Fig 2: Local infusion of the PI3-kinase inhibitor, wortmannin, blocks somatodendritic phosphorylation of rpS6 but not in the zone of activated synapses. (A) Immunostaining for p-ser235/236 ipsilateral to stimulation in a section distant from the infusion area. (B) High-magnification image of A. (C) Quantification of average OD across the dorsal blade of the dentate gyrus in ipsilateral sections distant from the infusion area (“Distant”), ipsilateral sections within the infusion area (“Infusion”), and contralateral, nonstimulated sections (“Contra”). (D) Statistical assessment at four sites along the dentate gyrus (denoted by the arrows), GCL, IML, MML, OML (ser235/236, N = 8), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a significant interaction (F(6,84) = 8.44, P < 0.0001), a significant main effect of treatment (F(2,84) = 114.30, P < 0.0001), and a significant main effect of region (F(3,84) = 42.36, P < 0.0001). Post hoc analysis with Bonferroni's multiple comparisons tests revealed that distant sections were statistically different than sections within the infusion area and contralateral sections in all regions. Notably, in the MML, sections within the infusion area were significantly different than contralateral sections (two-way ANOVA, P < 0.05). (E) Immunostaining of p-ser235/236 ipsilateral to the stimulation in a section within the infusion area in the presence of wortmannin (arrows denote infusion area). (F) High-magnification image of E. Note the decrease in rpS6 phosphorylation specifically in the GCL and nonactivated dendritic regions (IML and OML) and sparing of rpS6 phosphorylation in the zone of activated synapses (MML). (G) Immunostaining of p-ser235/236 contralateral to the stimulation. (H) High-magnification image of G. (I) Immunostaining of p-ser240/244 in a section distant from the infusion area. (J) High-magnification image of I. (K) Quantification of average OD across the dorsal blade of the dentate gyrus in distant, infusion, and contralateral sections. (L) Statistical assessment at four sites along the dentate gyrus (denoted by the arrows), GCL, IML, MML, OML (ser240/244, N = 5), error bars represent SEM. Statistical assessment by two-way ANOVA revealed a main effect of treatment (F(2,48) = 41.26, P < 0.0001) and a main effect of region (F(3,48) = 48.69, P < 0.0001), but no significant interaction (F(6,48) = 1.72, P = 0.137). (M) Immunostaining of p-ser240/244 ipsilateral to stimulation in a section within the infusion area in the presence of wortmannin (arrows denote infusion area). (N) High-magnification image of M. (O) Immunostaining of p-ser240/244 contralateral to the stimulation. (P) High-magnification image of O. Scale bars: A, 200 µm; B, 50 µm.

Fig 3: Protein kinase signaling and regulation of protein synthesis by ET-1 and insulin. Cardiomyocytes were exposed to 100 nM ET-1 or 50 mU/ml insulin or left unstimulated. A-C, Cardiomyocytes were exposed to agonists for 5 min (A) or 15 min (B and C). Protein extracts were immunoblotted with antibodies to phosphorylated ERK1/2 (A, upper panel), phosphorylated PKB/Akt (A, lower panel), phosphorylated mTOR (B, top panel), phosphorylated p70S6k (B, upper center panel), phosphorylated Rps6 (B, lower center panel) or total mTOR (B, lower panel). Each set of samples was prepared from a separate preparation of cardiomyocytes. C, Bands from images in panel B were quantified by scanning densitometry. Results are means ± SEM (n = 3). * p < 0.05, # p < 0.01, † p < 0.001 relative to controls (one-way ANOVA with Tukey post-test). D, Cardiomyocytes were exposed to agonists for the total time indicated and rate of protein synthesis was measured over the last 1 h of incubation using [3H]-Phe. Results are means ± SEM (n = 5 independent preparations of cardiomyocytes). * p < 0.05, # p < 0.01, † p < 0.001 relative to controls, ‡ p < 0.05 relative to 2 h (one-way ANOVA with Tukey post-test). E, Cardiomyocytes were unstimulated (Control) or exposed to agonists/inhibitors as follows: ET-1 (5 min), PD184352 (2 µM, 15 min), PD184352/ET-1 (10 min pretreatment with PD184352 before addition of ET-1 for 5 min), insulin (10 min), LY294002 (50 µM, 20 min), rapamycin (1 µM, 20 min), LY294002/insulin or rapamycin/insulin (10 min pretreatment with inhibitor before addition of insulin for 10 min). Extracts were immunoblotted for phosphorylated and total ERK1/2, PKB/Akt and p70S6K as indicated. Experiments were repeated with similar results. F, Cardiomyocytes were exposed for 2 h to 50 µM LY294005, 1 µM rapamycin, 2 µM PD184352, 100 nM ET-1 or 50 mU/ml insulin, or ET-1 or insulin in the presence of each inhibitor. The rate of protein synthesis was measured over the last 1 h of incubation using [3H]-Phe. Results are means ± SEM (n = 5 independent preparations of cardiomyocytes). # p < 0.01, † p < 0.001 relative to controls, * p < 0.05, ‡ p < 0.001 relative to ET-1 or insulin alone (one-way ANOVA with Tukey post-test).

Fig 4: A schematic summary of findings.Ribosomal protein S6 (rpS6) is directly phosphorylated by the kinases Rsk, S6K1 and PKA [11]. Rsk and S6K1 are activated by ERK1/2 and Akt-mTOR signaling respectively. These signaling pathways induce of rpS6 phosphorylation at S235/236 and enhance the translation of CXCL8 mRNA. The enhancement requires: 1) a 3’ UTR that is at least 300 bases; and 2) an AU base content exceeding fifty percent in the first hundred or so bases of the 3’ UTR immediately after the coding sequence, which we refer to as AU-rich proximal RNA cis-regulatory sequences (APS). This translational control mechanism contributes to the elevated CXCL8 protein expression in macrophages. On its own, the 5’ UTR of CXCL8 is a potent translation enhancer.

Fig 5: Ovulation-induced primordial follicle activation. Mice at P25 received one i.p. injection of PMSG and followed by another injection of hCG 48 h later. Ovaries were collected at the indicated time points (NC, PMSG 48 h, hCG 10 h, hCG 12 h, hCG 14 h and hCG 18 h). NC, control animals without any hormone injection. (a) Expression of NGF and p-rpS6 proteins by immunoblotting. (b) Proportions of p-rpS6-positive primordial follicles. Two serial sections from each ovary were used for p-rpS6 staining and at least three ovaries were chosen from each group. *P<0.05; **P<0.01 as compared with NC controls. (c) Expression of p-rpS6 in superovulated mouse ovaries 48 h post-PMSG (left panel) and 18 h post-hCG (right panel). Black arrows, p-rpS6-negative primordial follicles; red arrows, p-rpS6-positive primordial follicles. Bar=100 µm. (d) Schematic diagram showing selective activation of primordial follicles in mouse ovaries. Ovarian surgery or ovulation-induced injury immediately stimulated localized expression of NGF in interstitial cells (pink area). The stromal mTOR signaling pathway was then activated and this was followed with the sequential activation of the mTOR signaling pathway in pre-granulosa cells and toward oocytes. The primordial follicles were thus finally activated. However, those primordial follicles a distance from the stimuli maintained their dormancy (white area)