The Effect Of Hypoxia On LIMK1-mediated Cofilin Phosphorylation And Its Role In Intestinal Epithelial Barrier Dysfunction

It is well known that the intestinal epithelial barrier dysfunction and the increased permeability are the important pathophysiological changes at the early stage of severe burn injury, however the underlying molecular mechanisms remain uncertain. Previous studies have demonstrated that the tight junction(TJ) plays a pivotal role in the maintenance of intestinal barrier function integrity by the connection and signal exchange between TJ-associated proteins such as ZO-1, occludin, claudins and cytoskeletal actin. Under certain stimulus, the activity of cytoskeletal actin causes the cell contraction, dysfunction of tight junction proteins, and the disruption of tight junction, leading to the formation of intercellular space and the increase of permeability. Therefore, the interaction between TJ-associated proteins such as ZO-1, occludin, claudins and cytoskeletal actin affections may play important roles in the pathogenesis of intestinal epithelial barrier dysfunction following severe burn injury.It has been reported that cofilin, belonging to the family of actin-binding proteins, plays a key role in the regulation of actin polymerization and depolymerization. Upon activated, cofilin triggers the actin depolymerization, which in turn regulates the dynamic balance between F-actin and G-actin. Meanwhile, the activity of cofilin is regulated by LIM kinase(LIMK), an upstream molecule of cofilin and a class of dual activity of serine/threonine and tyrosine kinase. LIMK regulates the activity of cofilin by phosphorylating Ser3 site of cofilin, the substrate molecule of LIMK.Hence, we theoretically hypothesize that the increased oxygen free radicals, reduced ATP, changed cellular Ca2+ etc. occurring at the early stage of severe burn injury may cause the alteration of cofilin activity in intestinal epithelial cells by regulating LIMK, which in turn leads to the imbalance of actin dynamics, resulting in the intestinal epithelial barrier dysfunction and hyperpermeability. Accordingly, in this study, we developed an intestinal epithelial barrier model in vitro to investigate the role of LIMK1/cofilin/F-actin pathway in intestinal epithelial barrier dysfunction and permeability change under hypoxic condition, so as to further clarify the underlying mechanisms involved in the intestinal epithelial barrier dysfunction induced by severe burn injury.Methods1. The cultured human intestinal epithelial cell line Caco-2 was used to grown as cell monolayers on Transwell. The Caco-2 cell monolayers were randomly divided into control(0h), 1, 2, 6, 12 and 24 h post-hypoxia groups. The hypoxic treatment of cell monolayers was induced under 1% O2 condition.2. The transepithelial electrical resistance(TER) of Caco-2 monolayers was measured with a volt-ohmmeter, reflecting the barrier integrity of intestinal epithelial cell monolayers. The fluorescein isothiocyanate-labeled dextrane(FITC-dextran) probe was used to detect the permeability of Caco-2 monolayers.3. The indirect immunofluorescence assay was used to detect the distribution changes of tight junction proteins ZO-1, occludin and claudin-1, respectively. The changes in protein expression of ZO-1, occludin and claudin-1 were analyzed by Western blot assay.4. The fluorescence method was used to analyze the changes of F-actin distribution and the content ratio of F-actin/G-actin, respectively.5. The Western blot assay was used to detect the changes of protein expression of cofilin, p-cofilin, LIMK1 and p-LIMK1.Results1. Hypoxia caused significant decrease of TER and increase of permeability in Caco-2 cell monolayers, with the most significant change at 6h after hypoxia treatment.2. The protein expression of tight junction proteins occludin and claudin-1 was not markedly changed by hypoxic treatment of Caco-2 monolayers. But the protein expression of ZO-1 was significantly decreased after hypoxia. Hypoxia also caused an obvious change in distribution of tight junction proteins ZO-1, occludin and claudin-1 in Caco-2 monolayers.3. Hypoxia induced an obvious change in F-action distribution, cell retraction, and a significant decrease of F-actin/G-actin ratio in Caco-2 monolayers.4. The protein expression of cofilin was insignificantly changed by hypoxic treatment of Caco-2 monolayers. But hypoxia induced a significant decrease in the protein expression of p-cofilin, resulting in a significant decrease of cofilin phosphorylation.5. Hypoxia treatment did not affect the protein expression of LIMK1 in Caco-2 monolayers. However hypoxia caused a significant decrease in protein expression of p-LIMK1, leading to a significant decrease of LIMK1 phosphorylation in Caco-2 monolayers treated with hypoxia.ConclusionsIt is concluded that hypoxia induces the disruption of barrier function and increase of permeability in intestinal epithelial cells. The underlying molecular mechanisms are probably suggested as follows: hypoxia causes a decrease of cofilin phosphorylation and an increase of cofilin activity by inhibiting LIMK1 phosphorylation, which in turn leads to the depolymerization of F-actin and the disruption of dynamic balance between F-actin and G-actin, resulting in the changed distribution of tight junction proteins ZO-1, occludin and claudin-1 in intestinal epithelial cells challenged with hypoxia.