A.-C. Le Lamer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3819–3822
3821
Before performing our study on the expression of Nrf2 and
PPAR target genes in macrophages, we first assessed the toxicity
of the selected compounds LA, 8, 9 and 12 against these peritoneal
macrophages isolated from mice (Table 2). The results were com-
parable to those obtained with the preliminary cytotoxicity assay.
Altogether, these data revealed that induction of both PPAR
c
target
c
genes as Dectin-1 and CD36 and Nrf2 target genes as NQO-1 and
HO-1 by our lactones occurred through a direct activation of these
transcriptional factors. Comparison of their potency showed that
LA is not active or less potent than compound 8, which is itself
Thus, to investigate whether our MA are able to activate PPAR
Nrf2 pathways, murine peritoneal macrophages were treated with
subtoxic concentrations (1 M) of compounds LA, 8, 9 and 12. For
the quantification of the mRNA expression of PPAR and Nrf2 spe-
cific target genes, Dectin-1 and CD36 were studied as PPAR target
while heme oxygenase-1 (HO-1) and NAD(P)H quinone
oxidoreductase 1 (NQO-1) were selected as Nrf2 specific target
c
and
far less potent than both a-methylene-c-lactones 9 and 12. This
is in agreement with their MA reactivity (LA < 8 < 9 and 12),
thereby strengthening the hypothesis that these lactones act as
MA. Regarding the effect on Nrf2 target genes expression, this
observation was not surprising and was consistent with previous
reports that indicated the potency of phase II enzyme inducers par-
l
c
c
1
8,19
genes,
3
4–36
allels their efficacy in Michael reactions.
Indeed, it is now well
3
2
genes. Since PPARc is the target of the thiazolidinedione class
established that induction of Nrf2 signaling pathway involved
12
of anti-diabetic agents that include rosiglitazone (R) and Nrf2 is
activated by sulforaphane (SFN), we have used them as specific
activators. Figure 2 demonstrates that Dectin-1 mRNA was induced
by all these compounds, lactones 9 and 12 being more potent than
LA and lactone 8. Besides, the expression of CD36, NQO-1 and HO-1
mRNA was only induced by 9 and 12. Although ligand binding to
nuclear receptor is a key element of their activation, transcriptional
control is a multistep process. Mechanisms responsible for genes
transcription in the cell require a complex network of transcription
modification of cysteine thiols of the Keap1–Nrf2 complex, espe-
1
cially through oxidation or Michael addition. Likewise, our results
suggested that
lent ligands through a Michael addition with a cysteine residue
in the PPAR ligand binding domain.
Several sesquiterpene lactones featuring a
tone ring were claimed to be potent phase II enzymes induc-
a-methylene-c-lactone 9 and 12 may act as cova-
c
a-methylene-c-lac-
3
7–40
ers.
activator of PPAR
-methylene- -lactones 9 and 12, whatever the substituent in
b- and -position, revealed to be more potent activators than
Among them, parthenolide was described as a natural
4
1,42
c
and Nrf2.
It is noteworthy that in our model
3
3
factors and cofactors. These results suggest a subtle regulation of
Dectin-1 and CD36 promoters, probably involving different cofac-
a
c
c
tors, which would explain that the two PPAR
Dectin-1 and CD36) respond differently to LA and compound 8.
Next, to validate that these compounds have a specific effect on
the transcriptional activity of PPAR and Nrf2, the biological activ-
ity of these compounds was also evaluated in macrophages inval-
c
controlled genes
parthenolide (Data not shown). These results are in agreement
with other studies that demonstrate the biological activities of
(
simple
complex structures.
In conclusion, our study provided evidence that structurally
simple -methylene- -lactones might constitute interesting lead
compounds or biological probes able to activate both PPAR and
Nrf2 signaling pathways at non-toxic concentration. Recently, it
has been shown that PPAR and Nrf2 might reciprocally reinforce
the expression of one another, enhancing the transcription of
a-methylene-c-lactones analogues, compared to more
5
,38,43,44
c
À/À
À/À
idated for PPAR
c
or Nrf2 (isolated from PPAR
c
or Nrf2
mice,
a
c
respectively).
c
As shown in Figure 2, the upregulation of mRNA levels of
specific PPAR and Nrf2 target genes was completely abolished
in macrophages invalidated respectively for PPAR or Nrf2.
c
c
c
Figure 2. Compounds 9 and 12 induce both the expression of PPAR
c
and Nrf2 target genes through a direct activation. (A) Dectin-1 and (B) CD36 mRNA levels were evaluated
À/À
by qRT-PCR on murine peritoneal macrophages isolated from wild type mice (WT) or PPAR
R, Cayman Chemical) (5 M) or compounds LA, 8, 9 and 12 (1
isolated from wild type mice (WT) or Nrf2 knock-out mice (Nrf2 ) and treated during 5 h with compounds sulforaphane (SFN, Sigma Aldrich) (10
c
knock-out mice (PPAR
c
) and treated during 5 h with compounds rosiglitazone
(
l
lM). (C) NQO-1 and (D) HO-1 mRNA levels were evaluated by qRT-PCR on murine peritoneal macrophages
À/À
lM) or compounds LA, 8, 9
⁄
⁄⁄
⁄⁄⁄
and 12 (1
lM). Data are from a represented experiment performed in triplicate ±SD. p 6 0.05; p 6 0.01; p 6 0.005 compared with the control.