Figure 1.
Activation of PTH1R by fatty acids in (A, B) HEK293 and (C) MC3T3-E1 cells.
PTH(1–34) concentration in (A, B) was 100 nM (positive control). Fatty acids concentrations were 10 µM. HEK293 cells were transfected with PTH1R as described under Materials & Methods and treated for (A) 5 minutes or (B) 15 seconds. (C) PTH(1–34) concentration was 10 nM (positive control). Fatty acid concentrations were 1 µM. MC3T3-E1 cells were transfected with PTH1R as described under Materials & Methods. Cells were stimulated with PTH(1–34) or corresponding fatty acid in PBS for 5 minutes at 37°C. Western blots were used to determine ERK1/2 phosphorylation. Data represent mean of n≥6 experiments. Error bars indicate standard error of the mean (SEM).
Figure 2.
PTH1R antagonist inhibits ERK1/2 response to PTH(1–34) and fatty acids.
(A) Effect of 1 µM of PTH1R antagonist [Leu11,D-Trp12]PTH-rP(7–34) on pERK stimulated with: 10 nM PTH(1–34), 1 µM EPA and 1 µM DHA in DPBS for 5 minutes in HEK293 cells transfected with PTH1R. (B) Effect of 1 µM of PTH1R antagonist, [Nle8,18,Tyr34]PTH(3–34), on pERK stimulated with 10 nM PTH(1–34), 1 µM EPA, and 1 µM DHA in MC3T3-E1 cells expressing endogenous PTH1R. (C) Dose response to EPA at different concentrations of antagonist [Leu11,D-Trp12]PTH-rP(7–34) in HEK293 cells. (D) Schild plot of [Leu11,D-Trp12]PTH-rP(7–34) antagonism on EPA-stimulated PTH1R in HEK293 cells. Experiments were done 24 h after transfection of HEK293 cells with PTH1R in 12-well plates. Ratio of pERK to ERK was measured using western blots. Data represents the mean of at least four independent experiments.
Figure 3.
EPA modulates efficacy of PTH(1–34) on ERK1/2 activation.
(A) EPA increases the efficacy of PTH(1–34) activation in HEK293 cells. Experiments were done 24 h after transfection of HEK293 cells with PTH1R in 12-well plates. (B) Formal synergy analysis was quantified by combination index analysis versus magnitude of the effect. Combination index values that are less than 1 indicate synergistic interaction. (C) 10 nM PTH(1–34) did not increase the efficacy of 1 µM Linoleic Acid in PTH1R transfected HEK293 cells. (D) 10 nM PTH(1–34) increases the efficacy of 1 µM EPA activation in MC3T3-E1 cells expressing endogenous PTH1R. Ratio of pERK to ERK was measured using western blots. Data represents the mean of at least four independent experiments.
Figure 4.
Inhibition of PKA or PKC reduces PTH or fatty acid-stimulated ERK1/2 phosphorylation.
(A) HEK293 cells transfected with PTH1R and stimulated with PTH(1–34) (100 nM) for 5 minutes in the absence or presence of H-89 (20 µM), GFX (2.5 µM) or both. Cells were pretreated with inhibitors for 15 minutes. (B) Cells were stimulated with 10 µM EPA. (C) Cells were stimulated with 10 µM DHA. (D) PTH1R transfected cells in the presence of H-89, GFX or both. Values are expressed as ratio of pERK to ERK1/2 after 5 minute stimulation. Data represents the mean from at least three independent experiments.
Figure 5.
Response of PTH-CC FRET sensor and PM-CC (control FRET sensor) to stimulation by PTH, EPA, EPA and PTH1R inhibitor, [Nle8,18,Tyr34]PTH(3–34), or linoleic acid.
Experiments were done 24 h after transfection of PTH-CC FRET sensor or PM-CC, in HEK293 cells in chambered cover glass at 37°C. FRET ratio was defined as ratio of Citrine emission intensity at 525 nm to Cerulean emission intensity at 475 nm. The FRET response to PTH is from [50].
Table 1.
Summary of binding data.
Figure 6.
Fluorescence anisotropy of PTH1R membranes labeled with nM PTH(1–34)TMR as function of EPA or DHA concentration.
Displacement binding of 2 nM PTH(1–34)TMR with (A) EPA and (B) DHA after 4 hours incubation time. Data represents the mean of at least 15 independent experiments.
Figure 7.
EPA and DHA (A) stimulate Akt phosphorylation and (B) inhibit dexamethasone-induced osteoblast cell death.
(A) Phosphorylated Akt levels in MC3T3-E1 cells serum-starved for 4 h and then preincubated with 1 µM of PTH1R antagonist [Leu11,D-Trp12]PTH-rP(7–34) for 30 minutes followed by 1 µM of LCPUFAs or 10 nM PTH treatment for 5 minutes. (B) Mechanism of the suppressive effect of LCPUFA on cell death in culture of MC3T3-E1 cells. Cultures were maintained for 6 hours in the presence of dexamethasone (Dex) without or with preincubations with 1 µM PTH1R antagonist [Leu11,D-Trp12]PTH-rP(7–34) for 30 minutes followed by exposure to 1 µM of LCPUFA or 10 nM of PTH for 1 hour. Dead cells were enumerated by trypan blue staining. Data represents the mean of at least 6 independent samples.
Figure 8.
PTH1R siRNA inhibits ERK1/2 response to PTH(1–34) and fatty acids.
MC3T3-E1 cells transfected with 100 nM siPTH1R or scambled control siRNA 96 hours prior to treatment with 10 nM PTH(1–34), 1 µM EPA, or 1 µM DHA. Data represents the mean of at least 8 independent samples.