epithelial cells (RPTEC) (A, upper panels), and cells stimulated with 20ng/ml of TNFα for 48 hrs (A, lower panels) was performed by using anti-Trap1 antibodies (A, green) and by anti-DNaseI antibodies (A, red). No nuclear staining of DNaseI was observed in sham-stimulation of the cells (A, upper panels). After stimulation of RPTEC with TNFα, expression of DNaseI generally increased in staining intensity, and DNaseI translocated into the nucleus (A, lower panels). Nuclear location of DNaseI was confirmed by co-staining with DAPI (A, blue). The data demonstrate that DNaseI and DAPI were in confocus (violet in the merged picture, A, lower panel), thus confirming that DNaseI indeed was translocated to the nucleus. Correspondingly, by western blots both the 40 kDa and the 55 kDa bands increased in response to increasing TNFα stimulation (0, 10, 20ng/ml TNFα) (B). In the same cells, Trap 1 expression was reduced after TNFα stimulation, as determined by confocal microscopy (A, lower panels versus upper panels, green staining) and by western blot (B). Nuclear and cytoplasmic protein fractions were isolated from RPTEC after 48 hrs stimulation by TNFα (C). The 40 kDa and 55 kDa bands were detected in cytoplasmic fraction (C, lane 1), while weak 52 kDa band was observed in nuclear fraction of sham-stimulated RPTEC (C, lane 2). After 48 hrs stimulation with 20ng of TNFα, an increased DNaseI expression in cytoplasm (C, lane 3) and remarkably strong expression of the 52 kDa DNaseI in the nuclei was apparent (C, lane 4). Thus data from western blot analysis corresponded with data from confocal microscopy with respect to TNFα-induced nuclear translocation of DNaseI. Only nuclear fractions of RPTEC contained histone H1 protein as shown in D, confirming controlled separation of nuclear and cytoplasmic fractions in the cells. Actin was used as a loading control in all western blot analyses.
Mentions: RPTEC were stimulated for 24 hrs (S2A Fig) or for 48 hrs (Fig 5) by TNFα. In sham-stimulated RPTEC the anti-DNaseI antibody (Abcam) predominantly stained cytoplasm but not the nucleus (S2A Fig for 24h and Fig 5A for 48 hrs respectively, upper panels). In cells stimulated with 20ng/ml TNFα the anti-DNaseI antibody stained more strongly in cytoplasm and distinctly stronger in the nucleus (S2A Fig for 24 hrs and Fig 5A for 48 hrs respectively, lower panels). This nuclear translocation was consistent with an increased expression, not only of the 40 kDa protein, but also the approximately 55 kDa protein as demonstrated by western blot at 48 hrs of stimulation (Fig 5B). In agreement with data in Fig 1, Trap 1 expression was reduced concomitant with increased DNaseI expression (Fig 5B, see below). In order to characterize the sub-cellular origin of these proteins, we performed western blot analysis with anti-DNaseI antibodies on nuclear and cytoplasmic fractions of RPTEC (Fig 5C). The data demonstrated presence of a very weak 52 kDa band in the nuclear fraction of un-stimulated RPTEC (Fig 5C) but a very strong nuclear 52 kDa band after TNFα stimulation (Fig 5C). As is demonstrated, the 52 kDa protein was distinctly different from the 55 kDa protein in size.

Bottom Line: 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.Nuclear translocated DNaseI is shown to be enzymatically inactive, which may point at a new, yet unknown function of renal DNaseI.

View Article: PubMed Central - PubMed

Affiliation: RNA and Molecular Pathology Research Group, Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø, Norway.

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.

No MeSH data available.


Related in: MedlinePlus