Aminopeptidase N (APN)/CD13-dependent CXCR4 downregulation is associated with diminished cell migration, proliferation and invasion

Abstract

Aminopeptidase N (APN/CD13) is a 150 kDa membrane-bound ubiquitously expressed protease with a broad functional repertoire. It hydrolyzes small peptide mediators, modulates cell motility and adhesion to extracellular matrix and also acts as a viral receptor. In order to dissect the function of enzymatically active and inactive APN/CD13, substitutions of different enzymatic active amino acid residues were generated by site-directed mutagenesis and stably transfected into human embryonic kidney cells. All APN variants analyzed exhibited a complete loss of enzymatic activity, whereas wild type APN transfectants exerted a strong aminopeptidase-specific activity. Furthermore, wild type APN expression was associated with a significant decrease in proliferation, migration and also reduced anchorage-independent growth when compared to enzymatically inactive APN variants and controls. This appeared to be due to a downregulated mRNA and protein expression of the chemokine receptor CXCR4 and an inhibition of the stromal cell-derived factor (SDF)-1alpha/CXCL12-mediated migration. Thus, high APN enzyme activity may antagonize the cellular properties regulated by the CXCR4/SDF-1alpha system in embryonic kidney cells.

HER-2/neu-mediated Down-regulation of Biglycan Associated with Altered Growth Properties

“…Residual genomic DNA was digested with DNase I (Invitrogen) followed by transcription of 2 g of total RNA into cDNA using the Revert H Minus First Strand cDNA synthesis kit (MBI Fermentas, St. Leon-Roth, Germany) and oligo(dT) 18 primer according to the manufacturer's instructions. Comparative quantification of gene expression was performed as previously described (32). The target-specific primers used for quantitative PCR are listed in supplemental Table S1.…”

“…RNA Isolation and Real Time Quantitative RT-PCR-Total cellular RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Dueren, Germany) as previously described (32). Residual genomic DNA was digested with DNase I (Invitrogen) followed by transcription of 2 g of total RNA into cDNA using the Revert H Minus First Strand cDNA synthesis kit (MBI Fermentas, St. Leon-Roth, Germany) and oligo(dT) 18 primer according to the manufacturer's instructions.…”

“…Migration Assays-Migration assays were performed as previously described (32) Scratch Assay-The scratch assay mimicking the directional cell migration was employed. Briefly, cells were grown to density in 6-well plates.…”

Colorectal Carcinogenesis: Connecting K-RAS–Induced Transformation and CREB Activity In Vitro and In Vivo

“…cDNA synthesis, qPCR, and RT-PCR RNA was isolated using the NucleoSpin II kit (Macherey-Nagel) according to the manufacturer's instructions and then subjected to cDNA synthesis followed by qPCR or RT-PCR with target-specific primers (Supplementary Table S2A and S2B) as recently described (30). The PCR were performed in triplicates.…”

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

“…Quantitative realtime PCR (qPCR) or reverse transferase PCR (RT-PCR) was performed with target-specific primers (Supplementary Tables S1A and S1B) as recently described (24). For qPCR, the relative mRNA expression levels of the given target genes within the respective samples were normalized to the corresponding expression levels of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”