Limits...
The Hippo pathway is controlled by Angiotensin II signaling and its reactivation induces apoptosis in podocytes.

Wennmann DO, Vollenbröker B, Eckart AK, Bonse J, Erdmann F, Wolters DA, Schenk LK, Schulze U, Kremerskothen J, Weide T, Pavenstädt H - Cell Death Dis (2014)

Bottom Line: We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells.Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway.Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Muenster, Germany.

ABSTRACT
The Hippo pathway fulfills a crucial function in controlling the balance between proliferation, differentiation and apoptosis in cells. Recent studies showed that G protein-coupled receptors (GPCRs) serve as upstream regulators of Hippo signaling, that either activate or inactivate the Hippo pathway via the large tumor suppressor kinase (LATS) and its substrate, the co-transcription factor Yes-associated protein (YAP). In this study, we focused on the Angiotensin II type 1 receptor (AT1R), which belongs to the GPCR family and has an essential role in the control of blood pressure and water homeostasis. We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells. This shuttling of YAP is actin-dependent as disruption of the actin cytoskeleton inhibited dephosphorylation of LATS and YAP. Interestingly, in contrast to HEK293T cells, podocytes, which are a crucial component of the glomerular filtration barrier, display a predominant nuclear YAP localization in vivo and in vitro. Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway. Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis. Thus, our work indicates that the control of LATS activation and subsequent YAP localization is important for podocyte homeostasis and survival.

Show MeSH

Related in: MedlinePlus

The Angiotensin II receptor AT1R is an upstream regulator of the Hippo pathway. (a) Immunofluorescence staining showed homogenous cytoplasmic as well as nuclear localization of YAP (red) in unstimulated AT1R-overexpressing HEK293 cells; DAPI (blue) marks the nuclei and Alexa 488 Phalloidin (green) the actin cytoskeleton. Ang II stimulation (100 nM) for 15 min led to translocation of YAP to the nuclei. This effect was not seen in cells after prolonged Ang II treatment (100 nM, 24 h). Scale bars represent 10 μm. (b) Western blot analysis of sets of three independent extracts from AT1R-overexpressing HEK293 cells; unstimulated (left panel), stimulated (100 nM Ang II, 30 min, middle panel) or pretreated with the AT1R inhibitor Losartan (1 μM for 4 h) prior to stimulation with Ang II (right panel). Immunological detection revealed an Ang II-dependent strong increase of ERK phosphorylation, accompanied by the dephosphorylation of LATS1 on T1079 and its downstream target YAP on S127. The specific inhibition of Ang II signaling by the AT1R blocker Losartan demonstrated that phosphorylation (ERK) and dephosphorylation effects (LATS/YAP) are solely due to Ang II stimulation. (c) The ratio between phosphorylated and total amount of the protein is calculated and indicated (mean and S.D., t-test, **P<0.01; ns: not significant)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4260734&req=5

fig1: The Angiotensin II receptor AT1R is an upstream regulator of the Hippo pathway. (a) Immunofluorescence staining showed homogenous cytoplasmic as well as nuclear localization of YAP (red) in unstimulated AT1R-overexpressing HEK293 cells; DAPI (blue) marks the nuclei and Alexa 488 Phalloidin (green) the actin cytoskeleton. Ang II stimulation (100 nM) for 15 min led to translocation of YAP to the nuclei. This effect was not seen in cells after prolonged Ang II treatment (100 nM, 24 h). Scale bars represent 10 μm. (b) Western blot analysis of sets of three independent extracts from AT1R-overexpressing HEK293 cells; unstimulated (left panel), stimulated (100 nM Ang II, 30 min, middle panel) or pretreated with the AT1R inhibitor Losartan (1 μM for 4 h) prior to stimulation with Ang II (right panel). Immunological detection revealed an Ang II-dependent strong increase of ERK phosphorylation, accompanied by the dephosphorylation of LATS1 on T1079 and its downstream target YAP on S127. The specific inhibition of Ang II signaling by the AT1R blocker Losartan demonstrated that phosphorylation (ERK) and dephosphorylation effects (LATS/YAP) are solely due to Ang II stimulation. (c) The ratio between phosphorylated and total amount of the protein is calculated and indicated (mean and S.D., t-test, **P<0.01; ns: not significant)

Mentions: AT1R belongs to the group of GPCRs that act as upstream receptors of Hippo signaling. To analyze a putative influence of the AT1R on Hippo signaling, we used previously established AT1R-overexpressing HEK293 cells (kindly provided by Dr R Lefkowitz15). In an immunofluorescence experiment, the YAP distribution in these cells was analyzed after the Ang II treatment using a YAP-specific antibody. Stimulation with 100 nM Ang II for 15 min resulted in a strong nuclear accumulation of YAP, which is lost after a prolonged stimulation of 24 h (Figure 1a). Quantitative western blot analysis from cells stimulated with 100 nM Ang II for 30 min using phospho-specific antibodies revealed a decrease of LATS phosphorylation at Threonine 1079 (T1079) and of YAP at Serine 127 (S127) compared with the control without Ang II treatment (Figures 1b and c). This effect could be almost completely blocked by simultaneous treatment with the AT1R-specific inhibitor Losartan, proving that this effect is AT1R-dependent. Stimulation of the HEK293 cells with Ang II also showed an increased ERK phosphorylation, which is a well-known target of Ang II signaling.16, 17 The dephosphorylation of YAP was time-dependent and was not detectable after a stimulation of 24 h (Supplementary Figure 1). Therefore, Ang II stimulation inhibits Hippo signaling in HEK293 cells by dephosphorylation, and thereby inactivation of LATS kinase, resulting in a consecutive dephosphorylation and nuclear accumulation of YAP.


The Hippo pathway is controlled by Angiotensin II signaling and its reactivation induces apoptosis in podocytes.

Wennmann DO, Vollenbröker B, Eckart AK, Bonse J, Erdmann F, Wolters DA, Schenk LK, Schulze U, Kremerskothen J, Weide T, Pavenstädt H - Cell Death Dis (2014)

The Angiotensin II receptor AT1R is an upstream regulator of the Hippo pathway. (a) Immunofluorescence staining showed homogenous cytoplasmic as well as nuclear localization of YAP (red) in unstimulated AT1R-overexpressing HEK293 cells; DAPI (blue) marks the nuclei and Alexa 488 Phalloidin (green) the actin cytoskeleton. Ang II stimulation (100 nM) for 15 min led to translocation of YAP to the nuclei. This effect was not seen in cells after prolonged Ang II treatment (100 nM, 24 h). Scale bars represent 10 μm. (b) Western blot analysis of sets of three independent extracts from AT1R-overexpressing HEK293 cells; unstimulated (left panel), stimulated (100 nM Ang II, 30 min, middle panel) or pretreated with the AT1R inhibitor Losartan (1 μM for 4 h) prior to stimulation with Ang II (right panel). Immunological detection revealed an Ang II-dependent strong increase of ERK phosphorylation, accompanied by the dephosphorylation of LATS1 on T1079 and its downstream target YAP on S127. The specific inhibition of Ang II signaling by the AT1R blocker Losartan demonstrated that phosphorylation (ERK) and dephosphorylation effects (LATS/YAP) are solely due to Ang II stimulation. (c) The ratio between phosphorylated and total amount of the protein is calculated and indicated (mean and S.D., t-test, **P<0.01; ns: not significant)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260734&req=5

fig1: The Angiotensin II receptor AT1R is an upstream regulator of the Hippo pathway. (a) Immunofluorescence staining showed homogenous cytoplasmic as well as nuclear localization of YAP (red) in unstimulated AT1R-overexpressing HEK293 cells; DAPI (blue) marks the nuclei and Alexa 488 Phalloidin (green) the actin cytoskeleton. Ang II stimulation (100 nM) for 15 min led to translocation of YAP to the nuclei. This effect was not seen in cells after prolonged Ang II treatment (100 nM, 24 h). Scale bars represent 10 μm. (b) Western blot analysis of sets of three independent extracts from AT1R-overexpressing HEK293 cells; unstimulated (left panel), stimulated (100 nM Ang II, 30 min, middle panel) or pretreated with the AT1R inhibitor Losartan (1 μM for 4 h) prior to stimulation with Ang II (right panel). Immunological detection revealed an Ang II-dependent strong increase of ERK phosphorylation, accompanied by the dephosphorylation of LATS1 on T1079 and its downstream target YAP on S127. The specific inhibition of Ang II signaling by the AT1R blocker Losartan demonstrated that phosphorylation (ERK) and dephosphorylation effects (LATS/YAP) are solely due to Ang II stimulation. (c) The ratio between phosphorylated and total amount of the protein is calculated and indicated (mean and S.D., t-test, **P<0.01; ns: not significant)
Mentions: AT1R belongs to the group of GPCRs that act as upstream receptors of Hippo signaling. To analyze a putative influence of the AT1R on Hippo signaling, we used previously established AT1R-overexpressing HEK293 cells (kindly provided by Dr R Lefkowitz15). In an immunofluorescence experiment, the YAP distribution in these cells was analyzed after the Ang II treatment using a YAP-specific antibody. Stimulation with 100 nM Ang II for 15 min resulted in a strong nuclear accumulation of YAP, which is lost after a prolonged stimulation of 24 h (Figure 1a). Quantitative western blot analysis from cells stimulated with 100 nM Ang II for 30 min using phospho-specific antibodies revealed a decrease of LATS phosphorylation at Threonine 1079 (T1079) and of YAP at Serine 127 (S127) compared with the control without Ang II treatment (Figures 1b and c). This effect could be almost completely blocked by simultaneous treatment with the AT1R-specific inhibitor Losartan, proving that this effect is AT1R-dependent. Stimulation of the HEK293 cells with Ang II also showed an increased ERK phosphorylation, which is a well-known target of Ang II signaling.16, 17 The dephosphorylation of YAP was time-dependent and was not detectable after a stimulation of 24 h (Supplementary Figure 1). Therefore, Ang II stimulation inhibits Hippo signaling in HEK293 cells by dephosphorylation, and thereby inactivation of LATS kinase, resulting in a consecutive dephosphorylation and nuclear accumulation of YAP.

Bottom Line: We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells.Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway.Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Muenster, Germany.

ABSTRACT
The Hippo pathway fulfills a crucial function in controlling the balance between proliferation, differentiation and apoptosis in cells. Recent studies showed that G protein-coupled receptors (GPCRs) serve as upstream regulators of Hippo signaling, that either activate or inactivate the Hippo pathway via the large tumor suppressor kinase (LATS) and its substrate, the co-transcription factor Yes-associated protein (YAP). In this study, we focused on the Angiotensin II type 1 receptor (AT1R), which belongs to the GPCR family and has an essential role in the control of blood pressure and water homeostasis. We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells. This shuttling of YAP is actin-dependent as disruption of the actin cytoskeleton inhibited dephosphorylation of LATS and YAP. Interestingly, in contrast to HEK293T cells, podocytes, which are a crucial component of the glomerular filtration barrier, display a predominant nuclear YAP localization in vivo and in vitro. Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway. Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis. Thus, our work indicates that the control of LATS activation and subsequent YAP localization is important for podocyte homeostasis and survival.

Show MeSH
Related in: MedlinePlus