von Hippel‐Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation

Abstract

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is associated with the development of vascular tumors including renal cell carcinoma. Aside from the role played by the VHL protein (pVHL) in negative regulation of hypoxia-inducible factor, 41F-1␣, pVHL also takes part in cytoskeletal organization. Thrombin is a serine protease involved in angiogenesis and in cancer progression and its action is mediated by the protease-activated receptors (PARs). In several cell types, thrombin induces reorganization of the cytoskeleton along with RhoA activation. Thus, we conducted an investigation on the capacity of thrombin to regulate pVHL expression. Our results demonstrated that VHL mRNA and protein levels were increased by thrombin in cultured renal cancer cells. Cytoplasmic pVHL was redistributed to perinuclear regions and membrane fractions following thrombin treatments. Stimulation of Caki-1 cells with PAR1, PAR2 and PAR4 agonist peptides demonstrated that PAR1 was the receptor involved in thrombin-induced pVHL expression. Western blot analysis confirmed that these cells express PAR1 and that its expression was increased by thrombin. PAR1 activation by both thrombin and an agonist peptide stimulated renal cancer cell invasion through Matrigel. Interestingly, the upregulation of pVHL was dependent on RhoA because C3 exotoxin abolished pVHL induction. However, the pharmacological Rho kinase inhibitor, Y27632, did not influence pVHL expression in the presence of thrombin, suggesting that other RhoA effectors were involved in the process. Together, these results demonstrate that thrombin induces both pVHL expression via PAR1/RhoA activation as well as the stimulation of renal cancer cell invasion suggesting a role for thrombin in tumor invasion.

Modification in the von Hippel-Lindau Protein Is Involved in the Progression of Experimentally Induced Rat Glomerulonephritis

“…On the basis of this result, it is further hypothesized that a VHL inducer may accelerate and aggravate GN. Therefore, HEK293 cells were treated with 1 U thrombin, already reported as a VHL inducer in a specific cell line [10]. As shown in figure 4, thrombin slowly increased VHL expression in HEK293 cells with sustained elevation even after 24 h; in contrast, HIF-1α was not induced by thrombin (data not shown).…”

“…4 Thrombin induced VHL protein expression in HEK293 cells. The potency of thrombin, which has been reported to induce VHL in a specific cell line [10], to increase VHL protein expression was evaluated. Twenty-four hours later, VHL expression was induced by thrombin (1 U).…”

“…For comparison, AII (n = 9) or HV (n = 9) alone was administered after nephrectomy. For pre-induction of VHL, one unit of thrombin was administered intraperitoneally on day –1 (n = 15), during the nephrectomy, followed by the same protocol [10]. Rats were sacrificed on day 1 (n = 15) and day 2 (n = 6), and the kidneys were excised for further analysis.…”

“…The optimal concentration of HV was determined by the cell survival (MTT) assay, because the concentration (>10 µg/ml HV) was identified to be toxic to the cells. Cells were also incubated with thrombin (1 U), which has been reported to be an inducer of VHL recently [10]. …”

Protease-activated receptors in cancer: A systematic review

“…The ability of tumor cells to activate the coagulation system and to generate thrombin has been shown to enhance metastatic efficiency (105-108), while anticoagulant therapies interfere with metastatic disease in animal models and in humans (109). The prometastatic activity of thrombin in cancer has been well demonstrated (Table IV) (74,75,82,84,97,98,(110)(111)(112)(113). The principal thrombin receptor, PAR1, has been implicated in the promotion of these effects (Table IV).…”