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.