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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

NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells. CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. 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 NFAT5 determination) or 16 h (for S100A4 and AR determination). 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. siControl isoosmotic medium; *P < 0.05 vs. siControl 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 blot from 4 independent experiments is shown.
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Figure 2: NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells. CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. 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 NFAT5 determination) or 16 h (for S100A4 and AR determination). 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. siControl isoosmotic medium; *P < 0.05 vs. siControl 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 blot from 4 independent experiments is shown.

Mentions: To confirm that NFAT5 regulates the expression of S100A4 in CaKi-1 cells, we knocked down NFAT5 in CaKi-1 cells. Transfection of the cells with a NFAT5-specific siRNA construct resulted in an approximately 80% reduction of NFAT5 expression compared with cells transfected with an unspecific control siRNA (Figure 2). NFAT5 knockdown was accompanied by significantly reduced S100A4 expression, both under isosmotic and hyperosmotic conditions (Figure 2). Accordingly, the expression of the designated NFAT5 target gene AR was also significantly decreased in NFAT5-knockdown cells. We also performed siRNA-mediated knockdown of S100A4; as expected, this maneuver had no significant effect on expression of NFAT5 or AR. These results suggest strongly that S100A4 expression in CaKi-1 cells is under the control of NFAT5.


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

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

NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells. CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. 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 NFAT5 determination) or 16 h (for S100A4 and AR determination). 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. siControl isoosmotic medium; *P < 0.05 vs. siControl 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 blot from 4 independent experiments is shown.
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Figure 2: NFAT5-knockdown attenuates S100A4 expression in CaKi-1 cells. CaKi-1 cells were transfected with siRNA constructs for NFAT5 (siNFAT5), S100A4 (siS100A4) or with non-targeting siRNA (siControl) as indicated. 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 NFAT5 determination) or 16 h (for S100A4 and AR determination). 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. siControl isoosmotic medium; *P < 0.05 vs. siControl 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 blot from 4 independent experiments is shown.
Mentions: To confirm that NFAT5 regulates the expression of S100A4 in CaKi-1 cells, we knocked down NFAT5 in CaKi-1 cells. Transfection of the cells with a NFAT5-specific siRNA construct resulted in an approximately 80% reduction of NFAT5 expression compared with cells transfected with an unspecific control siRNA (Figure 2). NFAT5 knockdown was accompanied by significantly reduced S100A4 expression, both under isosmotic and hyperosmotic conditions (Figure 2). Accordingly, the expression of the designated NFAT5 target gene AR was also significantly decreased in NFAT5-knockdown cells. We also performed siRNA-mediated knockdown of S100A4; as expected, this maneuver had no significant effect on expression of NFAT5 or AR. These results suggest strongly that S100A4 expression in CaKi-1 cells is under the control of NFAT5.

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