Aim: Cellular senescence, leading to cell death through prevention of regular cell renewal, is associated with the upregulation of the tumor suppressor gene p16 INK4a. While this mechanism has been described as leading to progressive nephron loss, p16 INK4a upregulation in renal cell carcinoma has been linked to a disease-specific improved patient survival rate. While in both conditions endothelin-1 is also upregulated, the signaling pathway connecting ET-1 to p16 INK4a has not been characterized until this study. Main methods: Cell culture, qRT-PCR, Western Blot, immunoprecipitation (IP), proximity ligation assay (PLA), and non-radioactive electrophoretic mobility shift assay (EMSA). Key findings: In malignant renal proximal tumor cells (Caki-1), an activation of p16 INK4a and p21 waf1/cip1 was observed. An increased expression of E-26 transformation-specific (ETS) transcription factors was detectable. Using specific antibodies, a complex formation between ETS1 and extracellular signal-regulated kinase-2 (ERK2) was shown. A further complex partner was Mxi2. EMSA with supershift analysis for ETS1 and Mxi2 indicated the involvement of both factors in the protein-DNA interaction. After specifically blocking the endothelin receptors, ETS1 expression was significantly downregulated. However, the endothelin B receptor dependent downregulation was stronger than that of the A receptor. In contrast, primary proximal tubule cells showed a nuclear decrease after ET-1 stimulation. This indicates that other ETS members may be involved in the observed p16 INK4a upregulation (as described in the literature). Significance: ETS1, ERK2 and Mxi2 are important complex partners initiating increased p16 INK4a and p21w af1/cip1 activation in renal tumor cells.
Research Areas and Centers
- Academic Focus: Center for Brain, Behavior and Metabolism (CBBM)