Transient potential receptor channel 4 controls thrombospondin-1 secretion and angiogenesis in renal cell carcinoma.
Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.
Veliceasa
Dorina
D
Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Ivanovic
Marina
M
Hoepfner
Frank Thilo-Schulze
FT
Thumbikat
Praveen
P
Volpert
Olga V
OV
Smith
Norm D
ND
D000818:Animals; D015153:Blotting, Western; D022181:COP-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics; D002292:Carcinoma, Renal Cell / Q000098:blood supply* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002614:Chelating Agents / Q000494:pharmacology; D016510:Corneal Neovascularization / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000098:blood supply* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D002614:Chelating Agents; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D019700:Thrombospondin 1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D002118:Calcium