Procathepsin L secretion, which triggers tumour progression, is regulated by Rab4a in human melanoma cells

The switch of human melanoma cell phenotype from non to highly tumorigenic and metastatic is triggered by the increase of procathepsin L secretion, which modifies the tumour microenvironment. The aim of the present study was to identify components involved in the regulation of procathepsin L secretion in melanoma cells. We focused on Rab family members, i.e. Rab3A, Rab4A, Rab4B, Rab5A, Rab8A, Rab11A, Rab27A and Rab33A, which are involved in distinct regulatory pathways. From analysis of mRNA and protein expression of these Rab components and their knockdown by specific siRNAs (small interfering RNAs) it emerged that Rab4A protein is involved in the regulation of procathepsin L secretion. This result was strengthened as procathepsin L secretion was either inhibited by expression of a Rab4A dominant-negative mutant or increased by overexpression of the wild-type Rab4A. Rab4A regulation: (i) discriminates between procathepsin L secretion and expression of intracellular cathepsin L forms; (ii) did not modify other Rab proteins and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression, or IL-8 (interleukin-8) and MMP-2 (matrix metalloproteinase-2) secretion; and (iii) was still efficient during unglycosylated procathepsin L secretion. Thus down- or up-regulation of Rab4A expression or Rab4A function triggered inhibition or increase of procathepsin L secretion respectively. Furthermore, Rab4A regulation, by modifying procathepsin L secretion, switches the tumorigenic phenotype of human melanoma cells in nude mice.

Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells

We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156—197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.

Interleukin-4 and -13 promote basolateral secretion of H+ and cathepsin L by glomerular epithelial cells

Minimal change nephrosis (MCN) is characterized by massive proteinuria and ultrastructural alterations of glomerular visceral epithelial cells (GVEC). MCN has been associated with elevated production of interleukin (IL)-13 by circulating T lymphocytes and with T helper 2 lymphocyte-dependent conditions. We recently showed that GVEC express IL-4 and IL-13 receptors and that IL-4 and IL-13 increase transcellular ion transport over GVEC monolayers. We therefore hypothesized that IL-13 may directly injure GVEC. Here we demonstrate that IL-4 and IL-13 induce bafilomycin A1-sensitive basolateral proton secretion by cultured GVEC, indicating involvement of vacuolar H+-ATPase. The effects of IL-4 and IL-13 were accompanied by redistribution of the small GTPases Rab5b and Rab7, as shown by confocal immunofluorescence studies. Furthermore, Western blot analysis and assays for cysteine proteinase activity revealed basolateral secretion of the lysosomal proteinase procathepsin L by cultured GVEC, stimulated by IL-4 and IL-13. We speculate that IL-4 and IL-13 influence intracellular trafficking of proteins and promote proteolysis at the basolateral surface of GVEC, which may play a pathogenic role in altered glomerular permeability.