Study On The Regulation And Function Of Oligopeptide/Histidine Transporter PHT2 In Inflammation

The proton-coupled oligopeptide transporter(POT)family mediates the cellular uptake of di/tripeptides and many peptidomimetics utilizing an inwardly directed proton gradient and negative membrane potential.The family consists of 5 mammalian members,PEPT1(SLC15A1),PEPT2(SLC15A2),PHT1(SLC15A4),PHT2(SLC15A3)and SLC15A5.All these members are predicted to contain 12 transmembrane domains with both the N-and C-termini facing the cytosol.The majority of POT isoform characterization studies have focused on PEPT1 and PEPT2,they localized at apical membranes of the small intestine and proximal tubule of kidney,respectively,are responsible for the intestinal absorption and renal reabsorption of their substrates.In contrast,there is scant information on PHT1 and PHT2,which transport histidine in addition to di/tripeptide substrates and drugs.PHT1 is found primarily in brain and eye and PHT2 in lung,spleen and thymus,both are localized in the lysosomal membrane.Inflammation is an adaptive response that is triggered by noxious stimuli and conditions,such as infection and tissue injury,and is considered as a mechanism of innate immunity.The main trigger of inflammation is the recognition of microbes by receptors of the innate immune system.These receptors include the toll-like receptor(TLR)and the nucleotide oligomerization domain(NOD)-like receptor.A number of studies have implicated proton-coupled oligopeptide transporters(POTs)in the initiation and/or progression of inflammatory bowel disease and immune cell signaling.PEPT1 has been shown to mediate the transport of bacterially produced peptides like muramyl dipeptide(MDP)and L-Ala-y-D-Glu-meso-diaminopimelic acid(tri-DAP)which,when recognized by the intracellular pattern recognition receptor nucleotide-binding oligomerization domain 1(NOD1)and 2(NOD2),respectively,can initiate the immune response.PHT1 was found expressed abundantly in immune cells and studies suggest that PHT1 is associated with diabetes,inflammatory bowel disease and systemic lupus erythematosus,and promotes colitis through Toll-like receptor 9 and NOD1-dependent innate immune responses,which imply PHT1 may play an important role in innate immune system.The physiological role and pharmacologic relevance of PHT2,however,has remained largely unknown.PHT2 is predominately expressed in lymphatic system,like spleen,thymus and lung,and its subcellular localization was identified in the lysosomes.Our previous study found that lipopolysaccharide(LPS)can up-regulate the expression of PHT2 without affecting the expression of other transporters of POTs,which suggests the expression and regulation of PHT2 may be associated with inflammation.So,to study the regulation and function of PHT2 in inflammation may help us to understand the physiological role of PHT2 better.What’s more,it may provide more information for the pathogenesis and therapy of inflammatory disease,like inflammatory bowel disease.1.Regulation a-nd related mechanism of PHT2 in acute inflammation induced by lipopolysaccharide(LPS)The aim of this study was to elucidate the molecular expression of PHT2 in macrophages and mouse tissues,and to explore the regulation of PHT2 by lipopolysaccharide(LPS).The results showed relatively high expression of PHT2 in J774A.1 and THP-1 macrophage cells,mouse spleen and lung.Using an LPS-induced inflammatory cell model,we found that hPHT2 mRNA expression was up-regulated in THP-1 cells,and that the up-regulation was suppressed by specific inhibitors of NF-κB and MAPK signaling pathway respectively.Similar results were observed in mouse spleen during LPS-induced acute inflammation.These results suggested that PHT2 was expressed abundantly in macrophages,mice immune organs and lung,and can be up-regulated by LPS via the NF-κB and MAPK signaling pathway.2.Effects of dextran sodium sulfate(DSS)-induced mouse ulcerative colitis on the expression of POTs in colon tissue and its immune cellsA number of studies have implicated proton-coupled oligopeptide transporters(POTs)in the initiation and/or progression of inflammatory bowel disease and immune cell signaling.With this in mind,the objective of this study was to delineate the protein expression of PEPT1/2 and PHT1/2 in mouse colonic tissues and immune cells and characterize the potential role of these transporters,especially PHT2,in intestinal inflammation using a chemically derived colitis model.Initial studies examined the gene and protein expression of POTs using mouse colon,colonic cells and subtypes.We found that PHT1 and PHT2(but not PEPT1 and PEPT2)proteins were expressed in lamina propia mononuclear cells(LPMC)of the colon,and POT protein expression was not observed in colonic CDllb+ macrophages in normal mice.Moreover,PHT1 protein expression was very low in LPMC and down regulated by dextran sodium sulfate(DSS)-induced colitis in proximal and distal colon.In contrast,PHT2 protein was moderately expressed in colon and up regulated by DSS-induced colitis in distal colon and LPMC.PEPT1 protein was not expressed in colonic segments,cells or subtypes even during DSS-induced colitis.Later studies revealed that the immune response of LPMC in mice was not regulated by PHT1 or PHT2,at least when the bacterially derived components MDP,tri-DAP and LPS were presented to the cells exogenously,as opposed to exporting the degradation products of bacteria produced within lysosomes into the cytosol.3.Construction of cell model with stable expression of human oligopeptide/histidine PHT2 and investigation of its substrate propertiesThe aim of this study is to explore the substrate transport function of PHT2,in particular to examine whether the bacterial peptide MDP and tri-DAP are substrates of PHT2.PHT2 is localized in the lysosomal membrane,which causes the substrate research difficult.In order to study the transport function and substrate properties of PHT2,we mutated 19 and 20 leucine to alanine in protein sequence,so PHT2 can be localized to the cell membrane.MDCK cells were transfected with pcDNA3.1(+)-hPHT219,20AA-eGFP to get a stable transfect clone with hPHT2.Using MDCK-hPHT219,20AA cells to examine the POTs-related substrates,we found Gly-sar,valacyclovir,Gly-gly-gly and cefadroxil are substrates of hPHT2,5-ALA and captopril are not hPHT2 substrates.MDP is a substrate of hPHT2 and tri-DAP can be transported by PHT2 weakly The kinetic parameters of d3-L-Histidine,Gly-sar and valacyclovir in MDCK-hPHT219,20AA cells were estimated to be 1667+99,73.6±15,9,63.7±6.3(Vmax,pmol/mg protein/min);73.75+14,428+88,5350+1200(Km,μM);22.6,0.17,0.012(Clim,Vmax/Km).4.The interaction of PHT2 with bacterial muramyl dipeptide(MDP)The aim of this study is to explore the role of PHT2 in MDP-induced NOD2-dependent immune responses.RAW264.7 macrophages were transiently transfected with pcDNA3.I(+)-musPHT2-his tag plasmid to overexpress PHT2 protein.To treat RAW264.7-PHT2 cells or RAW264.7-Vector cells with MDP or LPS,and then examine the mRNA and protein expression of pro-inflammatory cytokine IL-6、TNFαand IL-1β,we found overexpression of PHT2 protein can significantly up-regulate the expression of pro-inflammatory cytokines induced by MDP,but not by LPS,which indicating that PHT2 promotes MDP-induecd inflammatory response through the transpot activity.We also study the effect of MDP on the expression of PHT2.By treating RAW264.7 macrophages with different concentration or different incubation time of MDP,we found Pht2 but not Phtl,was up-regulated significantly.The above results indicate that PHT2 has a role of promoting inflammation,and this effect is most likely to be achieved by its substrate properties of transporting bacterial peptide across the lysosomal membrane.In summary,we have revealed the regulation and related mechanisms of PHT2 in LPS-induced acute inflammation and ulcerative colitis,and demonstrated that PHT2 can promote the development of inflammation by transporting bacterial peptides.These findings may provide new ideas for pathogenesis and treatment of inflammation-related diseases.