A rapid and simple procedure is presented to obtain nearly pure populations of human neuron‐like cells from the SH‐SY5Y neuroblastoma cell line. Sequential exposure of SH‐SY5Y cells to retinoic acid and brain‐derived neurotrophic factor in serum‐free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5‐bromo‐2′‐deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain‐derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron‐specific enolase, and growth‐associated protein‐43 as well as neuronal polarity markers such as tau and microtubule‐associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large‐scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Vascular calcification commonly associated with several pathologies and it has been suggested to be similar to bone mineralization. The axis RANKL-OPG (receptor activator of nuclear factor κB ligand–osteoprotegerin) finely controls bone turnover. RANKL has been suggested to increase vascular calcification, but direct evidence is missing. Thus, in the present work, we assess the effect of RANKL in vascular smooth muscle cell (VSMC) calcification. VSMCs incubated with RANKL showed a dose-dependent increase in calcification, which was abolished by coincubation with OPG. To test whether the effect was mediated by signaling to its receptor, knockdown of RANK was accomplished by short hairpin (sh)RNA. Indeed, cells lacking RANK showed no increases in vascular calcification when incubated with RANKL. To further elucidate the mechanism by which RANK activation increases calcification, we blocked both nuclear factor (NF)-κB activation pathways. Only IKKα inactivation inhibited calcification, pointing to an involvement of the alternative NF-κB activation pathway. Furthermore, RANKL addition increased bone morphogenetic protein (BMP)4 expression in VSMCs, and that increase disappeared in cells lacking RANK or IKKα. The increase in calcification was also blunted by Noggin, pointing to a mediation of BMP4 in the calcification induced by RANKL. Furthermore, in an in vivo model, the increase in vascular calcium content was parallel to an increase in RANKL and BMP4 expression, which was localized in calcified areas. However, blood levels of the ratio RANKL/OPG did not change. We conclude that RANKL increases vascular smooth muscle cell calcification by binding to RANK and increasing BMP4 production through activation of the alternative NF-κB pathway.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRα1–α4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein–protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.