3.4 PB inhibited liver fibrosis through downregulation of GLI1
expression
Recent studies have reported that
a positive correlation exists
between HSC activation and GLI1 overexpression (Chung et al., 2016;
Guerrero-Juarez & Plikus, 2017; Zhang et al., 2017). Growing evidence
indicate that GLI1 is a critical regulator of adult liver repair and
hence, a potential diagnostic and/or therapeutic target in cirrhosis
(Chen et al., 2020; Seki, 2016; Zhang et al., 2017). Based on the above,
we investigated whether GLI1 was involved in the anti-hepatic fibrosis
activity of PB. To determine
whether PB regulated GLI1 activity, GLI-dependent luciferase activity
was monitored, and the results showed that
PB
strongly repressed GLI-luciferase
activities (Figure 4A). In addition, TGFβ1 stimulation significantly
increased GLI1 expression, while PB treatment decreased GLI1 expression
(Figure 4B, C). Similar results were also observed in PB treated mouse
pHSCs (Figure 4D, E). However, RT-qPCR results showed that PB scarcely
influenced the GLI2, GLI3 expression in LX-2 cells (Supplementary Fig.
2A). Furthermore, PB treatment downregulated GLI1 protein expression in
BDL-induced fibrotic liver tissues (Figure 4F).
Next, we wonder whether PB mediated anti-fibrosis effect dependent on
GLI1. First, we verified that knockdown ofGLI1suppressed the mRNA and protein expression of fibrogenic in LX-2 cells
(Figure 5A, B). These results indicate that GLI1 indeed plays an
important role in HSCs activation. Next, we measured Collagen I and αSMA
expression in PB treated LX-2 cells with or without GLI1knockdown. As shown in Fig 5C, PB could not further reduce the Collagen
I and αSMA expression in the context of GLI deficiency. In addition, the
similar results were achieved when the cells were treated with GANT61,
the most-widely used specific inhibitor of GLI1. GANT61 treatment
decreased GLI-luciferase activity and GLI1 mRNA levels (Supplementary
fig. 2B, C). However, the
combination of PB and GANT61 showed no additive effect on the reduction
of fibrogenic gene expression in LX-2 cells (Supplementary Fig. 2D).
Furthermore, overexpression of GLI1 promoted Collagen I and αSMA
expression, and reversed PB mediated inhibition of aforementioned
proteins (Figure 5D). These results illustrated that PB inhibited HSC
activation via downregulation of GLI1 expression.
3.5 PB blocked
the nuclear localization of GLI1 in HSCs
Previous
findings had shed light on GLI1 activity, which is tightly controlled
through the regulation of nuclear import and the modulation of protein
stability (Gulino, Di Marcotullio, Canettieri, De Smaele & Screpanti,
2012). GLI1 nucleus localization is critical for its growth-promoting
function.
Recently,
Zhang et al. reported that
GLI1
nuclear translocation leads to
HSC
contraction and cirrhotic portal hypertension (Zhang et al., 2020). We
next tested if PB
influenced
the GLI1 nuclear localization. Expectedly, PB treatment
blocked the translocation of GLI1
from the cytoplasm to the
nucleus
in a dose-dependent manner (Figure 6A). Nuclear and cytoplasmic
fractionation analysis for GLI1 distribution reciprocated the
immunofluorescence experiments in both LX-2 cells and mouse pHSCs
(Figure 6B, C). Given that nuclear translocation of GLI1 resulting in
increased GLI1 target gene expression (Kim, Kim, Cho, Kim, Kim &
Cheong, 2010), we then measured GLI1 downstream target genes. The mRNA
expression of well-known GLI1
downstream genes, including HHIP , CYCLIN D , CYCLIN
E and C-MYC were
dramatically down-regulated in
both PB-treated LX-2 cells and BDL-PB treated mouse liver samples
(Figure 6D, E). Similarly, GLI1 downstream genes were reduced in GANT61
treated LX-2 cells, whereas the combination of PB and GANT61 showed no
additive effect on the reduction of downstream genes expression in LX-2
cells (Supplementary Fig. 2E). Taken together, these results indicated
that PB repress the translocation
of GLI1 from the cytoplasm to the nucleus,
decreasing the expression of the
downstream target genes.
3.6 PB inhibited LAP2 α-HDAC1 mediated deacetylation of
GLI1
Published
literatures suggest that GLI1 is
temporarily inactive by acetylation (AcGLI1), therefore
acetylation is an important
modification to regulate cellular GLI1(Coni et al., 2017; Mirza et al.,
2019). Thus, we speculated that PB may affect the acetylation of GLI to
regulate its activity. Indeed, anti-acetylated lysine immunoprecipitates
indicated that GLI1 are
constitutively acetylated with PB
treatment (Figure 7A). Acetylation directly inhibits the expression of
transcription factors (Gurung, Feng & Hua, 2013), thus
PB may inhibit liver fibrosis by
upregulating the acetylation of GLI1, which inactivates it.
It has been reported that the acetylation of GLI1 is regulated by
HDAC1(Canettieri et al., 2010; Falkenberg et al., 2016) and LAP2α(Mirza
et al., 2019). Mechanistically,
LAP2α recruits HDAC1 to GLI1, physically interacts with HDAC1, and
scaffolds a complex with GLI1(Mirza et al., 2019). Next, we wonder
whether PB showed any effect on LAP2α and HADC1 expression. However,
no significant protein variations
were observed in both LAP2α and HADC1 after PB treatment in LX-2 cells
(Figure 7B). Given that LAP2α/HADC1 mediated GLI deacetylation play an
important role in GLI activity, we next wonder whether PB affect
LAP2α/GLI interaction or LAP2α/HADC1complex formation. As shown in Fig
7C, PB showed no effect on the
interaction between LAP2α/GLI1, however,
strongly inhibited the LAP2α/HDAC1
complex formation in HEK 293T cells (Figure 7 D). Congruently,
endogenous
LAP2α
and HDAC1 interaction was significantly disrupted by the PB treatment in
LX‐2 cells (Figure 7E), and this interaction was also reduced in
CCl4-induced mouse fibrotic liver tissue treated with PB
(Figure 7F).
Previous studies have demonstrated that nucleoplasmic complex
LAP2α-HDAC1 could protect GLI1 from acetylation and promote GLI1
activation(Mirza et al., 2019). As described in the above co-IP results,
confocal microscopy assay was used to determine the subcellular
localization of HDAC1 (red) and LAP2α (green) to confirm this resultin situ . Our results showed that PB had no influence on the
nuclear localization of LAP2α with or without TGFβ1 treatment. However,
PB obviously decrease the nuclear localization of HDAC1 after
TGFβ1
treatment (Figure 7G), indicating that
PB administration may disturb the
intracellular colocalization of LAP2α with
HDAC1. These results indicated
that, instead of interfering with LAP2α and GLI interaction,
PB inhibited LAP2α-HDAC1 complex
formation, which may responsible
for PB mediated GLI inactivation.
Next, we employed the histone deacetylase inhibitor vorinostat to
further validate the results. Vorinostat decreased the levels of
fibrogenic markers which is in consistent with previous published
results(Park et al., 2014), and no further reduction was observed when
combination with PB (Figure 8A). Immunofluorescence assay further
confirmed that PB and vorinostat downregulated GLI1 expression and
nuclear localization (Figure 8B). In addition,
PB
and vorinostat increased the acetylation of GLI1
(Figure
8C). Due to the protective effect of LAP2α on GLI1 deacetylation, we
next verify that whether LAP2α deficiency can attenuate HSC activation
by treating LX-2 cells with LAP2α siRNA. The fibrogenic markers were
suppressed in LAP2α siRNA treated LX-2 cells (Figure 8D, E). Similarly,
combination of LAP2α silencing and PB treatment blocked nucleus
translocation of GLI1 in LX-2 cells (Figure 8F). In addition,
LAP2α-silencing and PB treatment increased GLI1 acetylation (Figure 8G).
Taken together, our results indicate that PB interferes with LAP2α-HDAC1
complex formation, thereafter inhibits GLI1 deacetylation and downstream
signaling. PB could be a promising
therapeutic agent for liver fibrosis.