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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

Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (104 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H2O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls () were defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl hyperosmotic medium.
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Figure 3: Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (104 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H2O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls () were defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl hyperosmotic medium.

Mentions: S100A4 is known to stimulate proliferation and metastasis in renal carcinoma cells (Yang et al., 2013). In the knockdown experiments described above, we noticed decelerated growth of CaKi-1 cells transfected with NFAT5- or S100A4-specific siRNA, presumably due to downregulation of S100A4. To quantify this effect, we transfected CaKi-1 cells with NFAT5-specific siRNA, S100A4-specific siRNA or unspecific control siRNA, let the cells grow for 96 h in a 96-well plate and determined the number of viable cells by MTT assay. As shown in Figure 3A, NFAT5 siRNA and S100A4 siRNA had significant inhibitory effects on CaKi-1 cell proliferation.


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

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

Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (104 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H2O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls () were defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl hyperosmotic medium.
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Figure 3: Effect of NFAT5 and S100A4 knockdown on proliferation, migration and survival of CaKi-1 cells. (A) Proliferation. CaKi-1 cells (104 per case) were transfected with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods and seeded into one well of a 96-well plate. After 96 h, the cell number in each well was determined by MTT assay. The number of viable cells treated with control siRNA was defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl. (B) Migration. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. After reaching confluency, the cell layer was scratched with a 10 μl pipette tip. Shown are representative phase-contrast images of cells migrating into the wounded area, immediately after scratching (0 h) and after an incubation time of 24 h. (C) Cell survival. CaKi-1 cells were treated with NFAT5-specific (siNFAT5), S100A4-specific (siS100A4), or unspecific control (siControl) siRNA constructs as described in Methods. Confluent cells were kept in isoosmotic medium (; 300 mosm/kg H2O) or were exposed to hyperosmotic medium (■; 600 mosm/kg H2O) for 24 h. Thereafter, the cell number in each well was determined by MTT assay. Cell numbers in isosmotic controls () were defined as 100%. Data are means ± s.e.m. for n = 6; *P < 0.05 vs. siControl hyperosmotic medium.
Mentions: S100A4 is known to stimulate proliferation and metastasis in renal carcinoma cells (Yang et al., 2013). In the knockdown experiments described above, we noticed decelerated growth of CaKi-1 cells transfected with NFAT5- or S100A4-specific siRNA, presumably due to downregulation of S100A4. To quantify this effect, we transfected CaKi-1 cells with NFAT5-specific siRNA, S100A4-specific siRNA or unspecific control siRNA, let the cells grow for 96 h in a 96-well plate and determined the number of viable cells by MTT assay. As shown in Figure 3A, NFAT5 siRNA and S100A4 siRNA had significant inhibitory effects on CaKi-1 cell proliferation.

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