ETS-dependent p16 ^INK4a and p21 ^waf1/cip1 gene expression upon endothelin-1 stimulation in malignant versus and non-malignant proximal tubule cells

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.