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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.Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear 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

Nuclear DNaseI translocation in RPTEC was not associated with caspase 3 activation or DNA fragmentation.Western blot analysis was performed to detect presence of activated caspase 3 in cell lysates from RPTEC stimulated or non-stimulated with 20ng/ml of TNFα for 48 hrs (A). Resting Jurkat cells or cytochrome c treated Jurkat cells were used as a negative and positive control for activated caspase 3 (A, lane 1 and 2, respectively). As demonstrated in A, lane 4, TNFα-induced nuclear DNaseI translocation was not accompanied by activation of caspase 3 since only inactive pro-caspase 3 was found in RPTEC stimulated with TNFα. Only in cytochrome c treated cells activated caspase 3 was detected by western blot as shown in A, lane 2. Equal loading was controlled by staining with actin. Location of DNaseI in the nuclei of RPTEC did not result in chromatin fragmentation as demonstrated by DNA gel electrophoresis in B and TUNEL assay in C. DNA from murine spleen incubated with 5μM camptothecin for 6 hrs was used as a positive control for apoptotic DNA fragmentation (B, lane 2). No apoptotic DNA fragmentation was observed in DNA isolated from sham-stimulated RPTEC cells (B, lane 3) or in DNA isolated from RPTEC after stimulation with 20ng/ml of TNFα for 48 hrs (B, lane 4). TUNEL-positive staining in RPTEC cells was detected only after treatment with 10U of rhDNaseI (C, green). RPTEC cells stimulated with TNFα were TUNEL–negative as well as sham-stimulated cells as demonstrated in C. Lane 1 in B represent DNA size markers.
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pone.0129485.g007: Nuclear DNaseI translocation in RPTEC was not associated with caspase 3 activation or DNA fragmentation.Western blot analysis was performed to detect presence of activated caspase 3 in cell lysates from RPTEC stimulated or non-stimulated with 20ng/ml of TNFα for 48 hrs (A). Resting Jurkat cells or cytochrome c treated Jurkat cells were used as a negative and positive control for activated caspase 3 (A, lane 1 and 2, respectively). As demonstrated in A, lane 4, TNFα-induced nuclear DNaseI translocation was not accompanied by activation of caspase 3 since only inactive pro-caspase 3 was found in RPTEC stimulated with TNFα. Only in cytochrome c treated cells activated caspase 3 was detected by western blot as shown in A, lane 2. Equal loading was controlled by staining with actin. Location of DNaseI in the nuclei of RPTEC did not result in chromatin fragmentation as demonstrated by DNA gel electrophoresis in B and TUNEL assay in C. DNA from murine spleen incubated with 5μM camptothecin for 6 hrs was used as a positive control for apoptotic DNA fragmentation (B, lane 2). No apoptotic DNA fragmentation was observed in DNA isolated from sham-stimulated RPTEC cells (B, lane 3) or in DNA isolated from RPTEC after stimulation with 20ng/ml of TNFα for 48 hrs (B, lane 4). TUNEL-positive staining in RPTEC cells was detected only after treatment with 10U of rhDNaseI (C, green). RPTEC cells stimulated with TNFα were TUNEL–negative as well as sham-stimulated cells as demonstrated in C. Lane 1 in B represent DNA size markers.

Mentions: DNaseI can translocate into the nuclei upon apoptotic stimuli where it performs its canonical function- chromatin fragmentation [33,37]. However in RPTEC stimulated with TNFα or with IL-1β, nuclear translocation of DNaseI was not accompanied by induction of an apoptotic process or DNA degradation. As demonstrated in Fig 7A no activated caspase 3 was detected by western blot analysis in cell lysates of TNFα stimulated RPTEC. No DNA fragmentation was found in TNFα stimulated RPTEC with nuclear DNaseI location as shown by DNA agarose gel and by negative TUNEL assay (Fig 7B and 7C respectively). These data are in accordance with gel zymography analyses of murine nephritic kidneys, showing no endonucleolytic activity above 40 kDa (Fig 2D). These data indicate that DNaseI located in the nuclei after TNFα or IL-1β stimulations has another function than to serve as an endonucleolytic enzyme.


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)

Nuclear DNaseI translocation in RPTEC was not associated with caspase 3 activation or DNA fragmentation.Western blot analysis was performed to detect presence of activated caspase 3 in cell lysates from RPTEC stimulated or non-stimulated with 20ng/ml of TNFα for 48 hrs (A). Resting Jurkat cells or cytochrome c treated Jurkat cells were used as a negative and positive control for activated caspase 3 (A, lane 1 and 2, respectively). As demonstrated in A, lane 4, TNFα-induced nuclear DNaseI translocation was not accompanied by activation of caspase 3 since only inactive pro-caspase 3 was found in RPTEC stimulated with TNFα. Only in cytochrome c treated cells activated caspase 3 was detected by western blot as shown in A, lane 2. Equal loading was controlled by staining with actin. Location of DNaseI in the nuclei of RPTEC did not result in chromatin fragmentation as demonstrated by DNA gel electrophoresis in B and TUNEL assay in C. DNA from murine spleen incubated with 5μM camptothecin for 6 hrs was used as a positive control for apoptotic DNA fragmentation (B, lane 2). No apoptotic DNA fragmentation was observed in DNA isolated from sham-stimulated RPTEC cells (B, lane 3) or in DNA isolated from RPTEC after stimulation with 20ng/ml of TNFα for 48 hrs (B, lane 4). TUNEL-positive staining in RPTEC cells was detected only after treatment with 10U of rhDNaseI (C, green). RPTEC cells stimulated with TNFα were TUNEL–negative as well as sham-stimulated cells as demonstrated in C. Lane 1 in B represent DNA size markers.
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pone.0129485.g007: Nuclear DNaseI translocation in RPTEC was not associated with caspase 3 activation or DNA fragmentation.Western blot analysis was performed to detect presence of activated caspase 3 in cell lysates from RPTEC stimulated or non-stimulated with 20ng/ml of TNFα for 48 hrs (A). Resting Jurkat cells or cytochrome c treated Jurkat cells were used as a negative and positive control for activated caspase 3 (A, lane 1 and 2, respectively). As demonstrated in A, lane 4, TNFα-induced nuclear DNaseI translocation was not accompanied by activation of caspase 3 since only inactive pro-caspase 3 was found in RPTEC stimulated with TNFα. Only in cytochrome c treated cells activated caspase 3 was detected by western blot as shown in A, lane 2. Equal loading was controlled by staining with actin. Location of DNaseI in the nuclei of RPTEC did not result in chromatin fragmentation as demonstrated by DNA gel electrophoresis in B and TUNEL assay in C. DNA from murine spleen incubated with 5μM camptothecin for 6 hrs was used as a positive control for apoptotic DNA fragmentation (B, lane 2). No apoptotic DNA fragmentation was observed in DNA isolated from sham-stimulated RPTEC cells (B, lane 3) or in DNA isolated from RPTEC after stimulation with 20ng/ml of TNFα for 48 hrs (B, lane 4). TUNEL-positive staining in RPTEC cells was detected only after treatment with 10U of rhDNaseI (C, green). RPTEC cells stimulated with TNFα were TUNEL–negative as well as sham-stimulated cells as demonstrated in C. Lane 1 in B represent DNA size markers.
Mentions: DNaseI can translocate into the nuclei upon apoptotic stimuli where it performs its canonical function- chromatin fragmentation [33,37]. However in RPTEC stimulated with TNFα or with IL-1β, nuclear translocation of DNaseI was not accompanied by induction of an apoptotic process or DNA degradation. As demonstrated in Fig 7A no activated caspase 3 was detected by western blot analysis in cell lysates of TNFα stimulated RPTEC. No DNA fragmentation was found in TNFα stimulated RPTEC with nuclear DNaseI location as shown by DNA agarose gel and by negative TUNEL assay (Fig 7B and 7C respectively). These data are in accordance with gel zymography analyses of murine nephritic kidneys, showing no endonucleolytic activity above 40 kDa (Fig 2D). These data indicate that DNaseI located in the nuclei after TNFα or IL-1β stimulations has another function than to serve as an endonucleolytic enzyme.

Bottom Line: TNFα-stimulation resulted in 3 distinct effects; increased DNaseI and IL-1β gene expression, and nuclear translocation of DNaseI.Tubular cells stimulated with TNFα and simultaneously transfected with IL-1β siRNA resulted in increased DNaseI expression but no nuclear 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