ABSTRACT
The invasive nature of glioblastoma renders them incurable by current therapeutic interventions. Using a novel invasive human glioma model, we previously identified the neurotrophin receptor p75(NTR) (aka CD271) as a mediator of glioma invasion. Herein, we provide evidence that preventing phosphorylation of p75(NTR) on S303 by pharmacological inhibition of PKA, or by a mutational strategy (S303G), cripples p75(NTR)-mediated glioma invasion resulting in serine phosphorylation within the C-terminal PDZ-binding motif (SPV) of p75(NTR). Consistent with this, deletion (ΔSPV) or mutation (SPM) of the PDZ motif results in abrogation of p75(NTR)-mediated invasion. Using a peptide-based strategy, we identified PDLIM1 as a novel signaling adaptor for p75(NTR) and provide the first evidence for a regulated interaction via S425 phosphorylation. Importantly, PDLIM1 was shown to interact with p75(NTR) in highly invasive patient-derived glioma stem cells/tumor-initiating cells and shRNA knockdown of PDLIM1 in vitro and in vivo results in complete ablation of p75(NTR)-mediated invasion. Collectively, these data demonstrate a requirement for a regulated interaction of p75(NTR) with PDLIM1 and suggest that targeting either the PDZ domain interactions and/or the phosphorylation of p75(NTR) by PKA could provide therapeutic strategies for patients with glioblastoma.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , LIM Domain Proteins/genetics , Nerve Tissue Proteins/genetics , Receptors, Nerve Growth Factor/genetics , Transcription Factors/genetics , Animals , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Female , Humans , Mice , Mice, SCID , Neoplasm Invasiveness/pathology , Nerve Tissue Proteins/metabolism , PDZ Domains/genetics , Phosphorylation , RNA Interference , RNA, Small Interfering/genetics , Receptors, Nerve Growth Factor/metabolism , Signal Transduction/physiologyABSTRACT
Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.