Sequential and synchronized hypertonicity-induced activation of Rel-family transcription factors is required for osmoprotection in renal cells

Abstract

NF-κB and TonEBP belong to the Rel-superfamily of transcription factors. Several specific stimuli, including hypertonicity which is a key factor for renal physiology, are able to activate them. It has been reported that, after hypertonic challenge, NF-κB activity can be modulated by TonEBP, considered as the master regulator of transcriptional activity in the presence of changes in environmental tonicity. In the present work we evaluated whether hypertonicity-induced gene transcription mediated by p65/RelA and TonEBP occurs by an independent action of each transcription factor or by acting together. To do this, we evaluated the expression of their specific target genes and cyclooxygenase-2 (COX-2), a common target of both transcription factors, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) subjected to hypertonic environment. The results herein indicate that hypertonicity activates the Rel-family transcription factors p65/RelA and TonEBP in MDCK cells, and that both are required for hypertonic induction of COX-2 and of their specific target genes. In addition, present data show that p65/RelA modulates TonEBP expression and both colocalize in nuclei of hypertonic cultures of MDCK cells. Thus, a sequential and synchronized action p65/RelA → TonEBP would be necessary for the expression of hypertonicity-induced protective genes.

Keywords: Biochemistry; Cell biology; Molecular biology.

Fig. 1

Effect of hypertonicity on NF-κB expression, cellular distribution and activity in MDCK cells. MDCK cells were grown and subjected to 125 mM NaCl (∼512 mosm/kg H₂O) for different periods of time (0, 1.5, 3, 6, 12 and 24 h) as described in Methods. After treatment, cells were collected and subjected to total RNA isolation followed by RT-PCR for NF-κB (p65/RelA) and β-actin (Panel A), or by western blot analysis probing the membrane with mouse monoclonal p65-RelA antibody (1:500) and rabbit polyclonal β-tubulin antibody (1:5000) (Panel B). Each image is representative of three independent experiments eliciting similar pattern. In Panel C, MDCK cells were grown on sterile coverslips and after 16 h of treatment, cells were fixed and stained with mouse monoclonal p65-RelA antibody (1:100) and revealed by using a FITC-conjugated secondary antibody (1:200). Samples were mounted with Vectashield Mounting Medium. Fluorescence images were obtained with a Nikon Eclipse Ti with acquisition software Micrometrics SE Premium (Accu-Scope). The figure shows a representative image of three independent experiments. Panel D shows cell fractionation after hypertonic treatment followed by western blot analysis of nuclear and cytoplasmic fractions. Lamin A was used as nuclear marker while β-tubulin, as cytoplasmic marker. The nuclear fraction (p65-RelA/LaminA) to cytoplasmic fraction (p65-RelA/β-tubulin) ratios were calculated from the values obtained by the densitometry analysis of the western blot membranes from three independent experiments. The expression of MCP1, a target gene of NF-κB activity, was determined by RT-PCR (Panel E). The figure shows a representative image of three independent experiments. The results are expressed as the mean ± SEM of three independent experiments.

Fig. 2

Effect of hypertonicity on TonEBP expression, cellular distribution and activity in MDCK cells. MDCK cells were grown and subjected to 125 mM NaCl (∼512 mosm/kg H₂O) for different periods of time (0, 1.5, 3, 6, 12 and 24 h) as described in Methods. After treatment, cells were collected and subjected to total RNA isolation followed by RT-PCR for TonEBP and β-actin (Panel A) or subjected to western blot analysis by probing the membrane with rabbit polyclonal TonEBP antibody (1:500) and rabbit polyclonal β-tubulin antibody (1:5000) (Panel B). Each image is representative of three independent experiments eliciting similar pattern. In Panel C, MDCK cells were grown on sterile coverslips and after 16 h of treatment, cells were fixed and stained with rabbit polyclonal TonEBP (1:75) antibody and revealed by using a FITC-conjugated secondary antibody (1:200). Samples were mounted with Vectashield Mounting Medium. Fluorescence images were obtained with a Nikon Eclipse Ti with acquisition software Micrometrics SE Premium (Accu-Scope). The figure shows a representative image of three independent experiments. Panel D shows cell fractionation after hypertonic treatment followed by western blot analysis of nuclear and cytoplasmic fractions. Lamin A was used as nuclear marker while β-tubulin, as cytoplasmic marker. The nuclear fraction (TonEBP/LaminA) to cytoplasmic fraction (TonEBP/β-tubulin) ratios were calculated from the values obtained by the densitometry analysis of the western blot membranes from three independent experiments. The expression of AR, BGT1, SMIT and COX-2, TonEBP target genes, was determined by RT-PCR (Panel E). The figure shows a representative image of three independent experiments. The results are expressed as the mean ± SEM of three independent experiments.

Fig. 3

Effect of NF-κB pathway inhibitors on COX-2 protein and mRNA expression. MDCK cells were grown as described in Methods. Before NaCl addition, cells were treated with different concentrations of PDTC or parthenolide (Parthe) for 30 min, and then an aliquot of 5 M NaCl was added to achieve 125 mM final concentration (∼512 mosm/kg H₂O). After 24 h treatment, cell lysates were subjected to western blot analysis by probing PVDF membranes with rabbit polyclonal COX-2 antibody (1:250) and rabbit polyclonal β-tubulin antibody (1:5000) (Panel A shows a representative membrane and panel B shows bar graph representing the relative bands intensities ratios) or subjected to total RNA isolation followed by RT-PCR for COX-2, MCP1 and β-actin (Panel C shows a representative image and panels D and E show bar graphs representing the relative band intensities ratios). Each image is representative of three independent experiments eliciting similar pattern. The results are expressed as the mean ± SEM of three independent experiments.

Fig. 4

NF-κB and TonEBP activities under hypertonic conditions. MDCK cells were grown, treated with TonEBP siRNA or 10 μM Parthenolide (Parthe), and subjected to 125 mM NaCl (∼512 mosm/kg H₂O) as described in Methods. After 16 h treatment, cells were collected, and lysates were subjected to total RNA isolation followed by RT-PCR for TonEBP, NF-κB (p65/RelA), MCP1, IκB, COX-2, AR, BGT1, SMIT and β-actin. Panels A and B show the mRNA analysis of NF-κB target genes MCP1, IκB and COX-2. Panels C and D shows the mRNA analysis of TonEBP target genes SMIT, AR and BGT1. Panels E shows the mRNA analysis of TonEBP and panel F, the mRNA analysis of NF-κB (p65/RelA). Each image is representative of three independent experiments eliciting similar pattern. The results are expressed as the mean ± SEM of three independent experiments.

Fig. 5

Putative κB-sites in the TonEBP promoter. The scheme shows the location of the three putative κB-sites in the promoter of TonEBP predicted by TRANSFACT^®, with regards to the distance to the transcription start site (+1).

Fig. 6

NF-κB (p65/RelA) interacts with TonEBP under hypertonic conditions. Panel A shows western blot analysis of NF-κB (p65/RelA) protein immunoprecipitated by TonEBP antibody (upper image) and the western blot analysis of TonEBP protein immunoprecipitated by NF-κB (p65/RelA) antibody (lower image), from cells treated or not with hypertonic medium (125 mM NaCl, ∼512 mosm/kg H₂O) for 16 h. Panel B shows the confocal microscopy images of MDCK cells stained with rabbit polyclonal TonEBP (1:75) and mouse monoclonal NF-κB (p65/RelA) (1:100) antibodies and revealed by using a FITC- and Alexa 546- conjugated secondary antibody (1:200) as described in Methods. Samples were mounted with Vectashield Mounting Medium. Fluorescence images were obtained with Olympus FV300 Confocal Microscope (Model BX61), with acquisition software FluoView version 3.3 provided by the manufacturer and processed as described in Methods. The figure shows a representative image of three independent experiments.