Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-beta.

Neural progenitor cells isolated from the embryonic cerebral cortex are well known to differentiate into neurons and glial cells, but recent reports have demonstrated differentiation into smooth muscle cells (SMCs) under the influence of fetal bovine serum. In this study, we report that agonists for G protein-coupled receptors (GPCRs), including endothelin, lysophosphatidic acid and carbachol, effectively promote the expression of SMC-specific proteins in the presence of transforming growth factor-beta (TGF-beta). Incubation of neural progenitor cells with agonists for GPCRs or TGF-beta alone induced the expression of an SMC-specific protein, alpha-smooth muscle actin (SMA), and their combination resulted in incremental increase. Stimulation with combinations of each GPCR agonist and TGF-beta increased the numbers of large, flat cells with thick actin fibers and also caused expression of other SMC marker proteins. Endothelin and TGF-beta enhanced SMA promoter-luciferase reporter activity at different times after stimulation. The mutation of TGF-beta control element of SMA promoter constructs decreased TGF-beta-enhanced luciferase activity but not endothelin-stimulated activity. Transfection of active forms of RhoA and its effector, mDia, strongly enhanced SMA promoter activity, and a dominant negative form of RhoA inhibited endothelin-stimulated promoter activity but not TGF-beta-stimulated activity. Whereas endothelin consistently activated RhoA, TGF-beta did not, and a specific inhibitor of TGF-beta type I receptor blocked TGF-beta-enhanced SMA promoter activity, suggesting involvement of Smad phosphorylation. These results suggest that separate signaling pathways of G protein and TGF-beta cooperatively promote the expression of SMC-specific proteins in neural progenitor cells.

Gi2 signaling enhances proliferation of neural progenitor cells in the developing brain.

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.