Die Rolle des E-Cadherin/ß-Catenin Komplexes von TCF und ABCB1 bei der Cadmium-induzierten Apoptose, Proliferation und Zellüberleben von proximalen Tubuluszellen der Niere

• Antragsteller: Professor Frank Thévenod, Ph.D.
• Fachliche Zuordnung: Anatomie und Physiologie
• Förderung: Förderung von 2007 bis 2011
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 38273749

• Erstellungsjahr: 2011

Zusammenfassung der Projektergebnisse

The transition metal ion cadmium (Cd2+) is a significant environmental contaminant. Cd2+ damages the kidney proximal tubule (PT) in up to 10% of the exposed population. Chronic low Cd2+ exposure (CLCE) has emerged as a previously underestimated significant health hazard for human populations. With a biological half-life of ~20 years, Cd2+ accumulates in organs, particularly the kidney, which can result in failure, fibrosis or – with Cd2+ being a class 1 human carcinogen – cancer. We have investigated signaling pathways mediating stress and death, but also survival and proliferation (and eventually malignancy) of renal PT cells exposed to low micromolar Cd2+ concentrations. Cd2+ induces cell death in renal cells. We have identified Ca2+ and reactive oxygen species (ROS) as triggers of ER stress and early cell death, mediated by Ca2+-dependent calpain activation, while caspase- and AIF-dependent apoptosis are late events of calpain activation. Cd2+ also causes rapid de novo formation of the sphingolipid ceramide, which acts upstream to activate calpain. Adaptive and anti-apoptotic processes are initiated at different levels of the cell death program. 1) In an attempt to reduce the ER stress load and cell death, renal cells rapidly elicit an adaptive unfolded protein response (UPR) and up-regulate the putative Ca2+-dependent Cl- channel protein Bestrophin-3 (Best-3) via activation of ERK1/2 signaling. Best-3 prevents UPR-dependent induction of the pro-apoptotic transcription factor CHOP, thus blocking its apoptosis-inducing effect and prolonging adaptive cell survival. 2) Stress and apoptosis trigger further adaptive responses mediated by Wnt/-catenin/TCF4, which were observed both in cultured PT cells and in mouse kidney. Activation of Wnt signaling occurs when Cd2+ causes disruption of E-cadherin/-catenin complexes at adherens junctions, which damages the epithelial monolayer and promotes cell detachment, but also triggers canonical Wnt signaling. Interestingly, Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. We proved that E-cadherin over-expression decreases Wnt signaling and cell proliferation. Cd2+ also induced ROS dependent expression of pro-apoptotic CHOP, which is also a Wnt repressor. CHOP, however, did not overcome Wnt response and increased cell viability, possibly because Cd2+-induced TCF4 up-regulation may override CHOP effects. Wnt signaling elicited up-regulation of genes such as c-myc, cyclin D1 or Abcb1, which promote proliferation and apoptosis resistance of renal PT cells. Furthermore, Cd2+ increased the epithelial-to-mesenchymal transition (EMT) markers Twist, fibronectin and collagen I in mouse kidney. 3) We demonstrated that ABCB1 is a crucial factor in survival of renal cells by interfering with pro-apoptotic sphingolipid signaling through extrusion of pro-apoptotic ceramide and/or glucosyl-ceramide to evade Cd2+ apoptosis. 4) We also showed for the first time direct and -catenin-independent ABCB1 up-regulation in renal cancer cells by the paired-like homeodomain transcription factor 2 (PITX2), which has been recently associated with cancer progression. 5) Finally, it is known that G2/M arrest contributes to stabilization of p53-deficient mutated cells but its role and regulation in Cd2+-exposed and p53-deficient renal cells were unknown. We could show in p53-inactivated PT cells that Cd2+-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ATM/ATR to cause G2/M arrest. The Chk1/2 kinase inhibitor UCN-01, which relieves G2/M transition block, abolished Cd2+-induced G2 arrest and increased apoptosis. This was accompanied by prevention of Cd2+-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15. Hence Cd2+-induced G2/M transition block may also promote survival of pre-malignant PT cells and Cd2+ carcinogenesis. In summary, Cd2+ induces changes of renal epithelial properties to increase survival via evasion of apoptosis and sustained proliferation signalling towards fibrosis and malignancy.

Projektbezogene Publikationen (Auswahl)

• Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells. Toxicology and Applied Pharmacology 230:78-85 (2008). Toxicology and Applied Pharmacology, Vol. 230. 2008, Issue 1, pp. 78–85.
  Wolff, N.A., Lee, W.-K., Abouhamed, M., Thévenod, F.
  
• Cadmium and cellular signaling cascades: To be or not to be? Toxicol Appl Pharmacol 238: 221-239, 2009
  Thévenod F
  
• Multifaceted CFTR: novel role in ROS signaling and apoptotic cell death--a commentary on "CFTR mediates cadmium-induced apoptosis through modulation of ROS levels in mouse proximal tubule cells". Free Radic Biol Med 46: 1014-1016, 2009
  Thévenod F
  
• Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule. Am J Physiol Renal Physiol 296: F1504-1513, 2009
  Lee WK, Reichold M, Edemir B, Ciarimboli G, Warth R, Koepsell H, and Thévenod F
  
• Organic cation transporters: physiology, toxicology and special focus on ethidium as a novel substrate. Curr Drug Metab 10: 617-631, 2009
  Lee WK, Wolff NA, and Thévenod F
  
• Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells. Mol Cancer 9: 102, 2010
  Chakraborty PK, Lee WK, Molitor M, Wolff NA, and Thévenod F
  
• Cadmium-induced DNA damage triggers G(2)/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells. Am J Physiol Renal Physiol 298: F255-265, 2010
  Bork U, Lee WK, Kuchler A, Dittmar T, and Thévenod F
  
• Chronic cadmium exposure induces transcriptional activation of the Wnt pathway and upregulation of epithelial-to-mesenchymal transition markers in mouse kidney. Toxicol Lett 198: 69-76, 2010
  Chakraborty PK, Scharner B, Jurasovic J, Messner B, Bernhard D, and Thévenod F
  
• New perspectives in cadmium toxicity: an introduction. Biometals 23: 763-768, 2010
  Moulis JM and Thévenod F
  
• The role of Wnt/beta-catenin signaling in renal carcinogenesis: lessons from cadmium toxicity studies. Curr Mol Med 10: 387-404, 2010
  Thévenod F and Chakraborty PK
  
• ABCB1 protects kidney proximal tubule cells against cadmium-induced apoptosis: Roles of cadmium and ceramide transport. Toxicol Sci 2011
  Lee WK, Torchalski B, Kohistani N, and Thévenod F
  
• ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death of renal epithelial cells by reducing CHOP. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1823. 2012, Issue 10, pp. 1864–1876.
  Lee, W.-K., Chakraborty, P.K., Roussa, E., Wolff, N.A., Thévenod, F.