C10orf99 contributes to the development of psoriasis by promoting the proliferation of keratinocytes

Psoriasis is a chronic, relapsing inflammatory skin disease. The pathogenesis of psoriasis is complex and has not been fully understood. C10orf99 was a recently identified human antimicrobial peptide whose mRNA expression is elevated in psoriatic human skin samples. In this study, we investigated the functional roles of C10orf99 in epidermal proliferation under inflammatory condition. We showed that C10orf99 protein was significantly up-regulated in psoriatic skin samples from patients and the ortholog gene expression levels were up-regulated in imiquimod (IMQ)-induced psoriasis-like skin lesions in mice. Using M5-stimulated HaCaT cell line model of inflammation and a combinational approach of knockdown and overexpression of C10orf99, we demonstrated that C10orf99 could promote keratinocyte proliferation by facilitating the G1/S transition, and the pro-proliferation effect of C10orf99 was associated with the activation of the ERK1/2 and NF-κB but not the AKT pathways. Local depletion of C10orf99 by lentiviral vectors expressing C10orf99 shRNA effectively ameliorated IMQ-induced dermatitis. Taken together, these results indicate that C10orf99 plays a contributive role in psoriasis pathogenesis and may serve as a new target for psoriasis treatment.

kinase1/2 (ERK1/2) and NF-κB pathways. Blocking C10orf99 expression ameliorated epidermal hyperplasia, microangiogenesis and the infiltration of inflammatory cells in IMQ-induced psoriasis-like mice. Our results suggested that C10orf99 plays a contributive role in the pathogenesis of psoriasis and may serve as a potential therapeutic target for psoriasis.

Results
Expression of C10orf99 is elevated in psoriatic lesions. We first examined the expression of C10orf99 at protein level in skin samples obtained from psoriasis patients (n = 20) and healthy donors (n = 20) using immunohistochemical analysis. Staining of C10orf99 was mostly observed in cytoplasm (Fig. 1a). C10orf99 was mainly expressed in the basal layer of the epidermis in the normal skin; however, in psoriasis skin, C10orf99 was over-expressed throughout the thickened epidermis. Semi-quantitative analysis of the immunohistochemistry results indicated that the expression of C10orf99 is remarkably elevated in psoriatic skins compared to the normal controls (Table 1 and Fig. 1b). This observation was further confirmed by comparative western blot analysis of skin samples from 4 psoriasis patients and 4 healthy donors (Fig. 1c). In addition, we analyzed the expression of the mouse homolog of C10orf99, termed 2610528A11Rik, in the IMQ-induced mouse model 19 . Consistently, the expression of 2610528A11Rik was also significantly increased at the mRNA level in the skin lesions from IMQ-treated mice (Fig. 1d). Taken together, these data demonstrate an overexpression of C10orf99 in the psoriatic skin lesions. Enhanced keratinocyte proliferation is an important feature of psoriasis. Since C10orf99 regulates cell proliferation in cancer cells 13 and is significantly overexpressed in psoriatic skins, we determined whether C10orf99 may contribute to the development of psoriasis by regulating keratinocyte proliferation. We first tested its role in a well-established cell culture model of inflammation that recapitulates some features of psoriasis by stimulating HaCaT cells with a cocktail of cytokines M5 (including TNF-α, IL-17A, IL-22, IL-1α, and Oncostatin-M, 10 ng/ ml) 20,21 . As shown previously, expressions of TNF-α, IL-6, IL-8 and h-BD2 were markedly increased in HaCaT cells after 24 h treatment of M5 (Fig. 2a) 20 . More importantly, both western blot and qRT-PCR analysis showed that C10orf99 expression was greatly elevated in M5-stimulated HaCaT cells (Fig. 2a). Thus, HaCaT cells stimulated with M5 could serve as a cell culture model to investigate the role of C10orf99 in the proliferation of keratinocytes.
We then used two independent C10orf99-specific siRNAs to transiently decrease C10orf99 expression in M5-stimulated HaCaT cells. Western blot and qRT-PCR results confirmed the effective down-regulation of C10orf99 by both siRNAs (Fig. 2b). In the absence of the inflammatory cytokine cocktail M5, depletion of C10orf99 had none (siRNA-1) or very weak inhibitory effect (siRNA-2) on HaCaT cell growth (Supplementary Figure S5), presumably due to a very low basal expression level of C10orf99. Previous studies have shown that M5 treatment accelerates the growth of HaCaT cells by around 1.5 fold 22,23 . Notably, C10orf99 knockdown significantly reduced the growth rate of M5-stimulated HaCaT cells as demonstrated by the MTT assays (Fig. 2c). Cell cycle analysis by PI staining further revealed that C10orf99 knockdown led to a G1/S growth arrest accompanied by reduced expressions of G1-S progression regulators such as Cyclin D1 and Cyclin A (Fig. 2d,e). Thus, C10orf99 depletion inhibits the growth of keratinocytes under inflammatory conditions. C10orf99 downregulation has no effect on keratinocyte apoptosis. We also assessed the effect of C10orf99 downregulation on keratinocyte apoptosis by Annexin V/PI staining. However, no significant difference in apoptosis rate was observed between C10orf99-depleted and control cells, demonstrating that C10orf99 knockdown did not affect HaCaT cell apoptosis, ruling out a possibility that the reduced growth rate in C10orf99 knockdowned HaCaT cells was caused by increased apoptosis (Supplementary Figure S1).

Overexpression of C10orf99 promotes the proliferation of HaCaT cells.
To further confirm the role of C10orf99 in the regulation of KC proliferation, we over-expressed C10orf99 via a lentiviral vector in HaCaT cells without M5 treatment, which express low basal level of C10orf99. Lentivirus-mediated expression of exogenous C10orf99 was verified by qRT-PCR and western blot analysis (Fig. 3a,b). MTT assay showed that overexpression of C10orf99 increased the proliferation of HaCaT cells as compared with the blank or the empty virus control (Fig. 3c). PI cell cycle analysis revealed that overexpression of C10orf99 resulted in an increased S phase population with a concomitant decrease in the G1/G0 phase population (Fig. 3d). As expected, the expressions of the G1/S progression regulators, Cyclin D1 and Cyclin A were increased in C10orf99 over-expressed HaCaT cells versus the control cells (Fig. 3e). Therefore, C10orf99 overexpression promotes the proliferation of HaCaT cells by facilitating the G1/S progression.
C10orf99 activates the ERK1/2 and the NF-κB signaling. We initially evaluated phospho-p65, phosphor-ERK1/2 and phosphor-AKT expressions in M5-treated HaCaT cells by western blot. We observed that all these pathways were prominently activated after incubation with M5 for 15 to 30 min (Fig. 4a). To study the molecular mechanisms underlying the pro-proliferation function of C10orf99, we used C10orf99-specific siRNAs to decrease the expression of C10orf99 followed by treated with M5 for 30 min and analyzed several key proteins involved in the signaling pathways. The data demonstrated that knockdown of C10orf99 suppressed the activity of ERK1/2 and NF-κB signaling, as evidenced by the reduction of the levels of p-ERK1/2 and p-p65, but not the AKT pathway (Fig. 4b). Conversely, over-expression of C10orf99 increased the levels of p-ERK1/2 and p-p65 (Fig. 4c). Therefore, the pro-proliferation activity of C10orf99 is associated with the activation of the ERK1/2 and NF-κB pathways.

C10orf99 knockdown ameliorates imiquimod-induced dermatitis. To determine whether C10orf99
participates in the pathogenesis of psoriasis by promoting keratinocyte proliferation, we locally knocked down C10orf99 expression in mouse back skin by injecting lentiviral particles carrying the C10orf99 shRNA and examined the impact of C10orf99 depletion on the development of IMQ-induced psoriasis 24,25 . The knockdown efficiencies of three shRNAs were first verified in a mouse cell line C2C12 which expresses endogenous C10orf99. Out of three shRNAs, shRNA-1 showed the best knockdown efficiency and was therefore chosen for the in vivo experiment (Supplementary Figure S2). The in vivo knockdown efficiency of shRNA-1 was confirmed by qRT-PCR analysis on skin tissues from the virus-injected area (Fig. 5a). IMQ treatment induced sharply demarcated erythematous plaques covered with white silvery scale on mouse back. However, C10orf99 knockdown led to a significant decrease in plaque formation (Fig. 5b). Histological analysis of the skin sections revealed that  Table 1. Expression of C10orf99 in Normal skin and Psoriasis. Score 0, negative; score 1-4, weakly positive; score 5-8, moderately positive; score 9-12, strong positive. **P < 0.01, compared to normal skin.
the epidermal hyperplasia was ameliorated, the epidermal thickness was decreased and microangiogenesis and the infiltration of inflammatory cells were reduced in C10orf99-depleted mice compared to the control mice ( Fig. 5c,d). Prominent increases in p-p65, p-ERK1/2, p-AKT, Cyclin A and Cyclin D1 levels were observed after IMQ applied for 7 consecutive days (Fig. 5e). In addition, we found that C10orf99 knockdown down-regulated the expression of Cyclin D1 and the angiogenesis regulator VEGF (Fig. 5f). Moreover, consistent with the results in vitro, knock-down of C10orf99 by shRNA decreased the activity of ERK1/2 and NF-κB signaling, as demonstrated by downregulation of p-ERK1/2 and p-p65 (Fig. 5f). These data further supported that C10orf99 plays an important role in the development of psoriasis.

Discussion
C10orf99 was recently identified as a new human AMP 12 . Its gene is located on chromosome 10q23.1 and encodes a short basic peptide of 57 amino acid residues 12 . It has been found that C10orf99 mRNA has distinct tissue-specific expression patterns: highly expressed in colon, moderately expressed in tonsil and almost undetectable in other tissues 13 . Like other AMPs, such as LL-37 and h-BD2, C10orf99 also exhibits potent wide-spectrum antimicrobial activity against bacteria, fungi and viruses 12 . A study of large-scale mouse knockout library screen implicated C10orf99 in the immune regulation, for C10orf99 knockout mouse exhibits a decreased serum IgM level and an increased ratio of CD4+/CD8+ cells 26 . Similar to other AMPs, in situ gel-forming AP-57 peptide promotes cutaneous wound healing [27][28][29][30] . Recently, C10orf99 was speculated to participate in the pathogenesis of psoriasis without the support of any experimental data 15 . Therefore, it is of great interest to investigate the function of C10orf99 in the development of psoriasis.
In this work, we demonstrated that the expressions of C10orf99 and its ortholog were significantly up-regulated in the skin of psoriasis patients and IMQ-induced psoriatic mouse models respectively. We also showed that C10orf99 positively regulates keratinocyte proliferation. Paradoxically, C10orf99 was previously found to be growth-inhibitory in colon cancer cells 13 . The proliferation of keratinocyte is a complex process regulated by a variety of intracellular/extracellular agents including growth factors, neuropeptides, interleukins and inflammatory mediators, many of which may play versatile roles in different cellular/environmental contexts. It is not unusual that a protein performs opposite functions in the proliferation of two different epithelial cell lines. For instance, studies have shown that the S100A8/S100A9 complex, which has a broad range of intracellular and extracellular functions, suppresses keratinocyte proliferation, but promote the growth and metastasis of colorectal cancer cells 31,32 . How can the contradictory role of C10orf99 on cell proliferation be explained? The previous paper claimed that C10orf99 could inhibit colon cancer cell growth via interacting with SUSD2 13 . However, a transcriptome analysis demonstrated that SUSD2 was significantly down-regulated in psoriatic lesions 18 . In line with this, we observed that the expression of SUSD2 mRNA also decreased in M5-treated HaCaT cells (Supplementary Figure S3). Thus, it's likely that C10orf99 regulates keratinocyte proliferation through a distinct mechanism independent of the SUSD2 pathway. Furthermore, knockdown of C10orf99 in the colon cancer cell line DLD1 does not alter the ERK1/2 pathway activity (Supplementary Figure S4). Interestingly, recently studies found that C10orf99 is a natural ligand for the orphan receptor GPR15 33,34 . We speculated that, like other AMPs and chemokines, C10orf99 may function through different receptor pathways in different cellular contexts and exert versatile impacts on the proliferation of cells.
AKT, ERK1/2 and NF-κB signaling pathways play important roles in many biological functions. Studies have shown that the expression of all these protein kinases were significantly elevated in psoriatic skin and they play critical roles in the pathgenesis of psoriasis [35][36][37][38][39] . Our results in vitro showed that M5 greatly activated the AKT, ERK1/2 and NF-κB signaling pathways. C10orf99 signaling was previously speculated to involve the AKT pathway. Interestingly, our data showed that knockdown of C10orf99 decreased the level of p-ERK1/2, but not p-AKT, in the cell culture model of psoriasis. In addition, C10orf99 knockdown resulted in a significant reduction of p-p65, indicative of the down-regulation of the NF-κB pathway activity. Conversely, over-expression of C10orf99 promoted the activity of ERK pathway and NF-κB pathway. Thus, C10orf99 likely regulates the proliferation of keratinocyte by activating the ERK1/2 and the NF-κB signaling.
The regulatory role of C10orf99 in keratinocyte proliferation in vitro prompted us to assess its functionality in vivo. The IMQ-induced psoriasis-like mouse model recapitulates many features of human psoriasis and has been widely used to study the disease pathology. In the IMQ-treated mice, C10orf99 knockdown attenuated epidermal hyperproliferation, infiltration of inflammatory cells and microangiogenesis, which was accompanied by the decreased expression of VEGF. C10orf99 knockdown also decreased the expression of Cyclin D1, p-ERK1/2 and p-p65. However, the knockdown of C10orf99 had marginal effects on Cyclin A, which likely due to different cellular environments between in vitro and in vivo.
In summary, our results show that C10orf99 protein is highly expressed in psoriatic skin and that it regulates keratinocyte proliferation likely through activation of the ERK1/2 and NF-κB pathway. Furthermore, downregulation of C10orf99 ameliorates IMQ-induced psoriasis in mice. Thus, C10orf99 has a contributive role in psoriasis pathogenesis and may be a new target for psoriasis treatment.

Materials and Methods
Human skin tissue collection. A total of 40 paraffin-embedded tissues including 20 psoriasis tissues (11 male and 9 female, age range = 7-50 years) and 20 normal skin tissues (10 male and 10 female, age range = 10-43 years) for IHC were obtained from the tissue bank of the Department of Dermatology at the Second Affiliated Hospital of Xi'an Jiaotong University. In addition, we collected 8 fresh tissue samples including 4 psoriasis samples and 4 normal samples by punch biopsy under local lidocaine anaesthesia for Western blotting. The fresh tissue samples were snap frozen in liquid nitrogen and stored at −80 °C. Written informed consents were obtained from all patients. The study was performed in accordance with the declaration of Helsinki Principles and approved by the Research Ethics Board of the Second Affiliated Hospital of Xi'an Jiaotong University. All of the specimens were pathologically confirmed.
Immunohistochemistry. Immunohistochemistry was performed according to standard methods. The IHC results were scored independently by three experienced pathologists under microscope and quantified based on the following scoring system in a semiquantitative manner as previously reported 40,41 . The rate of positively stained cells was scored as follows: 0 (≤5%), 1 (6-25%), 2 (26-50%), 3 (51-75%), 4 (>75%). The staining intensity was graded as follows: 0 (colorless), 1 (light yellow), 2 (yellowish brown), 3 (chocolate brown). The score for each microscopic field was calculated by multiplying the two scores. The average score of five fields was taken as the final immunoreactivity score.
Histological analysis. The mouse back skin was collected, fixed in formalin and embedded in paraffin.
Sections were stained with haematoxylin and eosin. Epidermal hyperplasia (acanthosis) was quantified by measuring its thickness as described previously 42 . Firstly, Hamamatsu digital pathology system was used to scan the HE-stained sections. Then, the NDP.view software was used to evaluated the epidermis thickness and it measured the distance from the basal lamina to the bottom of the stratum corneum in HE-stained skin sections. Eight randomly-chosen fields of each section were measured and the mean was calculated.  Annexin V/PI staining. Cells were harvested by trypsinization without ethylenediaminetetraacetic acid (EDTA). The cells then were suspended by 500 μl binding buffer and incubated with 5 μl Annexin V-FITC (fluorescein isothiocyanate) solution and 5 μl Propidum Iodidum for 15 min at room temperature in dark. Flow cytometry was used to analyze cell apoptosis.

Mice.
Eight-week-old female BALB/c mice were purchased from the Animal Experiment Center of Xi'an Jiaotong University and they were kept under a 12 h light/dark cycle with specific pathogen-free conditions. All mice were housed at least 1 week prior to the study and provided with food and purified water ad libitum. All procedures involved in mice were performed in compliance with the Animal Care and Use Committee guidelines of Xi'an Jiaotong University School of Medicine.
IMQ-induced mouse model of psoriasis. Mice were anesthetizes with 200 ul 0.6% sodium pentobarbital and their backs were shaved with an electric clipper, then the mice were applied with a daily topical dose of 62.5 mg 5% IMQ cream (Mingxin, Chengdu, China) or Vaseline cream on their shaved back for 7 consecutive days. In the end, all mice were sacrificed. Back skin was isolated and half was fixed in 10% formaldehyde and the other half of the skin sample was finely chopped for RNA isolation and qRT-PCR analysis.
In vivo administration of C10orf99 shRNA lentivirus. Lentiviruses of C10orf99 shRNAs and control shRNAs were from GenePharma (Shanghai,China) and all the oligonucleotides were listed in supplementary  Table S2. shRNA-1 successfully knocked down the C10orf99 mRNA expression level and was used in animal experiment. Lentivirus particles (1.0 × 109 TU, 50 μL) encoding C10orf99 shRNA or negative control shRNA were injected intradermally into the shaved dorsal skin of mice. After three days, the mice started to be treated with IMQ as described above.
Statistical analysis. SPSS standard version 21.0 software (SPSS Inc, Chicago, IL) and GraphPad Prism 5.0 (La Jolla, CA, USA) were used for statistical analysis. Data were presented as mean ± SEM. The Mann-Whitney U test was used for immunohistochemistry analysis and the Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant. Data availability. All data generated or analysed during this study are included in this published article (and its Supplementary Information files).