The Mechanism Of Protein Kinase D2 Contributions To The Bleomycin-induced Localized Scleroderma In Mice

BackgroundScleroderma is a autoimmune disease characterized by micro-vascular damage and fibrosis of tissue and organ,it is mainly divided into systemic scleroderma and localized scleroderma(LS).LS is manifested as skin thickening limited to the areas distal to the elbows and knees with less internal organ involvement.And,several lines of evidence indicate that ?-SMA,collagen,inflammatory factors,activation of the immune system and vasculopathy have been shown to play pivotal roles in the pathogenesis of LS.In addition,inflammation causes severe fibrosis can be activate by activation of the signaling pathway(eg.NF-kB and MAPK signaling pathway).Protein kinase D(PKD)is a serine/threonine kinase and diacylglycerol(DAG)receptor protein family,with 3 different isoforms being identified,PKD1,PKD2 and PKD3.In the skin,Mohammad Rashel et al.demonstrated that PKD1 stimulates the reversal of keratinocyte differentiation in culture,suggesting a potential pro-proliferative role in epidermal adaptive responses.While,the role of protein kinase D2 in scleroderma is not clear.ObjectivesProtein kinase D2(PKD2)has been implicated in hyperproliferative skin disorders,however,its function in other skin-related diseases including localized scleroderma,has not been investigated.In this study,we investigated the effect of PKD2 catalytic activity bleomycin-induced localized scleroderma in mice.MethodsHomozygous kinase-dead PKD2 knock-in mice(PKD2SSAA/SSAA-KI mice,the feature of PKD2 catalytic deficient activity)were obtained through intercrossing mice heterozygous for PKD2S707A/S711A(PKD2SSAA).The wild-type and KI male mice were subjected to repeated subcutaneous injection of bleomycin to induce localized scleroderma.As controls,mice were injected with PBS.At the end of the experiment,mouse skin at the injection site was dissected,stained,and analyzed for morphological changes and expression of scleroderma biomarkers.PKD-regulated signaling pathways were examined by real-time RT-qPCR and western blotting.ResultsWe identified mice genotype by PCR,a 236-bp DNA product was amplified from wild-type mice;PKD2SSAA/SSAA-KI mice generated a 344-bp DNA product;PKD2WT/SSAA mice generated 236-bp and 344-bp DNA product.Histopathological analysis of stained the skin sections from the four groups of mice showed that in PKD2WT/WT mice the dermal thickness was increased over 2-fold in response to bleomycin treatment at the bleomycin injection site of PKD2WT/WT mice as compared to the vehicle-treated mice.However,this effect was largely inhibited in the PKD2SSAA/SSAA-KI mice treated with bleomycin.The dermal of mice in PKD2SSAA/SSAA-BLM group were arranged normally and the collagen fiber staining was weaker in the PKD2SSAA/SSAA-KI mice than that in the PKD2WT/WT mice in vehicle-treated control groups,and beomycin-induced collagen fiber expression was almost completely eliminated in PKD2SSAA/SSAA-KI BLM group as compared to the control PKD2WT/WT BLM group,implying a key role of PKD2 in bleomycin-induced collagen fiber expression.The result indicated that the vehicle-treated PKD2SSAA/SSAA-KI mice can significantly reduce the ?-SMA and collagen ?-?2 expression,as compared with the vehicle-treated wild type mice.It is prompted that PKD2 participated in ?-SMA and collagen ?-?2 expression.And,the knock-in of kinase-inactive PKD2 can significantly reduce the mRNA levels of ?-SMA,collagen ?,collagen ?-?1,collagen ?-?2,TGF-?1 and IL-6 in BLM-induced LS in mice.What's more,STAT3 expression and activity were evaluated by western blotting using STAT3 and p-STAT3(Tyr705)antibodies.STAT3 protein expression was not altered in four groups,but its phosphorylation at Tyr705 was significantly decreased in BLM-induced LS in PKD2SSAA/SSAA-KI mice compared to BLM-induced PKD2WT/WT mice.ConclusionProtein Kinase D2 plays a critical role in bleomycin-induced scleroderma in vivo.PKD2SSAA/SSAA-KI mice was resistant to bleomycin-induced localized scleroderma,exhibiting reduced dermis thickness,decreased ?-SMA,collagen expression and production of the involvement of inflammatory factors.