High Podocalyxin levels promote cell viability partially through up-regulation of Annexin A2.

Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein that is up-regulated in various types of tumors and whose expression levels positively correlate with tumor grade. We previously found Podxl to be highly expressed in murine tumorigenic neural stem/progenitor cells (NSPs). Here we investigated the effects of elevated Podxl levels in these cells. NSPs overexpressing Podxl did not form brain tumors upon intracranial transplantations, indicating that high levels of this gene alone are not sufficient for tumor initiation. However, Podxl overexpression had a positive effect on cell number, sphere formation and cell viability, indicating that it might in this way contribute to the development and/or maintenance of tumors. Proteome analyses of Podxl-overexpressing and control NSPs revealed increased levels of Annexin A2 (ANXA2). We also found increased transcript levels, indicating that PODXL stimulates expression of the Anxa2 gene. Lack of Anxa2 in Podxl-overexpressing NSPs resulted in reduced viability of these cells, suggesting that PODXL-mediated pro-survival effects can at least in part be explained by increased ANXA2 levels. Finally, our data indicate that Podxl overexpression activates the MAP kinase (MAPK) pathway which in turn up-regulates Anxa2 expression. Our data indicate a novel molecular connection between PODXL and ANXA2: both exert pro-survival effects in NSPs, and PODXL positively regulates ANXA2 expression through the MAPK pathway.

Myosin IXa regulates epithelial differentiation and its deficiency results in hydrocephalus.

The ependymal multiciliated epithelium in the brain restricts the cerebrospinal fluid to the cerebral ventricles and regulates its flow. We report here that mice deficient for myosin IXa (Myo9a), an actin-dependent motor molecule with a Rho GTPase-activating (GAP) domain, develop severe hydrocephalus with stenosis and closure of the ventral caudal 3rd ventricle and the aqueduct. Myo9a is expressed in maturing ependymal epithelial cells, and its absence leads to impaired maturation of ependymal cells. The Myo9a deficiency further resulted in a distorted ependyma due to irregular epithelial cell morphology and altered organization of intercellular junctions. Ependymal cells occasionally delaminated, forming multilayered structures that bridged the CSF-filled ventricular space. Hydrocephalus formation could be significantly attenuated by the inhibition of the Rho-effector Rho-kinase (ROCK). Administration of ROCK-inhibitor restored maturation of ependymal cells, but not the morphological distortions of the ependyma. Similarly, down-regulation of Myo9a by siRNA in Caco-2 adenocarcinoma cells increased Rho-signaling and induced alterations in differentiation, cell morphology, junction assembly, junctional signaling, and gene expression. Our results demonstrate that Myo9a is a critical regulator of Rho-dependent and -independent signaling mechanisms that guide epithelial differentiation. Moreover, Rho-kinases may represent a new target for therapeutic intervention in some forms of hydrocephalus.