María C. Castellanos

von Hippel-Lindau Tumor Suppressor Protein Regulates the Assembly of Intercellular Junctions in Renal Cancer Cells through Hypoxia-Inducible Factor–Independent Mechanisms

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.

Activation of HIF-prolyl Hydroxylases by R59949, an Inhibitor of the Diacylglycerol Kinase

Hypoxia-inducible factors (HIF) are heterodimeric (α/β) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-α subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-α for degradation. In the absence of oxygen, HIF-α hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-α protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2α protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-α in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-α was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

Multiple cis‐acting elements regulate the expression of the early T cell activation antigen CD69

CD69 is the earliest activation antigen expressed on T lymphocytes upon stimulation through the TCR, or with stimuli that mimic TCR triggering. Here we describe that the phorbol ester PMA and a calcium ionophore had a synergistic effect on both CD69 antigen expression and promoter activity in Jurkat cells, that was sensitive to cyclosporin A (CsA). CD69 promoter analysis indicated that the sequence − 78 to + 16 contained the elements responsible for PMA and PMA plus calcium ionophore induction, as well as CsA inhibition. Mutagenesis of two previously described AP‐1 motifs did not affect either the basal or the inducible promoter activities. Electrophoretic mobility shift assays allowed the identification of three novel inducible complexes composed by Egr‐1/Egr‐3, Egr‐1, and ATF‐3/Fos. Mutation of each sequence resulted in a partial reduction of the basal promoter activity, whereas the inducibility by PMA plus calcium ionophore remained almost unaffected. It was necessary to combine at least two mutations to obtain a significative or complete reduction of the response to the mitogenic stimulus. These results indicate that the inducible expression of CD69 gene by mitogenic signals is regulated by multiple cis‐acting elements and by the interplay of transcription factors of the AP‐1, EGR and ATF/CREB families.

Induction of Hypoxia-inducible Factor 1α Gene Expression by Vascular Endothelial Growth Factor

Transcriptional regulation of vascular endothelial growth factor (VEGF) is critically dependent on hypoxia-inducible factor 1 (HIF-1). However, not only hypoxia, but selected growth factors can induce HIF-1. High levels of both VEGF and HIF-1 coexist in certain conditions, e.g. tumors. Nonetheless, the possibility that the stimulatory relationship between HIF-1 and VEGF may be bi-directional has not been addressed up to date. The present study in endothelial cells analyzed whether HIF-1 is regulated by a product of its own transcriptionally activated genes, namely, VEGF. As a main finding, VEGF-A165 induced the increase of HIF-1α mRNA and HIF-1α protein and nuclear translocation. Autologous endothelial cell VEGF mRNA and protein were also increased upon exposure to exogenous VEGF. The signaling implication of reactive oxygen species was examined by comparison with H2O2 and hypoxanthine/xanthine oxidase and by the superoxide dismutase mimetic, MnTMPyP, the Rac1-NAD(P)H oxidase complex inhibitor, apocynin, transfection of a dominant negative Rac1 mutant, and transfection of a p67phox antisense oligonucleotide. Superoxide anion, largely dependent on Rac1-NAD(P)H oxidase complex activity, was the critical signaling element. The transductional functionality of the pathway was confirmed by means of a reporter gene flanked by a transcription site-related VEGF sequence and by quantitative PCR. In summary, the present results reveal a previously undescribed action of VEGF on the expression of its own transcription factor, HIF-1, and on VEGF itself. This effect is principally mediated by superoxide anion, therefore identifying a new, potentially relevant role of reactive oxygen species in VEGF signaling.

Inadequate Activation of the GTPase RhoA Contributes to the Lack of Fibronectin Matrix Assembly in von Hippel-Lindau Protein-defective Renal Cancer Cells

The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL(+) cells. We have previously reported that VHL(-) cells fail to form β1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.