Objectives: 

CCMs (Cerebral cavernous malformations) are vascular lesions located in the central nervous system characterized by enlarged vessels (caverns). CCMs affect over 0,5% of the population who present some clinical problems like intense headaches, seizures or bleeding. 80% of the cases are sporadic while the rest have a genetic basis, with three different CCM loci implicated: CCM1/KRIT1, CCM2/Malcavernin/OSM and CCM3/PDCD10. The latter, CCM3, whose participation in the disease is less studied, is subject of our interest since it was biochemically defined as an adaptor protein that can bind to a variety of different proteins like the GCK-III subfamily of Ste20 kinases. This binding could be important to prevent the development of cerebral cavernomas because under some stress situations it results in the phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins protecting cells from accidental cell death. Our aims are to find the pathways/mechanisms by which this ERM phosphorylation is dependent on CCM3 presence and its importance in the CCM3 pathobiology.

Materials: 

Cell culture, siRNA and shRNA-mediated knockdown, H202 and osmotic treatments, Immunoblotting, Fluorescence and Image Analysis, Viability assays, Protein Inhibitors.

Results: 

ERM phosphorylation is dependent on CCM3 presence under oxidative and osmotic stress models. By contrast, ERM phosphorylation is increased in untreated cells lacking CCM3. Studying the pathways implicated, and contrary on what could be expect, we have found that Rho signaling is not upstream of that phosphorylation, which we demonstrate using ROCK-1 and ROCK-2 inhibitors and siRNAs. Furthermore, known ROCK substrates such as LIMK2 are decreased in shCCM3 cells.

Conclusions: 

The product of the CCM susceptibility gene CCM3 participates in phosphorylation of the ERM cytoskeletal adaptor proteins under some circumstances. This adds to our understanding of the pathobiology of cerebral cavernomas.