Cathepsin L increased level upon Ras mutants expression: the role of p38 and p44/42 MAPK signaling pathways.

The involvement of Ras and three major Ras effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine nucleotide exchange factor in the regulation of lysosomal proteases cathepsin L and B in human fibroblasts was compared. We found that cathepsin L cell content was increased by active Ras overexpression through Raf- and PI3K-mediated signaling pathways, while cathepsin B processing was altered by active Ras overexpression. Cathepsin L increased level following active Ras overexpression correlates with an increase of p38 MAPK activation and content and with an increase of p44/42 MAPK activation, so we investigated the role of these signaling pathways using pharmacological inhibitors. Unexpectedly, the p38 MAPK inhibitor SB203580 produced an increase of cathepsin L content, while the p44/42 MAPK signaling cascade inhibitor U0126 produced a remarkable shift of cathepsin L processing in favor of procathepsin L. In both cases, cathepsin B level and processing were not affected. The analysis of CTSL1 gene transcript demonstrated that cathepsin L protein and transcript correlate both in fibroblasts expressing Ras mutants and in pharmacologically treated cells, thus indicating a transcriptional up-regulation.

Human lysosomal α-D-mannosidase regulation in promyelocytic leukaemia cells.

Lysosomal α-D-mannosidase is an exoglycosidase involved in the ordered degradation of N-linked oligosaccharides. It is ubiquitously expressed, although the main transcript is more abundant in peripheral blood leucocytes. Here we report that α-D-mannosidase enzyme activity is very high in the promyelocytic leukaemia cell lines HL60 and NB4, as compared with other leukaemic cell lines or cells from different human sources. The MAN2B1 transcript level correlates with enzyme activity, indicating a transcriptional up-regulation of the α-D-mannosidase gene. The promoter was then characterized in HEK-293 cells (human embryonic kidney 293 cells) and HL60 cells; regulatory sequences crucial for its activity were determined by reporter gene assay in HEK-293 cells and located in the region -101/-71 with respect to the first ATG codon. Supershift assay demonstrated that Sp1 (specificity protein 1) bound to this sequence both in HEK-293 and HL60 cells. However, 5'-RACE (5'-rapid amplification of cDNA ends) indicated the use of multiple upstream TSSs (transcription start sites) in HL60 with respect to HEK-293 cells and gel shift analysis of the sequence -373/-269 demonstrated a specific binding by NF-κB (nuclear factor κB) transcription factor in HL60 but not in HEK-293 cells. We concluded that despite the α-D-mannosidase promoter showing typical features of housekeeping gene promoters, α-D-mannosidase transcription is specifically regulated in HL60 by NF-κB transcription factor.

New perspectives for the diagnosis of Alzheimer's disease.

Alzheimer's disease is the most common cause of dementia in the elderly. Currently its clinical assessment is based on the exclusion of other forms of dementia and a definitive diagnosis requires a confirmation by examination of post-mortem brains. Therefore, there is a strong need to find easy measurable AD biomarkers that could facilitate the early diagnosis and monitoring the efficacy of the few therapies currently available. This would favor the development of further therapeutic approaches. Recently, dozens of biomarkers altered in peripheral tissues and body fluids have been patented by a variety of approaches, including transcriptomics, proteomics and peptidomics. However, assays for the routine laboratory diagnosis of AD are not available yet. The validation of these biomarkers is hindered by the fact that patient classification relies on clinical diagnosis that is not always accurate and this problem obstacles the enrollment of well characterized large patient cohorts needed for confirmation. This review provides an update of the status of research on AD peripheral biomarkers in the current post-genomic era, including recent patents in the field.