Functional Expression Of POTs In Prostate,Spleen And Macrophages

The proton-coupled oligopeptide transporters (POTs) mediate transport of short chain peptide and peptidemimetic into various cells utilizing the proton motion. POTs are encoded by SLC15A gene and are consisted by four members including PepTl (SLC15A1), PepT2(SLC15A2), PHT1(SLC15A4) and PHT2(SLC15A3). PepTl and PepT2are proton-coupled transporters for uptake of di/tri-peptide into cell. With low affinity and high capacity characteristic, PepT1is mainly distributed in intestine and mediated the absorption of di/tri-peptide from dietary digestion and peptide mimetic drugs, such as β-lactam antibiotics and angiotensin-convertion enzyme inhibitors. Different with PepTl, peptide transporter PepT2has high affinity and low capacity, mainly distributed in kidney, lung and choroid plexus. PHT1and PHT2were first cloned from brain and lymphatic system respectively. PHT1is primary expressed at brain and retina and play roles in the absorption of degradation product of neuropeptide and nerves mediator. PHT2has strong expression in spleen, lung and thymus gland, which indicated the important role in immune system. PHT1/2also mediates the transport of histidine in addition to di/tri-peptide. Physiological function and regulation of PHT1/2are still not well known. PepT2is very promising to be target of peptidemimetic drugs for its specific tissue distribution and high substrate affinity. We focused on the expression and regulation of POTs in the prostate in physiology and pathology. Moreover, function of PepT2and PHT2in spleen and macrophages and the role of PepT2in NODI and NOD2signal pathways were also clarified.1. Expression study of POTs in the prostatePepT1, PepT2, PHT1and PHT2expression in mouse prostate was studied with Western Blot and Real-time PCR methods. The results showed high mRNA expression of PepT2and PHT1in prostate while only PepT2protein immunity band has been detected. Immunohistochemistry study showed localization of PepT2mainly at the apical side of prostate epithelial cell. Expression of PepT2in the prostate of mice with different age was studied to clarify the physiological function of PepT2. The results demonstrated increased mRNA and protein expression of PepT2with prostate development in mice and reached a platform when sex matured (8week age), indicated the role of PepT2in prostate mature. The static analysis of about20samples of human prostate showed significantly lower mRNA and protein expression of PepT2in the prostate cancer (PCa) than benign prostatic hyperplasia (BPH). Lower mRNA expression of PHT1was also found in prostate cancer (PCa) samples, indicating down regulation of PepT2and PHT2by tumor microenvironment.mRNA expression of PepT2, PHT1and PHT2was detected in Human prostate epithelial cell (RWPE-1) and Human prostate cancer cell (PC-3). To demonstrate the effect of inflammation on POTs expression, RWPE-1and PC-3were stimulated by proinflammatory cytokine IFNy (50ng/mL) and TNFa (50ng/mL), and PepT2, PHT1 and PHT2mRNA expression was determined by Real-time PCR. In the RWPE-1cell, PHT2was extremely upregulated by IFNy (50ng/mL) and TNFa (50ng/mL) stimulation (P<0.001), but PepT2mRNA expression was significantly down regulated (P<0.01). While in PC-3cells, IFNr (50ng/mL) and TNFa (50ng/mL) stimulation for48h upregulated the PHT2mRNA expression significantly (P<0.001), but have no effect on mRNA expression of PepT2and PHT1. Basical mRNA expression of PHT2in RWPE-1and PC-3cells is very low, but the upregulation of PHT2mRNA expression by proinflammatory cytokines is so significantly, which indicated PHT2may have an important role in the immune response.2. Expression and function of POTs in mouse spleen and macrophages isolated from spleen.The expression of PepTl, PepT2, PHT1and PHT2in mouse spleen was investigated by Western Blot and Real-time PCR. High expression of PepT2and PHT2was demonstrated in mouse spleen. Lymphocytes and macrophages were isolated from mouse spleen for cell localization of PepT2and PHT2. The results showed that PepT2expressed much higer in macrophages than in lymphocytes while PHT2expressed in both cell types.The uptake of β-Ala-Lys(AMCA) in macrophages isolated from mouse spleen was significantly inhibited by POTs substrates such as carnosine, Gly-Sar and cephalexin, but not histidine which is substrate of PHT1and PHT2. Besides, pH dependent uptake of P-Ala-Lys(AMCA) was found in macrophages, with uptake increasing at more acidic extracellular pH. The uptake kinetic of P-Ala-Lys(AMCA) in macrophages followed Michealis equation with a Km value of75.5±14.3μM and Vmax of25.4±2.1pM/min per mg protein. Besides, uptake of Gly-Sar in macrophages was also inhibited by carnosine (5mM)(P<0.05) but not histidine (5mM). These results indicated that peptide transporter PepT2but not PHT2functioned as transporter of di/tripeptide in spleen macrophages.3. Studies on the role of PepT2in NOD1and NOD2signal pathways.The cell signal receptors NOD1and NOD2play an important role in innate immune system. Bacterial dipeptide r-iE-DAP and MDP are ligands of NOD1and NOD2respectively, when the ligands are recognized by NOD1or NOD2, NF-kB pathway can be activated and inflammatory cytokine will be produced. We studied the role of PepT2in the NOD1and NOD2signal pathway induced by r-iE-DAP and MDP in macrophages isolated from mouse spleen and THP-1macrophages. IL-6and TNFa mRNA expression was extremely induced by y-iE-DAP (lOμg/mL) and MDP (10μg/mL) treatment in mouse spleen macrophages, and the induction was diminished by pretreated with carnosine (5mM) or Gly-Sar (5mM)(P<0.05), but not affected by pretreatment of histidine (5mM). However, the increased IL-6and TNFa secretion in cell culture supernatant by MDP were not diminished by POTs inhibitors, which indicated other ways of MDP to across cell plasma membrane. POTs expression profile in THP-1, J774a.1and RAW264.7is very different. The four members of POTs besides PepTl were all detected in THP-1cell with moderate mRNA expression while only PHT2was detected in J774a.1cell. But for RAW264.7cells, mRNA expression level of all these POTs detected is very low. To further demonstrate the role of PepT2in NOD signal pathways, THP-1cells were used. And increased IL-6and TNFa mRNA was found by y-iE-DAP (l0μg/mL) but not MDP (l0μg/mL) in THP-1cells, and diminished significantly by pretreatment of carnosine (5mM) and histidine (5mM)(IL-6:P<0.01; TNFa:P<0.05). These results indicating PepT2play a role in NOD1signal pathway in THP-1macrophages, and the mechanism of inhibitory effect of histidine on inflammatory need to further studied. In conclusion, our results demonstrated the transporter role of PepT2for ligands in NOD1and NOD2signal pathways.4. Effect of LPS stimulated inflammation on the expression of POTs in macrophages and mouse.Expression of POTs is regulated by many factors, and in this chapter, effect of LPS stimulated inflammation on POTs expression in macrophages and mouse was studied. Extremely upregulation of PHT2was found in both THP-1and J774a.1cell when stimulated by LPS (lOng/mL) for2h (THP-1:P<0.01; J774a.1:P<0.001), but PepT2and PHT1expression was not affected by LPS stimulation. In addition, PHT2expression in spleen and lung tissue of mouse stimulated by LPS was found increased with a3-5fold change compared with control group. These results were very similary to that in prostate cells. Together, these observations indicated that PHT2may play an important role in immune responses. It is quite sure that PepTl and PepT2are plasma membrane transporters, while the localization of PHT1and PHT2is not so widely known but expression of PHT1and PHT2on the lysosomal membranes is generally accepted. The upregulated expression of PHT2in response to inflammatory stimulation may function to the transport of di/tri-peptide and histidine from lysosome into cytoplasm.