The Role Of PAR-2in α-Crystallin Protecting Retinal Ganglion Cells From Injury

Background：In mature mammals, injured axons of retinal ganglion cells (RGCs) are unable toregenerate and soon undergo cellular apoptosis and death. How to protect the RGCs andpromote axonal regeneration after optic nerve injury has always been a focus inneuroscience research. Previous work in our laboratory revealed that exogenousα-Crystallin was an important neural protection material from lens, which can promoteretinal ganglion cell survival and axon regeneration after optic nerve injury.Recent study found that the expression of endogenous α-Crystallin in RGCs wassignificantly increased in vitro and in vivo after injury, in addition, over-expression ofendogenous α-Crystallin significantly inhibited hypoxia-induced RGCs apoptosis in vitro.The above research suggests that α-Crystallin as one of heat shock protein family membersis involved in the endogenous neuroprotective effect, but its mechanism is unknown.The protease-activated receptor-2(PAR-2)，the second member of the PAR family,belongs to the G protein-coupled receptor (GPCR) family. The mRNA levels of PARs wereexpressed in developing rat eyes and up-regulated in adult rat retina following optic nervecrush injury. The latest study indicated that α-Crystallin interacted specifically only withPAR-2. Moreover, the PAR-2activation and over-expression of α-Crystallin reducedC2-ceramide-and staurosporine-induced cell death in astrocytes.Whether there exist co-expression and interaction between α-Crystallin and PAR-2inRGCs? Whether α-Crystallin can adjust the PAR-2activity, which starts the signaltransduction pathways to inhibit RGCs apoptosis? So far, the above issues have not beenreported.Objective:This study is to reveal the expression and function of PAR-2in RGCs, and to makesure the interaction between α-Crystallin and PAR-2. It will further investigate themolecular mechanism how α-Crystallin protects RGCs against injury through up-regulating the activity of PAR-2. As a result, it will establish the theoretical foundation for exploringthe new neuroprotective strategies for RGCs.Methods:PartⅡ1. The expression and location of PAR-2in RGCs were investigated by usingimmunohistochemistry, Q-PCR and Western blot methods in vivo (hypoxic injury) and invitro (optic nerve injury).2. PAR-2agonist-SLIGRL and antagonist–ENMD-1068were used to change PAR-2activation in our study. Whether PAR-2activation has a protective effect against thehypoxia-induced apoptosis of RGC-5cells was investigated by Cell Counting Kit-8assay,Hoechst33342staining and AnnexinV-FITC/PI assays, respectively. In addition, theexpression of the Bcl-2, Bax and the active subunit of Caspase-3were also analyzed bywestern blot method in order to explore the possible apoptosis way.PartⅢ1. The expression, location and interaction between α-Crystallin and PAR-2in retinawere investigated by using immunohistochemistry and co-immunoprecipitation methods in1,3,7,14and28days after optic nerve injury.2. The expression and interaction between α-Crystallin and PAR-2in RGC-5cellswere investigated by using immunocytochemistry and co-immunoprecipitation methods in6,12,24and48h after RGC-5cells hypoxic injury.PartⅣ1. Based on the known interaction of amino acid sequence between α-Crystallin andPAR-2, recombinant Ad-crystallinα vector without the120~154amino acids wasconstructed. The combining ability of mutant type α-Crystallin with PAR-2was detectedusing co-immunoprecipitation.2. RGC-5cells were cultured and infected by wild type and mutant type Ad-crystallinα.Apoptosis was evaluated using Hoechst33342staining and AnnexinV-FITC/PI assays,respectively. Calcium probe-rhod-2/AM was loaded into the cells to detect the intracellularCa2+transient by measuring the fluorescence intensity using confocal microscopy betweenwild type and mutant type Ad-crystallinα transfection groups. Results:PartⅡ1. In normal retina, PAR-2was distributed in inner nuclear layer, inner plexiform layerand retinal ganglion cells layer. After optic nerve injury, the expression of PAR-2wasincreased in the retina, which located mainly in the RGCs layers, inner plexiform layers,inner nuclear layers and outer plexiform layers. PAR-2was expressed in RGC-5cells andup-regulated at both mRNA and protein levels under hypoxic stress.2. CCK-8，Hochest33342staining and AnnexinV-FITC assays consistently confirmedthat PAR-2activation can significantly increase RGC-5cells survival and retard RGC-5cells apoptosis under hypoxia injury.3. The results of western blot showed PAR-2had a protective effect against thehypoxia-induced apoptosis of RGC-5cells by up-regulating the ratio of Bcl-2/Bax anddown-regulating the activation of Caspase-3.Part Ⅲ1. In normal retina, PAR-2was distributed in inner nuclear layer, inner plexiform layerand retinal ganglion cells layer, while a weak expression of α-Crystallin was found only inouter plexiform layer and retinal ganglion cells layer. After optic nerve injury, theexpressions of α-Crystallin and PAR-2were increased in the retina，which located mainly inthe RGCs layers, inner plexiform layers, inner nuclear layers and outer plexiform layers.Results of co-immunoprecipitation showed that there existed co-expression and interactionbetween α-Crystallin and PAR-2in the retina after injury.2. Positive staining of PAR-2was observed while negative staining of endogenousα-Crystallin was found in RGC-5cells under normaxia condition. After hypoxia injury, theexpressions of α-Crystallin and PAR-2were enhanced in the RGC-5cells. Results ofco-immunoprecipitation showed that there existed co-expression and interaction betweenα-Crystallin and PAR-2in the RGC-5cells under hypoxia condition.Part Ⅳ1. The successfully constructed mutant type recombinant Ad-crystallinα without120~154amino acids was identified with RT-PCR and gene sequence analysis.2. The constructed mutant α-Crystallin recombinant adenovirus and wild-typeα-Crystallin recombinant adenovirus were transfected to the target cells of RGC-5 respectively. Western Blot results showed the over-expression of both in the RGC-5.Mutant type α-Crystallin was further comfirmed to lose the ability to combine with PAR-2using co-immunoprecipitation.3. The wild type and mutant type Ad-crystallinα were transfected into RGC-5cells.Hochest33342and Annexin V-FITC assays consistently confirmed that over-expression ofwild type α-Crystallin could significantly retard hypoxia-induced RGC-5cell apoptosiscompared to mutant type Ad-crystallin. At the same time, Rhod-2/AM staining resultsshowed that calcium transient amplitude value significantly increased in wild typeAd-crystallinα transfection group, compared to mutant type Ad-crystallinα transfectiongroup in hypoxia-injury RGC-5cells. Two groups have statistically significant differences(P<0.01).Conclusions:1. PAR-2was expressed in RGCs and up-regulated after injury in vitro and in vivo.PAR-2activation has a protective effect against the hypoxia-induced apoptosis of RGC-5cells by up-regulating the ratio of Bcl-2/Bax and down-regulating the activation ofCaspase-3.2. There had been co-expression and interaction between endogenous α-Crystallin andPAR-2in RGCs after injury in vitro and in vivo.3. α-Crystallin recombinant adenovirus without the120~154amino acids wassuccessfully constructed.4. It confirmed that α-Crystallin could enhance PAR-2activity, which rescued RGC-5cells from hypoxia-induced apoptosis. It preliminarily disclosed the molecular mechanismof α-Crystallin protected against RGCs apoptosis.