Podocalyxin EBP50 Ezrin Molecular Complex Enhances the Metastatic Potential of Renal Cell Carcinoma Through Recruiting Rac1 Guanine Nucleotide Exchange Factor ARHGEF7

Abstract

Podocalyxin was initially identified in glomerular podocytes to critically maintain the structural and functional integrity of the glomerular ultrafiltrative apparatus. Lately, it has emerged as a malignant marker in tumors arising from a variety of tissue origins. By immunohistochemistry, we identified that 9.6% of renal cell carcinoma patients overexpress this protein. This subset of patients had significantly shorter disease-specific and overall survivals, and, importantly, we established podocalyxin overexpression as an independent prognostic factor for latent distant metastasis with multivariate analysis. Podocalyxin down-regulation by small interfering RNA led to defective migration in model renal tubular cells, which was corrected by re-expression of podocalyxin. The activity of the small GTPase Rac1, a wellcharacterized modulator of cell migration, was diminished by podocalyxin knock-down. Conversely , podocalyxin overexpression in human embryonic kidney cells up-regulated Rac1 activity, which depended on a complex formed by podocalyxin, ERMbinding phosphoprotein 50, ezrin, and ARHGEF7, a Rac1 activator. Therefore, podocalyxin can serve as a biomarker to identify renal cell carcinoma patients with higher metastatic potential for more aggressive intervention at earlier clinical stages. (Am J Pathol

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

“…We recently demonstrated that EBP50 interacts with b-PIX through its first PDZ domain, and b-PIX associates with the podocalyxin-EBP50-ezrin complex. 19 The interaction between EBP50 and b-PIX was confirmed by co-immunoprecipitation (Figure 5a). Both basal and MRC-5 conditioned medium-dependent Rac1 activation decreased upon b-PIX knockdown, confirming the involvement of b-PIX in the Rac1 activation, using MRC-5 conditioned medium (Figure 5b).…”

“…18 Previously, we identified a novel interaction between EBP50 and b-PIX, a specific GEF for Rac1 and Cdc42, and proposed that the podocalyxin-EBP50-ezrin complex recruited b-PIX and, thereby, enhanced the metastatic potential of renal cell carcinoma. 19 To confirm the role of EBP50 in Rac1 activation in living cells, we measured Rac1 activity in EBP50 knockdown MDCK cells in the presence of MRC-5 conditioned medium, which induces Rac1 activation via bioactive hepatocyte growth factor (HGF). 28 EBP50 knockdown not only resulted in diminished basal Rac1 activity but also significantly reduced the level of Rac1 activation compared with mock cells exposed to MRC-5 conditioned medium (Figure 4a).…”

“…18,19 Interactions with plasma membrane lipid PIP2 converts ezrin from an auto-inhibitory into an open active conformation, and subsequent PKCmediated phosphorylation of ezrin further stabilizes the activated state of ezrin. 36 Active ezrin then sequesters RhoGDI to release small GTPases for activation by exchanging GDP for GTP.…”

“…17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the accessibility of its PDZ domains for cognate ligands and the formation of EBP50 oligomers, both of which are required for EBP50 to act as a scaffold protein.…”

Expression of TNFRSF6B in kidneys is a novel predictor for progression of chronic kidney disease

“…Computerassisted quantitative analysis (Image Pro Plus 6.0, MediaCybernetics, SilverSpring, MD, USA) was performed as previously described. 22 Briefly, 5 glomeruli and 20 randomly selected nonoverlapping high-power fields of each renal cortical section were captured by a PROVIS AX80 microscope (Olympus, Tokyo, Japan) equipped with a 12-bit RETIGA 1300 digital camera (QImaging, Burnaby, Canada). The captured images were then analyzed by Image Pro Plus software.…”