PMC

FRET analysis in HEK293 cells. HEK293 were transiently transfected with plasmid of CKAR (A–C) or plasmids combined with CKAR and PTHR1 (D, E) or DESL (F); 72 hours later, FRET was measured after the application of 1 μM TPA (A), 10 μM 8-Br-cAMP (B), 100 nM–10 μM hPTH(1–34) (C), 100 nM hPTH(1–34) (D), and 1,000 nM GR(1–28) (E, F). In each experiment, 10 cells were included in the analysis under 40× magnified field. Results are expressed as mean values ±SEM of three independent experiments.

The role of cAMP/PKA signaling in PLC-independent PKC activation. HEK293 cells were stably transfected with PTHR1 and then transiently transfected with CKAR: 1,000 nM GR(1–28) (A) or 100 nM GR(1–34) (C) was applied to the cells and FRET was measured; 1.5 hours after the cells were treated with 10 μM Rp-cAMP, 1,000 nM GR(1–28) (B), or 100 nM GR(1–34) (D) was then added, followed by FRET measurement. In each experiment, 10 cells were included in the analysis under 40× magnified field. Results are expressed as mean values ±SEM of three independent experiments.

Active PKA subunits rescued the FRET response to GR(1–28) that was blocked by Rp-cAMP. HEK293 cells were stably transfected with PTHR1 and then transiently transfected with CKAR. After the cells were treated with Rp-cAMP for 1.5 hours, active subunits of PKA (including both 5 ng/mL PKA-α and 5 ng/mL PKA-β were added into the culture and at the same time, FRET was recorded. 0.1% TFA (vehicle) (A) or 1,000 nM GR(1–28) (B) was applied within two minutes after the addition of active PKAs. In each experiment, 10 cells were included in the analysis under 40× magnified field. Results are expressed as mean values ±SEM of three independent experiments.

PKC inhibition changed the effects of GR(1–28) and GR(1–34) on early apoptosis of MC3T3-E1 cells. MC3T3-E1 cells were cultured with α-MEM supplemented with 0.5% FBS and treated with peptides as a 4 hour/24 hour cycle. After three rounds of treatment, cells were suspended. The apoptosis was quantified by using Annexin V-EGFP Apoptosis Detection Kit (GenScript USA Inc., NJ, USA) per the manufacturer’s protocol. Data are expressed as means ± standard deviation (SD) of three independent experiments. Variables were analyzed using analysis of variance (ANOVA) and Bonferroni’s test for post hoc analysis. ^* p<0.05 versus control; ^# p<0.05 versus GR(1–28); ^@ p<0.05 versus GR(1–34) and GR(1–34)+Rp-cAMP.

PKC inhibition altered the effects of GR(1–28) and GR(1–34) on osteoblastic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were plated in 24-well plates coated with type I collagen in mineralization medium. PTH peptides incubated the cells in a 4 hour/48 hour cyclic style. Alkaline phosphatase (ALP) staining and ALP activity measurement at the second week, as well as the investigation of mineralized nodules and the calcium content of the cultures at the fourth week were performed. Data are expressed as means±standard deviation (SD). Variables were analyzed using analysis of variance (ANOVA) and Bonferroni’s test for post hoc analysis. ^* p<0.05 versus control; ^# p<0.05 versus GR(1–28) or GR(1–28)+Rp-cAMP; ^@ p<0.05 versus GR(1–34) and GR(1–34)+Rp-cAMP.

Effect of Go6983 on gene regulation by GR(1–28) or GR(1–34). After MC3T3-E1 cells in culture reached confluence, they were switched into DMEM containing 1% FBS for 12 hours. (A) Go6983 or the vehicle (control) was added into the culture for one hour, followed by treatment of 100 nM GR(1–28) for four hours. Expressions of indicated genes were quantified with real-time PCR. (B) MC3T3-E1 cells were cultured in 1% FBS for 12 hours, then Rp-cAMP (group of GR(1–28)+Rp-cAMP or GR(1–34)+Rp-cAMP) or vehicle (control) was added to the culture. 1.5 hours later, Go6983 (group of GR(1–34)+Rp-cAMP+Go6983) or vehicle were added to the medium before the treatment of 100 nM GR(1–28) or 10 nM GR(1–34) for 4 hours. Expressions of genes were quantified with real-time PCR. Data are expressed as means±standard deviation (SD). Variables were analyzed using analysis of variance (ANOVA) and Bonferroni’s test for post hoc analysis. ^* p<0.05 versus control; ^# p<0.05 versus GR(1–28) (A) or GR(1–28)+Rp-cAMP (B); ^@ p<0.05 versus GR(1–34)+Rp-cAMP.