ABSTRACT
The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd(mi149)), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.
Subject(s)
Cerebral Hemorrhage/genetics , Mutation , Protein Serine-Threonine Kinases/genetics , Zebrafish/genetics , Alternative Splicing , Animals , Cerebral Hemorrhage/embryology , Cerebrovascular Circulation/genetics , Chromosome Mapping , Embryo, Nonmammalian , Genes, Recessive , Genetic Variation , Polymorphism, Restriction Fragment Length , Protein Serine-Threonine Kinases/deficiency , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Zebrafish Proteins/genetics , p21-Activated KinasesABSTRACT
An siRNA directed against the extracellular calcium-sensing receptor (CaSR) was used to down-regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca(2+). In neither the siRNA-transfected cells nor the Ca(2+)-nonresponsive variant cells did inclusion of Ca(2+) in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca(2+) also failed to induce E-cadherin production or a shift in beta-catenin from the cytoplasm to the cell membrane. In mock-transfected cells and in a Ca(2+)-responsive variant line derived from the same parental CBS cells, Ca(2+) treatment resulted in growth-reduction. This was accompanied by increased E-cadherin production and a shift in beta-catenin distribution from the cytoplasm to the cell membrane. Additionally, down-regulation of c-myc and cyclin D1 expression was observed in mock-transfected cells and in the Ca(2+)-responsive variant line (along with reduced T cell factor transcriptional activation). Neither c-myc nor cyclin D1 was significantly down-regulated in the siRNA-transfected cells or in the Ca(2+)-nonresponsive variant cells upon Ca(2+) stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E-cadherin and beta-catenin was observed. Where CaSR expression was reduced, beta-catenin surface expression was likewise reduced.
