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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

Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells. CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H2O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; *P < 0.05 vs. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 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. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium.
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Figure 5: Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells. CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H2O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; *P < 0.05 vs. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 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. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium.

Mentions: We next analyzed the activation of signal transduction pathways to elucidate the molecular basis for the high basal NFAT5 activity in CaKi-1 cells, compared to HK-2 cells. Various signaling molecules are believed to mediate NFAT5 activity, among them focal adhesion kinase (FAK) (Neuhofer et al., 2014), the SRC kinase (Chen et al., 2011), often associated with FAK, and the MAP kinases p38, ERK1/2, and JNK (Tsai et al., 2007). We found that FAK and Src kinase are constitutively active in CaKi-1 cells (Figure 4), and pharmacological inhibition of these kinases decreased NFAT5 activity and expression in CaKi-1 cells (Figure 5). However, FAK and Src kinase are also constitutively active in HK-2 cells (Figure 4), indicating that FAK and Src are necessary, but alone not sufficient to account for the high NFAT5 activity in CaKi-1 cells. The MAP kinases p38, ERK1/2 (p44/42), and c-jun-terminal kinase (JNK or p54/46) also regulate NFAT5 activity. p38 and JNK are inactive under isosmotic conditions and are activated under hyperosmotic conditions in both CaKi-1 and HK-2 cells. In contrast, ERK1/2 is constitutively active in CaKi-1 cells, even under isosmotic conditions, but not in HK-2 cells. Inhibition of ERK1/2, but not of p38 or JNK, decreased NFAT5 activity and expression under isosmotic conditions in CaKi-1 cells (Figures 5A,C), while inhibition of p38 and JNK impaired osmolality-induced upregulation of NFAT5 activity and expression (Figures 5B,D). Accordingly, osmolality induced enhancement of S100A4 was attenuated by pharmacological inhibition of Src, FAK, ERK1/2, and p38 (Figure 5F). Surprisingly, JNK inhibition attenuated hyperosmolality-induced S100A4 expression only slightly, the reason for this is unclear. Taken together, these results indicate that constitutive activation of FAK and Src is a prerequisite for NFAT5 expression, osmolality-induced activation of p38 and possibly JNK stimulate NFAT5 activation under hyperosmotic conditions, and constitutive activation of ERK1/2 in CaKi-1 cells is probably responsible for the higher basal activity and expression of NFAT5 in these cells compared with HK-2 cells.


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

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

Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells. CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H2O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; *P < 0.05 vs. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 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. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium.
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Figure 5: Inhibition of FAK, Src, and MAP kinases attenuates NFAT5 activity in CaKi-1 cells. CaKi-1 cells were preincubated with the ERK1/2 inhibitor U0126 (U0; 10 μM), the p38 inhibitor SB202190 (SB; 10 μM), the JNK inhibitor SP600125 (SP; 10 μM), the FAK inhibitor PF-228 (PF; 5 μM), the Src inhibitor SrcI-1 (SrcI; 10 μM), or vehicle DMSO (Veh) for 30 min. Subsequently, cells were incubated at 300 or 500 mosm/kg H2O as indicated. (A,B) CaKi-1 cells were transfected transiently with a reporter construct in which the SEAP gene is under control of two TonE sites. After preincubation, the transfected cells were incubated for 24 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, SEAP activity was measured as described in Methods. Data are means ± s.e.m. for n = 4 per point; *P < 0.05 vs. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium. (C–F) After preincubation, CaKi-1 cells were incubated for 16 h at 300 or 500 mosm/kg H2O, as indicated. Subsequently, RNA was extracted and the abundance of NFAT5, S100A4 and β-actin mRNA transcripts was determined by qRT-PCR as described in Methods. Relative mRNA abundance of NFAT5 and S100A4 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. vehicle; #P < 0.05 vs. vehicle hyperosmotic medium.
Mentions: We next analyzed the activation of signal transduction pathways to elucidate the molecular basis for the high basal NFAT5 activity in CaKi-1 cells, compared to HK-2 cells. Various signaling molecules are believed to mediate NFAT5 activity, among them focal adhesion kinase (FAK) (Neuhofer et al., 2014), the SRC kinase (Chen et al., 2011), often associated with FAK, and the MAP kinases p38, ERK1/2, and JNK (Tsai et al., 2007). We found that FAK and Src kinase are constitutively active in CaKi-1 cells (Figure 4), and pharmacological inhibition of these kinases decreased NFAT5 activity and expression in CaKi-1 cells (Figure 5). However, FAK and Src kinase are also constitutively active in HK-2 cells (Figure 4), indicating that FAK and Src are necessary, but alone not sufficient to account for the high NFAT5 activity in CaKi-1 cells. The MAP kinases p38, ERK1/2 (p44/42), and c-jun-terminal kinase (JNK or p54/46) also regulate NFAT5 activity. p38 and JNK are inactive under isosmotic conditions and are activated under hyperosmotic conditions in both CaKi-1 and HK-2 cells. In contrast, ERK1/2 is constitutively active in CaKi-1 cells, even under isosmotic conditions, but not in HK-2 cells. Inhibition of ERK1/2, but not of p38 or JNK, decreased NFAT5 activity and expression under isosmotic conditions in CaKi-1 cells (Figures 5A,C), while inhibition of p38 and JNK impaired osmolality-induced upregulation of NFAT5 activity and expression (Figures 5B,D). Accordingly, osmolality induced enhancement of S100A4 was attenuated by pharmacological inhibition of Src, FAK, ERK1/2, and p38 (Figure 5F). Surprisingly, JNK inhibition attenuated hyperosmolality-induced S100A4 expression only slightly, the reason for this is unclear. Taken together, these results indicate that constitutive activation of FAK and Src is a prerequisite for NFAT5 expression, osmolality-induced activation of p38 and possibly JNK stimulate NFAT5 activation under hyperosmotic conditions, and constitutive activation of ERK1/2 in CaKi-1 cells is probably responsible for the higher basal activity and expression of NFAT5 in these cells compared with HK-2 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