Influence of RRM, RGG and Potential Phosphorylated Sites in Cold-Inducible Protein RBM3 on its Subcellular Localization and Neuroprotective Effects.

BACKGROUND: As a potent mediator of hypothermic neuroprotection, the cold-inducible protein RBM3 is characterized with one RNA-recognition motifs (RRM) and one arginine-glycine-rich (RGG) domain. It is known that these conserved domains are required for nuclear localization in some RNA-binding proteins. However, little is known about the actual role of RRM and RGG domains in subcellular localization of RBM3. METHODS: To clarify it, various mutants of human Rbm3 gene were constructed. Plasmids were transfected into cells and the localization of RBM3 protein and its varias mutants in cells and role in neuroprotection. RESULTS: In human neuroblastoma SH-SY5Y cells, either a truncation of RRM domain (aa 1-86) or RGG domain (aa 87-157) led to an obvious cytoplasmic distribution, compared to a predominant nuclear localization of whole RBM3 protein (aa 1-157). In contrast, mutants in several potential phosphorylated sites of RBM3, including Ser102, Tyr129, Ser147, and Tyr155, did not alter the nuclear localization of RBM3. Similarly, mutants in two Di-RGG motif sites also did not affect the subcellular distribution of RBM3. Lastly, the role of Di-RGG motif in RGG domains was further investigated. The mutant of double arginines in either Di-RGG motif-1 (Arg87/90) or -2 (Arg99/105) exhibited a higher cytoplasmic localization, indicating that both Di-RGG motifs are required for nucleic localization of RBM3. CONCLUSIONS: Our data suggest that RRM and RGG domains are both required for the nuclear localization of RBM3, with two Di-RGG domain being crucial for nucleocytoplasmic shuttling of RBM3.

Neural Cell Adhesion Molecule Regulates Osteoblastic Differentiation Through Wnt/ß-Catenin and PI3K-Akt Signaling Pathways in MC3T3-E1 Cells.

Neural cell adhesion molecule (NCAM) is involved in cell multi-directional differentiation, but its role in osteoblast differentiation is still poorly understood. In the present study, we investigated whether and how NCAM regulates osteoblastic differentiation. We found that NCAM silencing inhibited osteoblast differentiation in pre-osteoblastic MC3T3-E1 cells. The function of NCAM was further confirmed in NCAM-deficient mesenchymal stem cells (MSCs), which also had a phenotype with reduced osteoblastic potential. Moreover, NCAM silencing induced decrease of Wnt/ß-catenin and Akt activation. The Wnt inhibitor blocked osteoblast differentiation, and the Wnt activator recovered osteoblast differentiation in NCAM-silenced MC3T3-E1 cells. We lastly demonstrated that osteoblast differentiation of MC3T3-E1 cells was inhibited by the PI3K-Akt inhibitor. In conclusion, these results demonstrate that NCAM silencing inhibited osteoblastic differentiation through inactivation of Wnt/ß-catenin and PI3K-Akt signaling pathways.