PKCα/ERK/C7ORF41 axis regulates epidermal keratinocyte 1 differentiation through the IKKα nuclear translocation 2

Aberrant differentiation of keratinocytes disrupts the skin barrier and causes a series 2 of skin diseases. However, the molecular basic of keratinocyte differentiation is still 3 poorly understood. In the present study, we examined the expression of C7ORF41 4 using tissue microarrays by immunohistochemistry and found that C7ORF41 is 5 specifically expressed in the basal layers of skin epithelium and its expression is 6 gradually decreased during keratinocytes differentiation. Importantly, we corroborated 7 the pivotal role of C7ORF41 during keratinocyte differentiation by C7ORF41 8 knockdown or overexpression in TPA-induced Hacat keratinocytes. Mechanismly, we 9 first demonstrated that C7ORF41 inhibited keratinocyte differentiation mainly 10 through formatting a complex with IKKα in the cytoplasm, which thus blocked the 11 nuclear translocation of IKKα. Furthermore, we also demonstrated that inhibiting the 12 PKCα/ERK signaling pathway reversed the reduction of C7ORF41 in TPA-induced 13 keratinocytes, indicating that C7ORF41 expression could be regulated by upstream 14 PKCα/ERK signaling pathway during keratinocyte differentiation. Collectively, our 15 study uncovers a novel regulatory network PKCα/ERK/C7ORF41/IKKα during 16 keratinocyte differentiation, which provides potential therapeutic targets for skin 17 diseases. 18 we Our We also investigated the underlying mechanisms of C7ORF41 in keratinocytes differentiation, and found that C7ORF41 could regulate the nuclear translocation of IKKα. Moreover, we demonstrated the downregulation of C7ORF41 was mediated by PKCα/ERK signaling pathway. Taken together, our study identified a PKCα/ERK/C7ORF41/IKKα


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Epidermis, the outermost part of the skin, is a stratified squamous epithelium mainly subsequently migrate into the suprabasal layers and give rise to differentiated cells. 27 Generally, keratinocytes undergo three distinct stages including the spinous, granular 28 and stratum corneum layers in the process of differentiation [4], and express specific 29 differentiation markers at each layer [5]. As cells differentiation into the spinous layer, 30 they begin to express keratin 1 (KRT1) and KRT10 and simultaneously suppress the 31 expression of basal layer marker KRT5 and KRT14 [6,7]. At a more advanced stage, [7]. A finely-tuned balance between proliferation and differentiation must be tightly 38 controlled to maintain epidermal homeostasis. However, several evidences showed 39 that the dysregulation of keratinocytes differentiation is associated with skin diseases, 40 such as psoriasis, atopic dermatitis and squamous cell carcinomas [12][13][14]. Keratinocyte differentiation is a highly coordinated multistep process regulated by 2 intercellular and external signal stimuli. In the epidermis, an extracellular Ca 2+ 3 gradient is present from the basal layer to the cornified layer [15], and Ca 2+ signaling is 4 indeed involved in the keratinocytes terminal differentiation [16]. Studies indicated 5 that keratinocytes continued to proliferate when cultured in medium containing less 6 than 0.05 mM Ca 2+ and were induced to differentiate by elevating the extracellular 7 Ca 2+ concentration [15,17]. Ca 2+ induced PKC activation is implicated in the 8 keratinocyte proliferation and differentiation [2,18,19]. In addition, the activation of 9 PKC by TPA also stimulates keratinocytes differentiation [20,21]. Contrarily, 10 inhibition of the PKC activity with specific inhibitor GF109203X attenuated the 11 keratinocytes differentiation and stimulated proliferation [21,22]. The PKC family 12 contains more than 11 isozymes [23], of which only five different PKC isforms (α, δ, 13 ε, η, ξ) are expressed in epidermal keratinocytes [24,25]. (Classical) cPKCα is Ca 2+ 14 and phorbol ester dependent, while the (novel) nPKCs (PKCδ, ε, η) is Ca 2+ 15 independent; activation of the atypical PKCξ requires neither Ca 2+ nor phorbol ester 16 [18,26]. These specific PKC isoforms showed a characteristic expression pattern and 17 differential regulatory roles in the keratinocytes differentiation [2,21,25,27].

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How the PKC signaling pathway controls the balance between proliferation and 20 differentiation in keratinocytes is largely unknown. Several studies indicated that PKC 21 isoforms regulate keratinocytes differentiation by activation MAPK signaling, which 22 leads to the increase of AP1, SP1 and KLF4 transcription factors in the nucleus 23 [27][28][29]. Interestingly, IKKα, one of the catalytic subunits of the IKK complex, is also 24 a pivotal molecule in balancing growth and differentiation in the epidermis, which 25 does not depend on its kinase activity but requires IKKα nuclear translocation [30][31][32]. 26 Numerous studies indicated that the ablation of IKKα prevented the differentiation of 27 epidermal keratinocytes and enhanced their proliferative potential as well as skin 28 carcinogenesis [32,33]. IKKα expression in the skin has been reported to be elevated 29 after TPA or Ca 2+ treatment [34,35] In the present study, we studied the expression and function of C7ORF41 in epidermal 2 keratinocytes. Our results indicate that C7ORF41 is mainly present in basal layer of 3 epidermis, and its expression is gradually decreased during keratinocytes 4 differentiation. We also investigated the underlying mechanisms of C7ORF41 in 5 keratinocytes differentiation, and found that C7ORF41 could regulate the nuclear 6 translocation of IKKα. Moreover, we demonstrated the downregulation of C7ORF41 7 was mediated by PKCα/ERK signaling pathway. Taken together, our study identified 8 a novel PKCα/ERK/C7ORF41/IKKα regulatory network to mediate the 9 differentiation of epidermis. Alena Biological Technology Co., and were subjected to immunohistochemical 37 staining. Anti-C7ORF41 antibody was used to detect C7ORF41 expression as 38 previously described [43]. Briefly, immunohistochemistry of tissue microarrays was 39 performed as follows: 4 μm-thick sections were deparaffinized, rehydrated and 40 washed twice with PBS for 10, 5 and 5 min, respectively. After incubating with hydrogen peroxide for 10 min and antigen retrieval at 98℃ for 25 min, the slides 1 were held in blocking solution for 30 min at room temperature, and then incubated 2 with anti-C7ORF41 antibody at 4℃ overnight. Subsequently, after washed with PBS, 3 the slides were incubated with HRP-conjugated secondary antibody. The 4 immunohistochemical reaction was visualized with 3,3'-diaminobenzidine (DAB).

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Sections were removed from DAB solution upon confirmation of color development, 6 rinsed with PBS, and then counterstained with haematoxylin.     Master Mix (DBI-2044, Germany) with triplicates on the ABI step one plus real-time 34 PCR system. For each primer set, the Ct value was normalized to that of GAPDH as 35 inner control, which was further normalized to that of control sample. The results of 36 qRT-PCR was measured using the 2 -△△Ct method and presented as relative mRNA 37 level. To prepare total cell lysates, cells were washed with ice-cold PBS for twice and lysed 1 with RIPA buffer containing a complete protease and phosphatase inhibitor cocktail 2 (Roche Diagnostics, Germany) for 30 min on ice. The lysate was then centrifuged at 3 12000 rpm for 15 min at 4℃ and the supernatant was harvested for analysis. Protein 4 concentration was determined using BCA kit (Sigma) according to the manufacturer's 5 instructions. Equal amounts of sample protein (50μg/ well) were loaded and separated 6 by SDS-PAGE gels and transferred onto PVDF membrane (Millipore, USA). After 7 blocking, blots were incubated overnight at 4℃ with various primary antibody.

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Membranes were washed three times for 30 min in TBST followed by incubation with 9 the corresponding HRP-conjugated IgG secondary antibodies for 1 h at room 10 temperature. Membranes were washed and visualized with ECL (Millpore).  The vectors that contains C7ORF41 gene or shRNA targets to C7ORF41 were 31 constructed as described [37,39]. Human IKKα cDNA was cloned into pCAggs-HA 32 plasmid and the deletion mutation were generated by over-lap PCR method to amplify 33 pCAggs-HA-IKKα. All the constructed vectors were identified by DNA sequencing.  Student's t-test was performed to analyse the differences between two groups.

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Statistical differences among more than 2 groups were examined via one-way analysis 39 of variance (ANOVA) with post hoc Dunnett test. P ＜ 0.05 was considered 7 statistically significant. To understand the physiological role of C7ORF41 during keratinocyte differentiation, 5 we first applied TMA technology and immunohistochemistry to assess C7ORF41 6 protein expression in the normal skin tissue using the C7ORF41 antibody. As the 7 result shown in Fig.1A, C7ORF41 staining was not uniformly in the epidermis, but it 8 is primarily localized in the basal cell layer and dramatically reduced in the more 9 differentiated suprabasal layers. To further examine the expression of C7ORF41 in 10 terms of differentiation states, we measured its expression in the cultured Hacat 11 keratinocytes following the high concentrations of calcium for indicated times. We   Keratinocyte differentiation is often associated with cessation of proliferation. In 1 order to explore the role of C7ORF41 in keratinocytes proliferation, we examined the 2 potential of cell proliferation using CCK-8 in TPA-treated Hacat-C7ORF41 or 3 shC7ORF41-1# cells. As shown in Fig.3A-B, ectopic C7ORF41 expression 4 significantly promoted Hacat cells proliferation after TPA treatment; while C7ORF41 5 silencing dramatically inhibited cell growth when treated with TPA. In addition, to 6 determine the effect of C7ORF41 on the cell cycle, flow cytometric analysis was 7 performed. The results showed that the cells in the G0/G1 phase were dramatically 8 increased, while there was a significant decrease in the G2/M phase in 9 shC7ORF41-1# cells after TPA treatment for 48h, as compared with control group. On 10 the contrary, C7ORF41 overexpression increased the cells in the S phase ( Fig.3C-D).

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Taken together, all these data suggest that C7ORF41 regulates the cell cycle, and its  Hacat cells with or without TPA treatment. We found that TPA treatment significantly 30 promote IKKα nuclear translocation (Fig.4C). As expected, overexpression of 31 C7ORF41 dramatically reduced the nuclear IKKα (Fig.4D). To further examine 32 whether C7ORF41 blocked the nuclear translocation of IKKα through their 33 interaction with the nuclear localization sequence (NLS) within the kinase domain of 34 IKKα, we performed a co-IP experiments with indicated IKKα truncations. We 35 observed that C7ORF41 protein is immunoprecipitated by the full length IKKa and its 36 kinase domain, whereas the interaction is abolished when the NLS region of IKKα 37 was deleted (Fig.4E). Altogether, all these data suggested that C7ORF41 could form a 38 complex with IKKα in the cytoplasm to block the IKKα entering into the nucleus 39 through masking its NLS.

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In order to test whether IKKα is involved in C7ORF41-mediated keratinocytes 41 differentiation, we silenced IKKα using siRNA in TPA-induced shC7ORF41-1# cells.

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As shown in Fig.4F, silencing IKKα markedly reduced the keratinocytes differentiation, as evidenced by the expression of differentiation markers. Taken 1 together, these data indicated that C7ORF41 regulates the keratinocytes differentiation 2 through regulating IKKα nuclear translocation.  Interestingly, the expression of differentiation markers was dramatically decreased by 31 PD98059 and SC-514; whereas SP600125 markedly increased KRT10/FLG 32 expression ( Fig.5L-N), which is consistent with previously studies [48,49]. In order 33 to further explore whether the MEK/ERK pathway is also involved in regulating the 34 nuclear translocation of IKKα via C7ORF41, we silenced C7ORF41 using siRNA in

Discussion
Skin is the largest organ of the body and composed of the dermis and epidermis.

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Keratinocytes constitute the stratified epidermis, and these cells gradually 2 differentiate when they moved to the suprabasal layers and give rise to the cornified 3 layer at the surface of the skin. Skin, as the first line to defense the outside pathogen, 4 plays an important barrier role in protecting against harmful microorganisms, 5 chemical insults and retaining body fluids. Therefore, maintaining a balance between 6 keratinocyte differentiation and proliferation is necessary for epidermal homeostasis.  In the present study, we investigated the role of C7ORF41 in the regulation of 14 keratinocyte differentiation. We found C7ORF41 expression mainly localized in the 15 basal layer and its expression gradually decreased in the supabasal layer (Fig.1A). 16 Besides, we also found the expression of C7ORF41 significantly increased in 17 hyperproliferative skin squamous cell carcinoma (SCC) (supplementary 1E). Our 18 results showed that C7ORF41 overexpression inhibited the expression of 19 differentiation markers and promoted the proliferation of keratinocytes, while 20 C7ORF41 knockdown showed the opposite effects (Fig.2, Fig.3). These results 21 suggest that C7ORF41 plays an essential role for basal keratinocytes to keep in the 22 undifferentiated state and its reduction evokes differentiation to suprabasal layers.

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However, the precise mechanisms of C7ORF41 on keratinocyte function in the 24 epidermis have not been fully elucidated. Numerous studies reported that IKKα is a 25 critical regulator for keratinocyte differentiation as well as proliferation, which 26 requires IKKα nuclear translocation but does not depend on its kinase activity [30,31].

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Our previous studies confirmed that C7ORF41 could interact with IKKα/β in the 28 cytoplasm and regulate the activity of NF-κB [39,40]. In this study, we found that the  Fig.1H). We think the downregulation of 43 C7ORF41 in keratinocytes reduced the interaction with IKKα/β complex in the cytoplasm and the released IKKα/β complex exposed the kinase domain, which 1 caused the nuclear translocation of IKKα and the activation of IKKβ. Altogether, all 2 these factors promoted the keratinocyte differentiation.

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PKC isforms are also involved in keratinocyte proliferation and differentiation, and 4 individual isoforms have differential effects, even opposite in action to others [21,25].

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In this study, we found that silencing PKCα partially rescued the downregulation of All supporting data are included within the main article and its supplementary files.