1. ¹Department of Microbiology and Immunology, Marion Bessin Liver Research Center and Albert Einstein Cancer Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2. ²Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3. ³Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

Correspondence: RD Burk, Ullmann Bldg. Rm. 515, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: burk@aecom.yu.edu

Received 21 January 2003; Revised 14 March 2003; Accepted 20 March 2003.

Abstract

Mutations in the von Hippel–Lindau (VHL) gene are involved in the VHL family cancer syndrome and sporadic renal cell carcinoma. Previous studies indicated that VHL-induced growth arrest required high cell density and growth on extracellular matrix. In the present study, VHL protein (pVHL) levels were observed to be dramatically increased in cells grown to high cell density compared to cells grown at low cell density. Reverse transcription–polymerase chain reaction and Northern blot analysis indicated that VHL mRNA levels were equivalent in sparse and dense cells. The pVHL was rapidly degraded when cell–cell contact was disturbed by trypsinization or EDTA release. Treatment of cells with a proteasome inhibitor blocked the degradation of pVHL. Using a set of VHL deletions fused to GFP, a cell density-dependent region (CDDR) was identified and localized to the c-terminus of pVHL. In addition, other members of the VBC protein complex also showed a cell density-dependent regulation similar to pVHL. Cell density regulation of VHL did not require elongin binding and density-dependent regulation of other VBC components was not dependent on pVHL. In addition, hypoxia inducible factor-2 protein levels were elevated in sparse cells with low levels of pVHL and reduced or absent in confluent cells containing abundant VHL. These results indicate that pVHL levels and thus function are tightly regulated by cell–cell signaling. In addition, care must be taken when interpreting studies of VHL function and subcellular localization of cells grown at subconfluent conditions.