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NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

Küper C, Beck FX, Neuhofer W - Front Physiol (2014)

Bottom Line: In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis.Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity.Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Munich Munich, Germany.

ABSTRACT
The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.

No MeSH data available.


Related in: MedlinePlus

Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells. CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H2O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium.
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Figure 1: Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells. CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H2O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium.

Mentions: The cell line CaKi-1 was used as model for metastatic clear cell RCC. As non-cancerous control cells, the proximal tubule cell line HK-2 was used. Expression of NFAT5 in CaKi-1 and HK-2 cells was determined at both the mRNA (Figure 1A) and protein (Figure 1B) levels. NFAT5 levels were significantly higher in CaKi-1 cells compared to HK-2 cells. In both cell lines, NFAT5 expression increased during hyperosmotic stress (Figures 1A,B), whilst again expression levels were significantly higher in CaKi-1 cells. Expression of S100A4 was almost completely absent in HK-2 cells, while substantial amounts were present in CaKi-1 cells. Under hyperosmotic stress conditions, S100A4 expression in CaKi-1 cells increased further. As control, we also evaluated expression levels of the well-defined NFAT5 target gene AR. In accordance with the results above, expression levels of AR were also enhanced in CaKi-1 cells, presumably due to increased NFAT5 activity. Cellular activity of NFAT5 in CaKi-1 and HK-2 cells was assayed using a TonE-driven reporter vector. Cells, transiently transfected with the reporter construct, were incubated for 24 h in iso- or hyperosmotic medium. Basal NFAT5 activities under isoosmotic conditions were significantly higher in CaKi-1 cells (Figure 1C). Under hyperosmotic conditions, NFAT5 activity increased approximately 10 times in both cell types, and hence was significantly higher in CaKi-1 cells, which is in accordance with the higher expression levels of the NFAT5 target genes S100A4 and AR in these cells.


NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

Küper C, Beck FX, Neuhofer W - Front Physiol (2014)

Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells. CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H2O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium.
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Related In: Results  -  Collection

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Figure 1: Expression of NFAT5 and NFAT5 target genes in CaKi-1 and HK-2 cells. CaKi-1 cells, as model for clear cell renal cell carcinoma, or HK-2 cells, as proximal tubular control cells, were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 500 mosm/kg H2O). Medium osmolality was elevated by addition of NaCl. (A) Cells were incubated for 6 h (for determination of NFAT5 transcription) or 16 h (for determination of S100A4 and AR transcription). Thereafter, RNA was extracted and the abundance of NFAT5, S100A4, AR, and β-actin mRNA transcripts determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5, S100A4, or AR was normalized to that of β-actin to correct for differences in RNA input. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium. (B) Cells were incubated for 24 h and subsequently processed for immunoblotting to determine expression of NFAT5, S100A4, and AR as described in Methods. To demonstrate comparable protein loading, the blots were also probed for β-actin. Representative blots from 4 independent experiments are shown. (C) Cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After 24 h incubation in iso- or hyperosmotic medium, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; #P < 0.05 vs. HK-2 isoosmotic medium; *P < 0.05 vs. HK-2 hyperosmotic medium.
Mentions: The cell line CaKi-1 was used as model for metastatic clear cell RCC. As non-cancerous control cells, the proximal tubule cell line HK-2 was used. Expression of NFAT5 in CaKi-1 and HK-2 cells was determined at both the mRNA (Figure 1A) and protein (Figure 1B) levels. NFAT5 levels were significantly higher in CaKi-1 cells compared to HK-2 cells. In both cell lines, NFAT5 expression increased during hyperosmotic stress (Figures 1A,B), whilst again expression levels were significantly higher in CaKi-1 cells. Expression of S100A4 was almost completely absent in HK-2 cells, while substantial amounts were present in CaKi-1 cells. Under hyperosmotic stress conditions, S100A4 expression in CaKi-1 cells increased further. As control, we also evaluated expression levels of the well-defined NFAT5 target gene AR. In accordance with the results above, expression levels of AR were also enhanced in CaKi-1 cells, presumably due to increased NFAT5 activity. Cellular activity of NFAT5 in CaKi-1 and HK-2 cells was assayed using a TonE-driven reporter vector. Cells, transiently transfected with the reporter construct, were incubated for 24 h in iso- or hyperosmotic medium. Basal NFAT5 activities under isoosmotic conditions were significantly higher in CaKi-1 cells (Figure 1C). Under hyperosmotic conditions, NFAT5 activity increased approximately 10 times in both cell types, and hence was significantly higher in CaKi-1 cells, which is in accordance with the higher expression levels of the NFAT5 target genes S100A4 and AR in these cells.

Bottom Line: In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis.Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity.Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Munich Munich, Germany.

ABSTRACT
The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration.

No MeSH data available.


Related in: MedlinePlus