Discussion
A significant body of evidence indicates that LPA plays a crucial role
in the growth and differentiation of intestine as well as the protection
of intestinal mucosa from a variety of noxious conditions (Konno et al.,
2019; Li et al., 2005; Lin et al., 2010; Thompson et al., 2018; Yun &
Kumar, 2015). However, the role of LPA in the mechanism of protection of
the colonic mucosal barrier function is poorly understood. In this new
study, we provide evidence that LPA2 receptor activation
is radioprotective in the colon by demonstrating that the
LPA2 receptor agonist RP1 protects the intestinal
epithelium against radiation-induced disruption of apical junctional
complexes and prevent or alleviate barrier dysfunction.
The results of in vitro studies in transformed Caco-2 and
non-transformed m-ICC12 epithelial monolayers showed
that radiation induces a rapid disruption of intestinal epithelial tight
junctions in a dose-dependent manner (Fig.1B). In our previous study, we
have reported that radiation causes disruption of mouse colonic
epithelial tight junctions in vivo as early as 2 hours
post-irradiation (Shukla et al., 2016). The present in vitro data
demonstrate that radiation directly affects the intestinal epithelium
without the systemic influences. The protective effect of
LPA2 in irradiated Caco-2 and m-ICC12cell monolayers indicate that LPA directly interacts with the epithelial
cells to attenuate radiation-induced tight junction disruption.
Irradiation of Caco-2 cell monolayers was associated with a reduction in
the inactive form levels of cofilinpS3. This decrease
in cofilinpS3 levels without change in total cofilin
indicates that radiation causes cofilin activation. Disruption of the
actin cytoskeleton was previously shown to disrupt tight junctions,
which is a common mechanism involved in tight junction disruption
mediated by various injurious factors (Madara, Moore & Carlson, 1987).
Therefore, activation of cofilin, an actin severing protein, is likely
involved in radiation-induced tight junction disruption and disruption
of the actin cytoskeleton. Cofilin is known to be inactivated by LIM
kinase-mediated phosphorylation on S3 residue (Chen & Macara, 2006),
and LPA activates the Rho-ROCK-LIM kinase pathway (Pandey, Goyal &
Siess, 2007). Abrogation of LPA-mediated protection of tight junctions
by the Rho-GTPase inhibitor Toxin-B suggests that LPA likely promotes
cofilin phosphorylation by Rho-Rock-LIM kinase pathway.
Previous studies indicated that the intestinal mucosal protective
effects of LPA are mediated by activation of LPA2receptor (Deng, Balazs, Wang, Van Middlesworth, Tigyi & Johnson, 2002;
Deng et al., 2015; Deng et al., 2007; Elias et al., 2009; Khurana et
al., 2008; Kuo et al., 2018; Li et al., 2005; Lin, Lai, Makarova, Tigyi
& Lin, 2007; Thompson et al., 2018; Yoshida, He & Yun, 2016). Our data
in the present study show that radiation-induced disruption of tight
junction and adherens junction is more severe in LPA2deficient mice compared to that in WT mice, suggesting that
LPA2 activity exerts a protective effect on the
intestinal epithelial tight junctions and adherens junctions in
vivo . The role of LPA2 receptor in the protection of
tight junction was further determined by evaluating the effect of an
LPA2-selective agonist, RP1 (Patil et al., 2014), on the
radiation-induced disruption of intestinal junctions. The prevention of
radiation-induced redistribution of tight junction and adherens junction
proteins from the intercellular junctions by RP1-pretreatment indicates
that RP1 and LPA2 receptor activation blocks
radiation-induced disruption of AJC in the mouse colon.
Radiation-induced disruption of tight junction was associated with an
increase in mucosal permeability to inulin, indicating the
radiation-induced barrier disruption in the mouse colon in vivo .
Furthermore, barrier dysfunction was associated with an increase in the
levels of plasma LPS in irradiated mice, demonstrating radiation-induced
endotoxemia. Prophylactic administration of RP1 blocked
radiation-induced colonic mucosal permeability and endotoxemia.
Therefore, data from this part of the study indicates that
LPA2 activation via prophylactic RP1-treatment prevents
radiation-induced disruption of intestinal epithelial AJC, mucosal
barrier dysfunction, and endotoxemia.
In order to determine whether RP1 can mitigate radiation-induced
intestinal barrier dysfunction, we evaluated the effect of RP1 when
administered at 24 hours post-irradiation in mice in vivo .
Results of this study showed that RP1 completely reversed
radiation-induced redistribution of occludin, ZO-1, claudin-3,
E-cadherin and β-catenin, indicating the reversibility of
radiation-induced disruption of tight junctions and adherens junctions
and the presence of functioning LPA2 in the irradiated
enterocytes. RP1 also reversed the radiation-induced increase in ileal
and colonic mucosal permeability to inulin and endotoxemia. Data
demonstrates that the LPA2 is an effective target for
the treatment of radiation injury to the gut, and RP1 is effective in
alleviating radiation-induced intestinal damage. Our previous study
indicated that radiation caused oxidative stress in the colonic mucosa
and that N-acetylcysteine, the antioxidant, effectively blocked
radiation-induced disruption of colonic epithelial tight junctions and
barrier dysfunction (Shukla et al., 2016). In the present study, we
examined the effect of RP1 on radiation-induced oxidative stress by
measuring the levels of antioxidant genes in the colonic mucosa.
Significant reduction of mRNA for Gpx1 , SOD1 ,Prdx1 , CAT (catalase), and Nrf2 indicated that
radiation suppresses antioxidant gene expression in colonic mucosa. Data
also showed that RP1 completely reversed radiation-induced suppression
of Gpx1 , SOD1 , and Prdx1 expression, and partially
reversed the effect of radiation on Nrf2 expression; RP1 showed
no significant impact on catalase expression. Therefore RP1-mediated
activation of the LPA2 receptor provides significant
protection against oxidative damage by irradiation.
All of the above studies applied the TBI model to evaluate the effects
of RP1. To mimic the conditions of the GI-ARS, we assessed the effect of
RP1 in the PBI-BM5 model in which the shielded bone marrow expands and
allows for the survival of the animal proviso protection of the gut
mucosa. At 28 hours post-irradiation, the junctional distribution of
tight junction and adherens junction proteins were unaffected,
indicating that PBI-BM5 did not affect intestinal tight junctions and
adherens junctions until 28 hours post-irradiation. This PBI effect is
in contrast to TBI effects, which disrupted tight junctions within 2
hours post-irradiation. However, by 52 hours post-irradiation, PBI-BM5
caused a severe loss in the junctional distribution of occludin, ZO-1,
E-cadherin, and β-catenin, which was sustained at least until 76 hours.
RP1 (3 mg/kg daily, s.c.) treatment beginning 24 hours post-irradiation
completely alleviated PBI-induced disruption of tight junctions and
adherens junctions both at 52 and 76 hours post-irradiation. These data
demonstrate that LPA2 receptor activation via RP1
administered 24 hours post-irradiation can alleviate PBI-induced damage
to the intestinal epithelial junctions. Conventionally, PBI-BM5 is
characterized by higher dose (>5 Gy) radiation exposure and
sparing of 2.5-5.0% of bone marrow; this is considered as the ideal
model for GI-ARS. TBI, characterized by ablation of 100% of bone
marrow, is considered as hematopoietic acute radiation syndrome (H-ARS).
Interestingly, in the present study, we show that PBI-BM5 does not
affect colonic epithelial tight junction for at least 28 hours
post-irradiation; tight junction disruption was observed at 52 hours and
sustained at least until 100 hours post-irradiation. This is distinctly
different from the effect of TBI. TBI caused tight junction disruption
in less than 2 hours post-irradiation. This observation suggests that
the 5% bone marrow protected by shielding during irradiation and the
bone marrow-derived factors may have delayed the damage to colonic tight
junction and barrier function. A new avenue of studies is necessary to
understand the protective role of bone marrow in the intestinal
epithelium.
Dramatic reduction of the levels of reduced-protein thiols accompanied
by elevation of oxidized-protein thiols in the colon indicate that
PBI-BM5 induces oxidative stress in colonic mucosa. Here we demonstrated
for the first time that RP1-treatment significantly attenuates
PBI-BM5-induced oxidative stress. PBI-BM5-induced oxidative stress was
associated with modulation of Nrf2 and antioxidant gene
expression. NRF2 is a transcription factor critical for the expression
of antioxidant genes (Cameron, Sekhar, Ofori & Freeman, 2018; Jaiswal,
2004). Immunofluorescence imaging, immunoblot analysis, and Nrf2mRNA measurement by RT-qPCR indicated that PBI-BM5 caused a significant
reduction of Nrf2 expression, which was prevented by RP1 at 24
hours post-irradiation. Considerable decreases in the levels of mRNA forGpx1 , SOD1 , Prdx1 , Trx1 , and CAT by
PBI-BM5 and prevention of these effects by RP1indicate that in the
PBI-BM5 model of irradiation the antioxidant gene expression is
suppressed and that RP1 alleviates this effect of PBI. Therefore,
induction of oxidative stress is likely one of the mechanisms involved
in PBI-BM5-induced intestinal barrier dysfunction. Furthermore, it
underlines the antioxidant action of RP1-mediated activation of
LPA2 receptors in the mechanism of its protective
effects.
To determine whether disruption of the actin cytoskeleton and loss of
its interaction with tight junction and adherens junction are involved
in the mechanism of PBI-BM5-induced disruption of the apical junctional
complex, we measured the levels of detergent-insoluble fractions of
tight junction and adherens junction. The detergent-insoluble fraction
predominantly consists of the actin cytoskeleton. In the intact
epithelium and non-disrupted tight junctions, the junctional proteins
are bound to the actin cytoskeleton; therefore, they are recovered in
the actin-rich detergent-insoluble fractions (Seth, Sheth, Elias & Rao,
2007). Disruption of the actin cytoskeleton leads to disruption of tight
junctions and loss of detergent-insoluble fractions of tight junction
proteins (Madara, Moore & Carlson, 1987). The present study shows that
the detergent-insoluble fractions of claudin-3, E-cadherin, and
β-catenin were significantly reduced by PBI-BM5, which was blocked by
RP1-treatment. Data indicate that γ-irradiation disrupts the association
between the actin cytoskeleton and the junctional proteins. Furthermore,
it suggests that preservation of the integrity of the actin cytoskeleton
may be involved in the mechanism of RP1-mediated protection of AJC in
irradiated mice. The results of this study also show that γ-irradiation
decreases the levels of F-actin, likely through activating cofilin in
the colonic mucosa, and RP1 treatment blocks this effect via
LPA2 receptor-mediated activation of the Rho-Rock-LIM
kinase pathway.
In summary, this study demonstrates that γ-irradiation disrupts AJC in
the intestinal epithelium, induces mucosal barrier dysfunction, and
causes endotoxemia, likely by inducing oxidative stress and disrupting
the actin cytoskeleton. Furthermore, our data demonstrate that
activation of the LPA2 receptor prevents and mitigates
radiation-induced intestinal barrier dysfunction and endotoxemia.
Therefore, LPA2 agonists like RP1 via the protection of
the AJC of the colonic mucosa could have therapeutic benefits in the
treatment of diseases associated with the disruption of the intestinal
barrier.