Polycystin-1 regulates the stability and ubiquitination of transcription factor Jade-1.

Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.

Tumor suppressor von Hippel-Lindau (VHL) stabilization of Jade-1 protein occurs through plant homeodomains and is VHL mutation dependent.

The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor alpha transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL beta domain was sufficient for Jade-1 binding, both the alpha and beta domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167Trp, did not stabilize Jade-1 fully. Therefore, loss of Jade-1 stability may correlate with renal cancer risk. Endogenous Jade-1 in stable renal cancer lines also exhibited VHL mutation-dependent regulation. As in the cotransfections, VHL truncations did not increase endogenous Jade-1 abundance, whereas the VHL missense mutations tested partially increased Jade-1 expression. Additional studies with non-PHD proteins indicated that Jade-1 stabilization by VHL is highly specific. Fibronectin was not stabilized like Jade-1 by VHL, nor were candidate VHL interactors from a yeast screen. Thus, protein stabilization likely reflects the biological activity of largely intact VHL protein on the PHD-extended PHD module of Jade-1. Dysregulation of the VHL protein stabilization pathway or of Jade-1 itself may therefore contribute to VHL renal disease and renal cancer pathogenesis.

Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor pVHL is lost in the majority of clear-cell renal cell carcinomas (RCC). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in approximately 30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here, using kinase arrays, we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Furthermore, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify 2 mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. In addition, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC.

Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.

The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting the degradation of hypoxia-inducible HIF-alpha transcription factors; additional mechanisms have been proposed. pVHL also stabilizes the plant homeodomain protein Jade-1, which is a candidate renal tumour suppressor that may correlate with renal cancer risk. Here we show that Jade-1 binds the oncoprotein beta-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type beta-catenin but not a cancer-causing form of beta-catenin. Whereas the well-established beta-catenin E3 ubiquitin ligase component beta-TrCP ubiquitylates only phosphorylated beta-catenin, Jade-1 ubiquitylates both phosphorylated and non-phosphorylated beta-catenin and therefore regulates canonical Wnt signalling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of beta-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL downregulates beta-catenin in a Jade-1-dependent manner and inhibits Wnt signalling, supporting a role for Jade-1 and Wnt signalling in renal tumorigenesis. The pVHL tumour suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

Jade-1, a candidate renal tumor suppressor that promotes apoptosis.

Medical therapies are lacking for advanced renal cancer, so there is a great need to understand its pathogenesis. Most renal cancers have defects in the von Hippel-Lindau tumor suppressor pVHL. The mechanism by which pVHL protein functions in renal tumor suppression remains unclear. Jade-1 is a short-lived, kidney-enriched transcription factor that is stabilized by direct interaction with pVHL. Loss of Jade-1 stabilization by pVHL correlates with renal cancer risk, making the relationship between Jade-1 and renal cancer compelling. We report that Jade-1 expression was barely detectable in all tested renal cancer cell lines, regardless of VHL status. Strikingly, proteasome inhibitor treatment increased endogenous Jade-1 expression up to 10-fold. Jade-1 inhibited renal cancer cell growth, colony formation, and tumor formation in nude mice. Intriguingly, Jade-1 also affected the pattern of cell growth in monolayer culture and 3D culture. Jade-1 increased apoptosis by 40-50% and decreased levels of antiapoptotic Bcl-2. Antisense Jade-1-expressing cells confirmed these results. Therefore, Jade-1 may suppress renal cancer cell growth in part by increasing apoptosis. Jade-1 may represent a proapoptotic barrier to proliferation that must be overcome generally in renal cancer, perhaps initially by pVHL inactivation and subsequently by increased proteasomal activity. Therefore, Jade-1 may be a renal tumor suppressor.

von Hippel-Lindau partner Jade-1 is a transcriptional co-activator associated with histone acetyltransferase activity.

Jade-1 was identified as a protein partner of the von Hippel-Lindau tumor suppressor pVHL. The interaction of Jade-1 and pVHL correlates with renal cancer risk. We have investigated the molecular function of Jade-1. Jade-1 has two zinc finger motifs called plant homeodomains (PHD). A line of evidence suggests that the PHD finger functions in chromatin remodeling and protein-protein interactions. We determined the cellular localization of Jade-1 and examined whether Jade-1 might have transcriptional and histone acetyltransferase (HAT) functions. Biochemical cell fractionation studies as well as confocal images of cells immunostained with a specific Jade-1 antibody revealed that endogenous Jade-1 is localized predominantly in the cell nucleus. Tethering of Gal4-Jade-1 fusion protein to Gal4-responsive promoters in co-transfection experiments activated transcription 5-6-fold, indicating that Jade-1 is a possible transcriptional activator. It was remarkable that overexpression of Jade-1 in cultured cells specifically increased levels of endogenous acetylated histone H4, but not histone H3, strongly suggesting that Jade-1 associates with HAT activity specific for histone H4. Deletion of the two PHD fingers completely abolished Jade-1 transcriptional and HAT activities, indicating that these domains are indispensable for Jade-1 nuclear functions. In addition, we demonstrated that TIP60, a known HAT with histone H4/H2A specificity, physically associates with Jade-1 and is able to augment Jade-1 HAT function in live cells, strongly suggesting that TIP60 might mediate Jade-1 HAT activity. Thus, Jade-1 is a novel candidate transcriptional co-activator associated with HAT activity and may play a key role in the pathogenesis of renal cancer and von Hippel-Lindau disease.

The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.