Role of NRF2 and PI3K/Akt in the immunoregulatory response of
macrophages to 4HT and TAM
It is known that xenobiotics activate NRF2 and regulate immune gene
transcription, including a reduction of IL1β mRNA. We thus asked whether
NRF2 was involved in 4HT and TAM activity and used the ARE-luc2 reporter
mice, which carry the luciferase gene linked to an AREs-containing
promoter (Rizzi et al., 2018), to readily acquire evidence for this
hypothesis. Notably, 4HT increased the luciferase activity in peritoneal
macrophages obtained from ARE-luc2 mice (Figure 5A), while similar
concentrations of E2 were inactive. NRF2 activation generally derives
from the reduced proteasomal degradation and consequent increased
stability of NRF2. Consistent with this, Western blot analyses showed
increased NRF2 protein levels following 4HT treatment, while E2 was
again ineffective (Figure 5B). These effects were not mediated by
increased NRF2 expression, as NRf2 mRNA levels did not change in
response to 4HT (see Supplementary Figure 2C). We then assessed the
expression of endogenous NRF2-target genes and observed that 4HT and TAM
increased the mRNA levels coding the antioxidant enzyme Hmox1, both in
wild-type and ERαKO macrophages (Figure 3C). Moreover, a strong increase
in Hmox-1 protein levels was observed following TAM and 4HT treatments,
providing a biological evidence for NRF2 mediated effects of these
drugs. Positive effects were also observed for other NRF2 target genes,
namely NADP dehydrogenase quinone 1 and the metabolic enzyme
Transaldolase-1 (See Supplementary Figure 2D). These results support the
hypothesis that 4HT and TAM induce macrophage responses through the
activation of NRF2 and NRF2-mediated regulation of gene expression.
The PI3K/AKT pathway has been associated with induction of NRF2
stability and transcriptional activity. We thus assessed the involvement
of this pathway in NRF2 activation by SERMs. We observed that the
positive effect of 4HT and TAM on Hmox1 mRNA was completely abolished
when assayed in the presence of the PI3K inhibitor, LY294,002, used at
10 µM concentration for 30 min before SERMs addition (Figure 5E). The
effects on VEGFα expression were also significantly decreased. To
further sustain the involvement of the PI3K pathway, we evaluated the
presence of the phosphorylated form of Akt (pAkt), a downstream mediator
of PI3K signaling. Indeed, pAkt was detected shortly after 4HT and TAM
treatments (see Figure 5F). On the other hand, PI3K inhibition did not
modify E2 action on VEGFα mRNA. Moreover, Akt phosphorylation was not
detected in macrophages treated with this hormone, thus supporting the
evidence that PI3K and NRF2 activation are specific events induced by
4HT and TAM in macrophages. Altogether, these results demonstrated that
the transcriptional response of macrophages to 4HT and TAM is mediated,
at least in part, by NRF2 activation and involves the PI3K/Akt signaling
pathway.