Abstract
Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.
Highlights
The von Hippel-Lindau disease tumour-suppressor gene (VHL) mRNA and protein are widely expressed, analysis of the differential expression of VHL mRNA in the kidney during human embryogenesis is compatible with a specific role in normal renal development (Richards et al, 1996). These findings suggest that the VHL gene would appear to be intimately involved in the control of growth and differentiation of normal renal tubular cells
As parathyroid hormone (PTH) stimulus but not vasopressin exposure would result in enhanced cyclic AMP production in proximal tubular epithelial cells (PTECs) cells, a functional assay using parathyroid hormone (PTH)-sensitive but vasopressin-insensitive cyclic AMP measurements was performed using the Amersham Biotrak cAMP [1251] assay system (Amersham International, Little Chalford, Buckinghamshire, UK) to further ensure that the cultured cells obtained represented PTECs
Our investigations have confirmed the striking growth, differentiation and morphological effects that HGF/SF exposure has on cultured normal renal tubule cells, and has yielded useful initial information regarding the spectrum of transient changes in gene expression induced during PTEC mitogenesis
Summary
Isolation and culture of normal renal tubule cells. Proximal tubule epithelial cells (PTEC) were isolated from the cortex of normal human donor kidneys not used for transplantation because of technical reasons, using an adaptation of the methods of Detrisac et al (1984). The cell monolayers obtained in the second to fourth subcultures were characterized using various monoclonal antibodies (MAbs) directed against cytokeratin and MAbs specific to proximal tubule brush border enzymes, URO-3 (F23) and URO-4 (S27). A MAb against membrane proteins specific to distal tubular cells URO-5 (T16) was used as negative control. Desmin and Thy 1 antibodies were used as negative controls to ensure that no contamination with mesangial cells had occurred. As parathyroid hormone (PTH) stimulus but not vasopressin exposure would result in enhanced cyclic AMP (cAMP) production in PTEC cells, a functional assay using parathyroid hormone (PTH)-sensitive but vasopressin-insensitive cyclic AMP (cAMP) measurements was performed using the Amersham Biotrak cAMP [1251] assay system (Amersham International, Little Chalford, Buckinghamshire, UK) to further ensure that the cultured cells obtained represented PTECs
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