Interferon-gamma Regulated HIF-1α Pathway Involves In The Regulation Of Epithelial Barrier Function In A Hypoxia Model

Background and objectivesIschemia of the gastrointestinal tract has tremendous clinical implications. A largenumber of patients each year suffer from an ischemic insult to the gastrointestinal tract,resulting in a high degree of morbidity and mortality. In fact, the loss of intestinal epithelialbarrier function (EBF) may be a major contributing factor to the morbidity of this condition. Anumber of other medical conditions may also contribute to a loss of intestinal EBF, including:burns, trauma, major surgery, inflammatory bowel disease (IBD) and administration of totalparenteral nutrition (TPN). This loss of intestinal mucosal barrier function may inducesystemic inflammatory response syndrome, leading to multiple organ failure, and death;however, the mechanisms of loss of barrier function are still not well understood.Recent studies have shown that the regulation of intestinal mucosal barrier function isclosely related to local mucosal hypoxia under different pathological conditions and this isrelated to the unique anatomic structure of the intestinal mucosa. Hypoxia is a commoncondition in many diseases; local mucosal hypoxia may be the causative factor resulting in theloss of intestinal barrier function. That rationale has been confirmed in a variety of acute andchronic pathological conditions such as septic shock, toxemia, ischemia-reperfusion andinflammatory bowel disease which result in hypoxia of local intestinal mucosa. At the sametime, local mucosal hypoxia synergizes with the inflammatory reaction, resulting in theproduction of a large number of inflammatory factors which aggravate the loss of intestinalmucosal barrier function. Recent study shows that the increase of intraepithelial lymphocytes(IEL) derived interferon-γ (IFN-γ) expression caused by hypoxia results in the loss ofintestinal barrier function. Study has shown that acute intestinal ischemia-reperfusionconditions may also activate IEL and increase the IEL derived IFN-γ expression. This rise inIFN-γ expression may increase the intestinal permeability with increased intestinal hypoxia.IFN-γ is always thought to be related to the damage of tight junctions between intestinalepithelial cells under the condition of hypoxia. Inflammtory bowel disease which is characteristic of cronic inflammation and increased permeability is related to IFN-γ tightly.The expression of IFN-γ and claudin-2which increases the permeability of intestinal barrier isincrease in Crohn's disease. Study in vitro also certifies that IFN-γ could depressphosphatidylinositol3-kinase signal pathway, decreasing the expression of occludin, whichinfluences intestinal barrier function.Recent evidences show that Hypoxia-inducible factor-1(HIF-1) is the key factor inmucosal hypoxia induced intestinal mucosal barrier dysfunction.HIF-1is a central transcription regulatory factor which is involved in a number ofpathways responsible for the expression of barrier protection factors, which including mucin,intestinal trefoil factor, ecto-5'-nucleotidase (CD73), p-glycoprotein and vasodilatorstimulated phosphoprotein (VASP) and in the implementation of stress and pathologicalconditions of the intestinal barrier regulating mechanisms.The HIF-1is a heterodimeric transcription factors composed of an oxygen-sensitiveα-subunit and a constitutively expressed β-subunit. normal oxygen tension, along with thepresence of iron and2-oxoglutarate, allows for PHD-mediated hydroxylation of α-subunit attwo proline residues (402and564) within the ODD domain. Hydroxylation of the α-subunitacts as a recognition signal for pVHL leading to ubiquitination and subsequent degradation.IFN-γ may inhibit the acitvation of HIF-1indirectly. Recently, a study showed that IFN-γ,through a STAT1-dependent manner in endothelial cells, increases prolyl hydroxylase3(PHD3) expression, while the PHD3can regulate HIF-1degradation; Another study foundthat IFN-γ can downregulate HIF-1transcriptional activity by inhibiting the activity of NF-кB.NF-кB may be an upstream regulator of HIF-1expression and, at the same time, PHD can bea negative regulator of the NF-кB pathway.However, the mechanism of IFN-γ regulates HIF-1is not well understood. In this study,we investigated the regulation between IEL-derived IFN-γ and HIF-1, especially HIF-1expression and transcriptional activity, utilizing transepithelial resistance (TER) measure,Western blot, Chromatin Immunoprecipitation Assay, gene transfection and so on. This studywill be helpful to understand the mechanisms responsible for the loss of intestinal epithelialbarrier functionThis may lead to new methods to prevent this loss. Methods1．To determine the alter of the TER of intestinal epithelial cell caused by hypoxia andIFN-γ, Human intestinal epithelial cell line HT-29hypoxia and IFN-γ interfence model wasset up. TER was measure by Trans-well Assay.2．To explore the influence of IFN-γ on HIF-1induced intestinal barrier protect factors,including ITF, mucin-3, CD73, CD55and platelet activating factor receptor (PAFR), themRNA levels of these factors were analyzed by using RT-PCR method under normoxic,hypoxic and IFN-γ interfence conditions in HT-29.3．To investigate the influence and relationship between IFN-γ and HIF-1, mRNA,protein levels and transcriptional activity were analyzed by using RT-PCR, Western Blot andChromatin Immunoprecipitation.4． PHD inhibitor Dimethyloxaloylglycine (DMOG) was uesd to inhibit HIF-1αO2/PHDs/pVHL degradation signal pathway. PHD1, PHD2, PHD3shRNA interfenceplasmids were constructed by using gene recombination technics and transfected into HT-29cells. subsequently, the mRNA and protein expression levels of PHD1, PHD2, PHD3andHIF-1α were detected respectively by semi-quantification RT-PCR and Western blot and therelationship between HIF-1α and PHDs was further analyzed at the level of proteins.Results1．IFN-γ is involved in the loss of epithelial mucosal barrier function in hypoxia.TER of Hypoxia and IFN-γ group (Hx+I group) is lower than nomoxia group (Nx group)and hypoxia group (Hx group)(p<0.05). From the0h, TER of Nx group changed slightly. ButTER of Hx group and Hx+I group were descending. After48h, TER of Nx group was(47.467±2.696) Ohms, TER of Hx group was (43.867±2.651) Ohms, TER of Hx+I group was(31.414±4.239) Ohms.2．IFN-γ down-regulates HIF-1induced intestinal barrier protect factors (ITF, mucin-3,CD73, CD55) in hypoxia.Hypoxia group mRNA levels of ITF, mucin-3, CD73,CD55and PAFR were riser thanNx group(p<0.05). Hx+I group's mRNA levels of ITF, mucin-3, CD73and CD55were lowerthan Hx group's(p<0.05), but There is no difference between Hx+I group and Hx groupmRNA level of PAFR(p>0.05). 3．IFN-γ decreases the expression of HIF-1α in hypoxia.There is no difference between Hx+I group and Hx group mRNA level ofHIF-1α(p>0.05). Nx group mRNA level of HIF-1α was (1.011±0.124), Hx group was(4.514±0.575), Hx+I group was (4.862±0.334).Hx group protein levels of HIF-1α (4.694±1.373) was riser than Nx group. But Hx+Igroup protein levels of HIF-1α (1.307±0.377) was depressed (p<0.05).4．No effect of IFN-γ on the regulation of regulate the transcriptional activity of HIF-1αas a transcriptional factor.PCR Analysis of Chromatin Immunoprecipitation showed that input of Hx group andHx+I group (1.053±0.074) were not different obviously. There was no band without HIF-1αantibody process. No significant differences were found between Hx group with HIF-1αantibody process (0.732±0.196) and Hx+I group (0.771±0.053)(p>0.05).5．PHD2degradate pathway is important in depression of the expression of HIF-1αcaused by IFN-γ.HIF-1α protein expression (5.495±0.736) was higher after100μg/ml DMOG treated.However, no significant differences of HIF-1α protein expression were found among DMOGtreated and both DMOG and IFN-γ treated (5.789±0.657)(P>0.05).shRNA RNAi expression vectors with report gene eGFP were successfully constructedand transfected into HT-29cells. HIF-1α protein expression level in (siR-PHD2+IFN-γ)group (5.275±0.248) was similar to that of siR-PHD2group (5.156±0.246)(P>0.05).Conclusions1．IFN-γ is involved in loss of intestinal mucosal barrier function in hypoxia.This loss ofintestinal mucosal barrier function is related to the inhition of HIF-1induced intestinal barrierprotect factors (ITF, mucin-3, CD73, CD55) tightly.2．IFN-γ decreases the expression of HIF-1α in hypoxia, due to restraining of HIF-1αtranslation activity or promoting its degradation. IFN-γ doesn't regulate the transcriptionalactivity of HIF-1α as a transcriptional factor in hypoxia.3．DMOG could inhibit the decrease of exprssion of HIF-1α in hypoxia caused by IFN-γ.Silencing the expression of PHD2by RNAi also prohibit this decrease of exprssion of HIF-1α.But Silencing the expression of PHD1and PHD3don't affect the decrease of HIF-1α obviously, implying PHD2degradate pathway is important in depression of the expression ofHIF-1α caused by IFN-γ.