von Hippel–Lindau protein complex is regulated by cell density

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 densitydependent 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 densitydependent 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-2a 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.

hnRNP A2/B1 Modulates Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines

“…Previous reports have indicated that VHL affects hnRNP A2/B1 expression, and VHL is also regulated by cell density (42,43). According to the reported observations, we suggest a link between cell density, hnRNP A2/B1 expression, and EMT regulation in nonepithelial cancer cells.…”

VHL Promotes E2 Box-Dependent E-Cadherin Transcription by HIF-Mediated Regulation of SIP1 and Snail

“…Interestingly, the expression of VHL is strictly regulated by cell density where the steady-state amount of VHL in human renal proximal tubule epithelial cells was shown to increase more than 100-fold in dense cultures relative to sparse cultures (3). In addition, other components of the VHL E3 ligase complex showed a similar cell density-dependent regulation (44). Importantly, HIF-2␣ level was elevated in sparsely growing cells with low levels of VHL and significantly reduced or undetectable in confluent cells containing abundant VHL (44).…”

Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma

“…Because the increase in pVHL under hypoxic conditions displayed the greatest stimulation at cell confluence in serum-free medium, our subsequent experiments were carried out under these conditions, where the level of proliferation was low to reduce the contribution of the cell cycle. Also, previous studies indicated that functional VHL induced growth arrest at high cell density (36) and regulated cyclin D1 expression (2,3). Second, the level of pVHL expression was examined by Western blot analysis for various periods of time in normoxic or hypoxic conditions (Fig.…”