Role Of Hedgehog Signaling Pathway In Psoriasis

Background:Psoriasis is a common chronic inflammatory skin disease, which affects□125million people worldwide, characterized by hyperproliferation of keratinocytes and prominent immune infiltrates with complex etiopathogenesis. The hyperproliferation of epidermal keratinocytes is driven by cytokines secreted by activated resident immune cells, as well as the keratinocytes themselves.Hedgehog (Hh) signaling not only plays crucial roles during embryonic development and adult homeostasis, but abnormal activation of the Hh pathway has also been implicated in the pathogenesis of various diseases in adults, including a variety of different malignancies such as basal cell carcinoma (BCC), pancreatic carcinoma, retinoblastoma, and medulloblastoma.During physiologic Hh signaling, Hh ligands bind to the cell surface receptor Patched1(PTCH1), thereby releasing Smoothened (SMO) from PTCH-mediated inhibition. SMO activation then triggers a series of intracellular events, culminating in alterations in gene expression mediated by the GLI family of transcription factors (GLI1, GLI2, and GLI3;). In the absence of Hh ligands, PTCH1represses SMO activity, and the GLI transcription factors (GLI2and GLI3) are proteolytically cleaved into repressors within the primary cilium. The cleavage requires the activities of suppressor of fused (Sufu) and kinesin family member7(Kif7). Three different Hh homologues, Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh), were characterized in mammals. Shh is the predominant Hh ligand in the skin. In skin, Shh is involved in the growth of hair follicles and Ihh has been reported to regulate growth of sebocytes.Previous reports have suggested that the Hh pathway is activated in lesional psoriatic skin, and that pharmacological inhibition of this pathway using an inhibitor of Hh signaling, cyclopamine, may lead to rapid resolution of the disease. In contrast, Gudjonsson et al. demonstrated that there was absence of elevated Hh target genes expression in lesional psoriatic skin. Taken together, these data raises questions regarding the suggestion that whether Hh pathway is activated in the pathogenesis of psoriasis and whether Hh pathway can be manipulated as therapeutic antipsoriatic methods.Objective:To investigate Hh pathway activity in psoriasis, the functional changes of cultured human primary keratinocytes after stimulation or inhibition of Hh pathway, and the in vivo amelioration of psoriatic lesions after intraperitoneal cyclopamine treatment.Method:In the first part:One1-cm surgical biopsy of untreated lesional skin from the arm or back was taken in all patients. Biopsies were also taken from age-matched healthy volunteers, who served as controls. Then epidermis was peeled off from one part of the skin. Total RNA and protein was extracted from the epidermis, the whole skin, and also the cultured primary epidermal keratinocytes. The mRNA and protein level of Hh pathway molecules was determined by reverse transcript polymerase chain reaction (RT-PCR) and Western-blot, separately. We further localized these transcript factors by immunofluorescence (IF) staining in the epidermis of surgical skin specimens. The Hh pathway ligands:Shh, Ihh and Dhh in the serum of healthy and psoriasis patients were determined by enzyme linked immunosorbent assay (ELISA).In the second part:The effect of activation and inhibition of Hh pathway on proliferation, apoptosis, cell cycle arrest, senescence, generation of ROS, migration and KC differentiation was observed by treatment of recombinant Sonic Hedgehog and cyclopamine. The mRNA, protein and subcellular localization of the Hh pathway transcript factors GLIs was observed after overexpression of Sufu in cultured keratinocytes by lentivirus infection. In addition, protein levels related to keratinocytes differentiation were determined after Sufu overexpression.In the third part:C57BL/6mice were treated with5%Imiquimod to induce psoriasis model, and then treated with2dosages of intraperitoneal injection of cyclopamine. The inhibition of cyclopamine to psoriasis was observed by PASI index and H&E staining.Results:I Activation of Hh pathway in psoriasis at transcriptional level:1. The mRNA level of GLI3was significantly lower in psoriatic lesional epidermis than in healthy controls, while Shh, SMO, Sufu and GLI1were not significantly different;2. The mRNA level of Sufu was significantly lower in psoriatic lesional whole skin than in healthy controls, while Shh, SMO, GLI1and GLI3were not significantly different;3. The mRNA level of GLI3was significantly lower in cultured psoriatic lesional keratinocytes than in healthy controls, while Shh, SMO, Sufu and GLI1were not significantly different.II Activation of Hh signaling in psoriasis at protein levels:1. The Shh, Ihh and Dhh levels in the serum of psoriasis patients were significantly higher than in healthy controls;2. The protein level of GLI3, Sufu, PTCH1and PTCH2in cultured psoriatic lesional keratinocytes was significantly lower than in healthy controls, GLI2was slightly elevated without significant difference, while Shh and GLI1were not significantly different;3. In both normal and psoriatic skin, GLI1, GLI2, GLI3and Sufu strongly labeled the whole epidermis except for the stratum corneum by immunofluorescence. The subcellular immunostaining of a transcriptional repressor, GLI3, appeared to be very different between normal epidermis and psoriatic epidermis. In normal skin, intracellular GLI3was localized mostly in the cellular nuclei of epidermal keratinocytes, and signals for nuclear fluorescence were stronger than for cytoplasmic fluorescence. Interestingly, in psoriatic skin, nuclear fluorescence for GLI3was lower than cytoplasmic fluorescence. III Stimulation with recombinant human Shh alters the phenotype of keratinocytes:1. Recombinant human Shh with various concentrations (0-1000ng/mL) for24h induced the transcription of the Hh target gene GLI1in a dose dependent manner, while the changes of GLI3and Sufu were not significant;2. Shh increased the proliferation of keratinocytes in a dose-dependant manner;3. Shh increased the ROS generation of keratinocytes.IV Stimulation with cyclopamine alters the phenotype of keratinocytes:1. Cyclopamine inhibited the proliferation of keratinocytes in a dose-dependant manner;2. Cyclopamine induced the apoptosis of keratinocytes in a dose-dependent manner, while Shh significantly inhibited keratinocytes apoptosis;3. Cyclopamine induced cell cycle arrest at G1phase of keratinocytes, while TNF-a can reverse this effect;4. Psoriatic keratinocytes have a less senescent phenotype in comparison to healthy controls. Cyclopamine significantly induced cell senescence, clobetasol17-propionate significantly decreased cell senescence, TNF-a had no significant effect, while cyclopamine can significantly reverse the decreased senescence by clobetasol17-propionate;5. Cyclopamine significantly decreased ROS generation of cultured human primary keratinocytes, clobetasol17-propionate also significantly decreased ROS generation, TNF-a significantly increased ROS generation, while cyclopamine significantly inhibited the ROS increase by TNF-a;6. Cyclopamine significantly decreased migration of cultured human primary keratinocytes, Shh significantly increased migration, clobetasol17-propionate significantly decreased migration, TNF-a had no significant effect.V Overexpression of Sufu alters the phenotype of keratinocytes:1. Sufu was confirmed to be overexpressed both at mRNA and protein levels in cultured human primary keratinocytes;2. The protein level of GLI1and GLI2was decreased after Sufu overexpression in cultured human primary keratinocytes;3. The level of proteins related to keratinocytes differentiation was increased after Sufu overexpression in cultured human primary keratinocytes;4. GLI3transloated from cytoplasm to nucleus after Sufu overexpression in cultured primary psoriatic keratinocytes.VI Cyclopamine ameliorated the psoriasis-like skin lesions in an IMQ-induced mouse model:1. Cyclopamine decreased PASI index of the psoriasis-like skin lesions in an IMQ-induced mouse model;2. Cyclopamine ameliorated the psoriasis-like skin lesions in an IMQ-induced mouse model by H&E staining. Conclusion:Ⅰ. Hh pathway is activated in psoriasis both at mRNA and protein level;Ⅱ. Shh can increase the proliferation, inhibit the apoptosis, induce ROS generation and increase migration of keratinocytes;Ⅲ. Cyclopamine can inhibit the proliferation of keratinocytes, and this may due to apoptosis and cell cycle arrest; Cyclopamine can induce cell senescence, inhibit ROS generation and inhibit migration of keratinocytes;Ⅳ. Sufu overexpression can inhibit the activation of Hh pathway, and then increase the differentiation of psoriatic keratinocytes;Ⅴ. Cyclopamine can ameliorate the psoriasis-like skin lesions in an IMQ-induced mouse model.