Regulation of lysophosphatidic acid receptor-stimulated response by G-protein-coupled receptor kinase-2 and beta-arrestin1 in FRTL-5 rat thyroid cells.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that activates a variety of biological activities including cell proliferation. Three mammalian LPA receptor (LPAr) subtypes have been identified by molecular cloning, named lp(A1), lp(A2) and lp(A3), that are coupled to heterotrimeric G-proteins for signal transduction. The LPAr are endogenously expressed in the rat thyroid cell line FRTL-5 and we used the FRTL-5 cells permanently transfected to obtain moderate overexpression of G-protein-coupled receptor kinase-2 (GRK2) or beta-arrestin1 to study whether GRK2 and beta-arrestin1 desensitise LPAr-mediated signalling and regulate LPA-stimulated functional effects. Using RT-PCR we documented that lp(A1), lp(A2) and lp(A3) receptors are all expressed in FRTL-5 cells. We then analysed the signal transduction of the LPAr in FRTL-5 cells. Exposure to LPA did not stimulate inositol phosphate formation nor cAMP accumulation but reduced forskolin-stimulated cAMP. LPA was also able to stimulate MAP kinase activation and this effect was abolished by pertussis toxin pretreatment. These results suggest that LPAr are mainly coupled to a pertussis toxin-sensitive G-protein in FRTL-5 cells. In order to investigate whether GRKs and arrestins are involved in the regulation of LPAr-mediated signalling, we used the FRTL-5 cell line permanently transfected to overexpress GRK2 (named L5GRK2 cells) or beta-arrestin1 (L5betaarr1 cells). The ability of LPA to inhibit forskolin-stimulated cAMP accumulation was blunted in L5GRK2 and more markedly in L5betaarr1. The MAP kinase activation was also blunted in L5GRK2 and in L5betaarr1B cells. Exposure to 20 microM LPA increased the phosphorylation of extracellular signal-regulated kinases ERK1/2 by approximately 3-fold in L5pBJI cells (FRTL-5 cells transfected with the empty vector pBJI) while it induced a modest increase in L5betaarr1 and was ineffective in L5GRK2. We measured [3H]thymidine uptake in L5betaarr1B and in L5 GRK2 cells to test whether GRK2 and beta-arrestin1 could have a role in the regulation of LPAr-mediated cell proliferation. The mitogenic response induced by 35 microM LPA was substantially blunted in L5betaarr1 (-69+/-6%) and in L5GRK2 (-69.8+/-4.5%) cells as compared with L5pBJI. Our findings document that the receptor-mediated responses elicited by LPA are regulated by GRK2 and beta-arrestin1 in FRTL-5 cells and indicate that this mechanism is potentially important for the control of the LPA-stimulated proliferative response.

Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by a cAMP-dependent protein kinase A-independent mechanism.

The involvement of mitogen-activated protein (MAP) kinases in the mitogenic effect of thyrotropin (TSH) is not fully elucidated. In FRTL-5 cells, we found that the MAP kinase kinase (MEK) inhibitors UO126 and PD98059 substantially decreased TSH-induced DNA synthesis, indicating that MAP kinases are involved in the TSH-stimulated proliferative response. Accordingly, TSH, forskolin (FSK) and 8-bromo-cAMP induced a rapid (3 min) and transient activation of ERK1/2, as assessed by phosphorylation of myelin basic protein and ERK1/2. This effect was cAMP-dependent and protein kinase A (PKA)-independent. The activation of Rap1 and B-Raf was involved in the mechanism of MAP kinase stimulation by TSH. TSH induced rapid (3 min) GDP/GTP exchange and activation of Rap1. After a 3-min exposure to FSK, B-Raf was recruited to a vesicular compartment, where it colocalized with Rap1. Both activation of Rap1 and translocation of B-Raf were PKA-independent. The Rap1 dominant negative Rap1N17 significantly reduced TSH-stimulated but not insulin-like growth factor 1-stimulated ERK1/2 phosphorylation, whereas the Ras dominant negative RasN17 inhibited the effect of both agonists. In conclusion, our results document that TSH increases intracellular cAMP, which rapidly stimulates MAP kinase cascade independent of PKA. This novel mechanism could integrate other pathways involved in TSH-stimulated proliferative response.

Nutritional antioxidants as antidegenerative agents.

In this study, primary cultures of cerebellar granule neurons were prepared from eight-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. All experiments were performed with fully differentiated neurons (eight days). To induce apoptosis, culture medium was replaced with a serum-free medium (containing 5 mM KCl) eight days after plating. In another series of experiments, apoptosis was induced by application of glutamate (50 microM) to the cell cultures. Apoptosis was measured by flow cytometry, the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling) method, and by the classical method of DNA fragmentation. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by both low K(+) concentrations and glutamate, a series of natural antioxidants and a red wine lyophilized extract (which is rich in antioxidant compounds) were tested in our experimental model. It was found that ascorbic acid (30 microM) and a red wine lyophilized extract (5 microgram/ml) were capable of blocking the apoptotic process. Addition of the following natural antioxidants did not have any protective effect on apoptosis induced by low K(+) concentrations: trans- and cis-resveratrol (5-200 microM), alpha-tocopherol (100-200 microM), reduced glutathione (100-400 microM), 3-hydroxytirosol (25-100 microM), epicatechin (25-100 microM), or quercetin (25-50 miroM). It is concluded that only a limited number of natural antioxidants are provided with antiapoptotic activity in cultured cerebellar granule neurons. This effect is probably exerted by reducing ROS formation, and by blocking caspase-3 activity.