Independent and synergistic roles of MEK-ERK1/2 and PKC pathways in regulating functional changes in vascular tissue following flow cessation

Abstract

Background: The MEK-ERK1/2 and PKC pathways play critical roles in regulating functional changes in tissues, but their interplay remains poorly understood. The vasculature provides an ideal model to study these pathways, particularly under conditions of flow cessation, which is highly relevant to ischemia and other cardiovascular diseases. This study examined the independent roles, additive effects, and time-dependent dynamics of MEK and PKC pathway inhibition in functional receptor upregulation.

Methods: Rat basilar arteries were cultured for 48 h with selective inhibitors targeting MEK (Trametinib), PKC (RO-317549) and their downstream ERK (Ulixertinib) and NF-kB (BMS 345541). Functional changes in ET_B receptor responses were assessed via wire myography following stimulation with Sarafotoxin 6c (S6c). Western blot analysis quantified ERK phosphorylation, and the effects of inhibitor timing and combination treatments were evaluated.

Results: MEK inhibition reduced ERK phosphorylation and ET_B receptor-mediated contractility, whereas PKC inhibition had no effect on ERK phosphorylation but significantly reduced ET_B receptor function. Combining MEK and PKC inhibitors produced an additive effect, resulting in greater suppression of functional changes compared to single treatments. At 6 h following flow cessation, PKC inhibition effectively suppressed ET_B receptor function, while MEK inhibition had minimal effects when introduced at this delayed time point.

Conclusions: The MEK and PKC pathways independently drive functional changes in vascular tissue, particularly following flow cessation. MEK inhibition is effective early, while PKC inhibition remains effective when applied later. The additive effects observed with combined MEK and PKC inhibition indicate parallel and functionally independent pathway activation during ET_B receptor upregulation.

Keywords: ETB upregulation; Flow cessation; Ischemia; MEK-ERK1/2 pathway; PKC pathway; Vasoconstriction.

Graphical abstract

Fig. 1

Comparison of fresh BAs and BAs after 48 h of incubation in organ culture regarding ET_Breceptors: (a) Immunofluorescence of ET_B receptors of the smooth muscle cell layer in fresh rat BAs and BAs after organ culture (48H) (b) Quantification of ET_B immunofluorescence, compared with Student's t-test (c) Contraction (mN) to 60 mM K⁺, compared with Student's t-test (d) Log concentration-response curves in response to S6c, and values compared by repeated measures two-way ANOVA, with Sidak's post-test (e) Representative sample traces from LabChart. Effects of S6c Data show the contractile responses in rat BAs to cumulative contractions of sarafotoxin (S6c). Arrows indicate the times at which different concentrations were applied (Biological replicate; n = 5–9). * = p < 0.05.

Fig. 2

Effects of the MEK inhibitor, Trametinib, and PKC inhibitor, RO-317549, on ERK and NF-κB phosphorylation. BAs were incubated in OC for 6 h. (a) Western blot analysis of total ERK and p-ERK, (b) Quantitative data of ERK, (c) Western blot of total NF-κB and pNF-κB (d) Quantitative data of NF-κB Data are shown as mean ± SEM (Biological replicate; n = 5) and protein expression was compared by one-way ANOVA, with Dunnett's post-test. * = p < 0.05. Please note that the order of the inhibitor is not the same across the figures, due to the original experimental setup.

Fig. 3

Comparison of the effects of the MEK inhibitor Trametinib and PKC inhibitor, RO-317549 to the S6C-mediated contraction of BAs after a 48-h OC. (a) Log concentration-response curves in response to S6c, following incubation with 10⁻¹⁰–10⁻⁶ M Trametinib, (b) E_max(S6c) responses of BAs incubated with different concentrations of Trametinib, (c) Log concentration-response curves in response to S6c, following incubation with 10⁻⁸–10⁻⁵ M RO-317549 [16], (d) E_max(S6c) responses of BAs incubated with different concentrations of RO-317549, (e) E_max(S6c) responses of BAs incubated with different concentrations of Trametinib, and combination of RO-317549 10⁻⁶ M with different concentrations of Trametinib. The Trametinib curve is the same as shown in panel b. The HillSlope was fixed at −1.727, (f) Representative Sample Traces from LabChart. CTRL refers to BAs incubated for a 48-h in OC with DMSO/Vehicle. Data are shown as mean ± SEM (Biological replicate; n = 5–18). Values were compared with a Student's t-test with a multiple correction (Holm-Sidak). * = p < 0.05.

Fig. 4

The effect of different ERK inhibitors on S6c-mediated contraction of BAs incubated in OC for 48 h. (a) Log concentration-response curves in response to S6c, following incubation with the ERK inhibitors Ravoxertinib, (GDC-0994), Temuterkib (LY3214996) and Ulixertinib (BVD-523) at 10⁻⁶ M, the E_max was compared by a multiple t-test with a Holm-Sidak correction, (b) Log concentration-response curves in response to S6c, following incubation with 10⁻⁹–10⁻⁵ M Ulixertinib the E_max was compared by a multiple t-test with a Holm-Sidak correction (c) E_max(S6c) responses to S6c of BAs incubated with different concentrations of Ulixertinib, (d) E_max(S6c) responses to S6c of BAs incubated with the combinations of Ulixertinib 10⁻⁷ M and RO-317549 (10⁻⁶ M), and Ulixertinib (10⁻⁷ M) and Trametinib (10⁻⁸ M). E_max compared by one-way ANOVA, with Dunnett's post-test. Data are shown as mean ± SEM (Biological replicate; n = 4–18). * = p < 0.05.

Fig. 5

The effect of NF-κB inhibitors in the contractility of BAs incubated for 48 h in OC. (a) Log concentration-response curves in response S6c, following incubation with the NF-κB inhibitors SP100030, BMS 345541, IMD 0354 (10⁻⁶ M) the E_max was compared by a multiple t-test with a Holm-Sidak correction (b) Log-concentration-response curves in response to S6c following incubation with 10⁻⁵ M and 10⁻⁶ M BMS 345541, the E_max was compared by a multiple t-test with a Holm-Sidak correction (c) E_max(S6c) contractions of RO317549 10⁻⁶ M, BMS 345541 10⁻⁶ M and their combination, compared with Trametinib 10⁻⁸ M and its combination with BMS 345541 10⁻⁶ M. E_max were compared by one-way ANOVA, with Dunnett's post-test. Data are shown as mean ± SEM (Biological replicate; n = 4–18). * = p < 0.05.

Fig. 6

Time-dependent effects of inhibitor addition (a) Addition of the different inhibitors at different time points 0 h or delayed 6 h. (b) Combination of 10⁻⁸ M Trametinib and 10⁻⁶ M RO-31 at time 0 or delayed 6 h, the control experiment is in panel a. Data are shown as mean ± SEM (Biological replicate; n = 4–18). The E_max were compared by Student's t-test. * = p < 0.05.

Fig. 7

Signaling pathways involved in ET_Breceptor upregulation and VSMC contraction. Extracellular stimuli such as flow cessation activate the MAPK/ERK pathway, which is disrupted by MEK inhibition (Trametinib) or ERK inhibition (Ulixertinib). In parallel, intracellular Ca²⁺ increase leads to delayed PKC activation, promoting NF-κB signaling via the IKK complex. PKC and NF-κB inhibition (RO-317549 and BMS345541, respectively) remain effective when applied up to 6 h post-stimulus. Combined inhibition further suppresses ET_B upregulation on VSMCs and reduces receptor-mediated contraction, indicating that both pathways contribute independently and are required for full functional response. Abbreviations: ET_B: endothelin type B receptor; VSMC: vascular smooth muscle cell; NF-κB: nuclear factor. Created in BioRender. Kazantzi, S. (2025) https://BioRender.com/u89c445