Background
Pathological left ventricular cardiac hypertrophy is a major risk factor for cardiovascular mortality and morbidity. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the ERK1/2 dimerization dependent autophosphorylation ERK(T188) in particular were found to mediate pathological hypertrophy. Thus, inhibition of pERK(T188) is a possible therapeutic strategy to target maladaptive ERK1/2 signaling while avoiding the cardiotoxic effects of global ERK1/2 inhibition.
Aim of the study
Immune cells and particularly macrophages were found to play an important role in the pathogenesis of heart disease and remodeling processes following cardiac injury. As ERK1/2 have been shown to impact cell signaling in macrophages, we analyzed different ERK1/2 targeting approaches for phagocytosis and efferocytosis in macrophages in vitro to evaluate the potential impact on cardiac macrophages. These macrophage functions are essential for clearance in tissue homeostasis.
Methods
We investigated different effector functions in macrophages such as cytokine production following lipopolysaccharide (LPS) stimulation, phagocytosis of E. coli particles, and efferocytosis of human apoptotic ventricular myocytes. These were studied in macrophage-like cells of the human cell line THP-1 that were pretreated with trametinib (MEK1/2 inhibitor), ulixertinib (ERK1/2 inhibitor) or DEL-22379 (DEL; ERK1/2 dimerization inhibitor), as well as in murine bone marrow derived macrophages (BMDMs) with altered pERK(T188) signaling: T188A (phosphorylation deficient mutant), T188D (phosphorylation simulating mutant) and EDI (constitutive expression of the ERK1/2 dimerization inhibitor EDI).
Results
Pre-treatment of THP-1 macrophages with trametinib showed a tendency to attenuate LPS-induced mRNA expression of the pro-inflammatory cytokine TNFα and the anti-inflammatory cytokine IL-10. DEL significantly decreased the expression of TNFα and induced IL-10 at baseline and following LPS stimulation. Trametinib, but not ulixertinib nor DEL, negatively affected the protein expression and secretion of the pro-inflammatory cytokine IL-1β.
DEL, but not trametinib, decreased phagocytosis and efferocytosis in THP-1 macrophages. In BMDMs, DEL attenuated phagocytic efficiency, i.e., the amount of phagocytosed particles per cell, but not the proportion of macrophages that took up particles. Altered pERK(T188) signaling in T188A, T188D and EDI BMDMs did not affect phagocytosis.
Conclusion
This study showcases another advantage of inhibiting ERK1/2 dimerization over global ERK1/2 inhibition – the attenuation of pro-inflammatory signaling and cytokine expression while simultaneously inducing anti-inflammatory IL-10. This is especially of interest for a condition such as cardiac hypertrophy which is associated with inflammation believed to exacerbate the disease. Neither EDI, ulixertinib nor trametinib had a negative effect on phagocytosis or efferocytosis, indicating the impact of DEL to be an off-target effect as well as revealing these effector functions to be independent from ERK1/2 dimerization. Therefore, inhibition of ERK1/2 dimerization and subsequent phosphorylation of ERK(T188) are relevant therapeutic options for cardiac hypertrophy, targeting pathological ERK1/2 signaling without inducing cardiotoxicity, and attenuating the pro-inflammatory macrophage response without impacting efferocytosis.
