Pro‐inflammatory cytokine IL‐6 regulates LMO4 expression in psoriatic keratinocytes via AKT/STAT3 pathway

Abstract The transcription factor LIM‐only protein 4 (LMO4) is overexpressed in the psoriatic epidermis and regulates keratinocyte proliferation and differentiation. High LMO4 expression levels are induced by interleukin‐23 (IL‐23) to activate the AKT/STAT3 signaling pathway. Interleukin‐6 (IL‐6) is mainly involved in regulating T cell functions and development in patients with psoriasis. However, whether LMO4 expression is regulated by IL‐6 remains unclear. Therefore, the purpose of this study is to explore the role and molecular mechanisms of IL‐6 in regulating LMO4 expression. The interleukin‐6 (IL‐6) levels in human plasma were determined using a chemiluminescence immunoassay system. A psoriasis‐like mouse model was established using imiquimod induction. Epidermal keratinocytes (HaCaT) were cultured in defined keratinocyte‐serum‐free medium and stimulated by IL‐6 alone or with inhibitors. The proteins of interest were detected using western blot analysis, immunofluorescence, and immunohistochemistry. The 5‐ethynyl‐2′‐deoxyuridine assay was used to detect cell proliferation. The results revealed that IL‐6 levels were markedly increased in the plasma of patients with psoriasis, compared to healthy control. The high expression of LMO4 was consistent with high levels of IL‐6, p‐AKT, and p‐STAT3 in the lesions of both psoriasis patients and imiquimod‐induced psoriasis‐like mice. IL‐6 activates the AKT/STAT3 signaling pathway, followed by LMO4 high‐expression in HaCaT cells. IL‐6 induces HaCaT proliferation and differentiation via AKT/STAT3 signaling pathway activation. We think that the high expression of LMO4 in psoriatic keratinocytes requires IL‐6 to activate the AKT/STAT3 signaling pathway and leads to epidermal keratinocytes abnormal proliferation and differentiation.

differentiation via AKT/STAT3 signaling pathway activation.We think that the high expression of LMO4 in psoriatic keratinocytes requires IL-6 to activate the AKT/STAT3 signaling pathway and leads to epidermal keratinocytes abnormal proliferation and differentiation.
Therefore, we hypothesized that LMO4 overexpression in the psoriatic epidermis may be caused by IL-6 induction, resulting in psoriatic keratinocyte hyperproliferation and mis-differentiation.We showed that IL-6 levels are significantly increased in the plasma of psoriasis patients versus healthy donors.LMO4 overexpression was consistent with increased IL-6 levels in both human psoriatic lesions and imiquimod-induced psoriasiform dermatitis.IL-6 induces LMO4 expression by activating the AKT/STAT3 signaling pathway to regulate keratinocyte proliferation and differentiation.Thus, the IL-6/AKT/STAT3/LMO4 pathway is a potential therapeutic target for psoriasis treatment.

| Clinical specimens
Skin biopsies and peripheral blood samples were obtained from outpatients at the Affiliated Anhui Provincial Hospital of the Anhui Medical University.Informed consent for the experiments was not required because Chinese laws consider the human tissues and plasma left from surgery or clinical examination discarded materials.The use of pathological specimens and the review of all pertinent patient records were approved by the Ethics Review Board of the Affiliated Anhui Provincial Hospital of the Anhui Medical University.We've acquired psoriatic lesions and surrounding tissues from 6 patients with psoriasis, and foreskin from 4 healthy donors.These specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned.Human plasma was frozen at −80℃ for subsequent experiments.Interleukin-6 (IL-6) levels in plasma of 18 donors and 26 patients with psoriasis were determined using a chemiluminescence immunoassay system (Immunlite 1000; Siemens Healthineers).

| Animals model
A total of 12 BALB/c mice (6-8-week old male) were purchased from the Center for Laboratory Animal Science of Anhui Medical University.The mice were housed at a constant temperature and humidity with a 12-h light/dark cycle and allowed a free diet.Twelve mice were randomly and evenly divided into control and imiquimod groups.A 2 × 2 cm 2 area was shaved on the back of each mouse 48 h before experiment.A daily dose of 50 mg imiquimod cream (5% IMQ; MedShine) or Vaseline (V8230; Solarbio) was used on the back of each mouse for six consecutive days. 36,37On the sixth day, the mice were killed, and the skin of the shaved backs was fixed with 4% paraformaldehyde at least 24 h, dehydrated with a series of graded alcohol, embedded with paraffin, and sectioned using HistoCore AUTOCUT (Lecia Biosystems).All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and the ethical guidelines of Anhui Medical University (NO.20200007).All procedures were performed in accordance with the China Council on Animal Care and Use guidelines.

| Western blot analysis
Total protein was extracted from HaCaT cells using RIPA lysis buffer (Biosharp; Cat#BL504A), and the total concentration of sample proteins were measured using the BCA Protein Assay Kit (Biosharp; Cat#BL521A).The total protein was separated using FuturePAGE (ACE Bio; Cat#ET15420Gel), transferred onto a polyvinylidene fluoride membrane (Millipore; Cat#IPVH00010), and blocked with 5% nonfat milk in PBST.

| Statistical analysis
All experiments set up at least three duplicate wells.The statistical analysis was performed with GraphPad Prism 8 (version 8.3).Values are expressed as mean ± standard error of the mean (SEM).Mann-Whitney U was used in Figure 1A to compare the difference between normal and psoriasis group.Unpaired two-sided Student's t-test was used in Figure 3B to compare the proliferation between control and IL-6 group.A one-way analysis of variance (ANOVA) was used in Figure 4B to compare the proliferation between groups.Statistical significance was set at p < .05.

| Expression profile of IL-6 and LMO4 in clinical samples
A chemiluminescence immunoassay was used to detect plasma levels of IL-6 in healthy donors and patients with psoriasis.IL-6 levels were significantly increased in the plasma of patients with psoriasis versus healthy controls (Figure 1A) (***p < .001).Paraffin-embedded skin specimens from patients with psoriasis and imiquimodinduced psoriasis-like mice were collected to determine the expression profiles of IL-6 in the epidermal tissues.An H&E staining analysis of psoriatic lesions showed classic characteristics including epidermal hyperplasia, parakeratosis, acanthosis, elevated dermal papilla, and inflammatory infiltration.The pathological changes in the psoriasis-like murine skin resembled those in patients with psoriasis (Figure 1B).
To detect the expression profiles of IL-6 and LMO4 in the skin tissues, paraffin-embedded skin specimens were subjected to immunohistochemical analysis.As shown in Figure 1C and 1D, IL-6 was expressed at lower levels in healthy skin and LMO4 was expressed only in the basal layer keratinocytes of the epidermis.Increased IL-6 expression was observed in every layer of the hyperplastic epidermis.LMO4 was highly expressed in the dermis and epidermis, consistent with IL-6 expression in perilesional and psoriatic lesions, and was only expressed in keratinocytes located in the basal layer of the epidermis of healthy skin.
Furthermore, to investigate whether IL-6 induces LMO4 expression in keratinocytes, HaCaT cells were treated with 15 ng/mL IL-6 for 0, 12, 24, and 48 h.A western blot analysis analysis showed that LMO4 expression was induced by IL-6 in a time-dependent manner (Figure 1E).

| IL-6-induced LMO4 expression in keratinocytes requires AKT/STAT3 signaling pathway activation
To investigate whether IL-6-induced LMO4 expression is required for AKT/STAT3 signaling pathway activation within psoriatic lesions, immunohistochemical staining was performed to examine the p-AKT and p-STAT3 levels.The p-Akt and p-Stat3 levels were markedly increased in the human psoriatic epidermis versus the perilesional tissues and normal epidermis (Figure 2A).Consistent with human psoriatic lesions, the AKT/TAT3 signaling pathway was also abnormally activated in the psoriasiform lesions of mice (Figure 2B).
To determine whether IL-6 regulates LMO4 expression via AKT/STAT3 signaling pathway activation in keratinocytes, HaCaT cells were cultured for 0, 12, 24, and 48 h in D-KSFM containing IL-6.Immunoblotting analysis showed a significant increase in p-AKT and p-STAT3 levels in HaCaT cells after exposure to IL-6, consistent with the upregulation of LMO4 expression (Figure 2C).And then HaCaT cells were then treated with the JAK2 inhibitor (10 μM AG490) or AKT inhibitor (10 μM MK-2206) plus IL-6 to block the signaling pathway, and we found that LMO4 expression was markedly decreased, consistent with the inhibition of AKT and STAT3 activities (Figure 2D,E).Furthermore, a substantial decrease in p-STAT3 levels and LMO4 expression were observed after exposure to a STAT3 inhibitor (80 μM NSC 74859) (Figure 2F).

| IL-6-induced LMO4 expression regulates keratinocytes proliferation and differentiation via AKT/STAT3 pathway activation
To understand the roles of IL-6-induced LMO4 highexpression in regulating keratinocyte proliferation and differentiation, HaCaT cells were cultured in D-KSFM for 5 days to maintain the primary keratinocyte state and then treated with IL-6 for 0, 1, 2, and 3 days.Compared with PBS treatment, IL-6-treated HaCaT cells formed tight junctions and presented a cobblestone-like morphology (Figure 3A).EdU incorporation assays were performed to determine the effect of IL-6 on keratinocyte proliferation, the gating strategy showed in Figure S1, the percentage of EdU-positive cells was used for analysis of cell proliferation.The results showed that keratinocyte growth increased after exposure to IL-6 versus PBS (Figure 3B).Furthermore, both immunoblotting and immunofluorescence staining analyses were performed to detect molecular markers of differentiated epidermal cells.Involucrin, keratin 5, and keratin 1 levels were markedly increased in HaCaT cells after exposure to IL-6 consistent with upregulated LMO4 expression (Figure 3C,D).
To further investigate whether keratinocyte proliferation and differentiation is induced by AKT/STAT3 signaling pathway activation, inhibitors were used to block the AKT/STAT3 signaling pathway activated by IL-6.The EdU incorporation assay showed that JAK2, AKT, and STAT3 inhibitors attenuated cell growth compared to IL-6 treatment (Figure 4A,B).As shown in Figure 4C, IL-6-induced involucrin, keratin1, and keratin 5 expressions dramatically decreased in keratinocytes after exposure to JAK2 inhibitors.Notably, both the STAT3 inhibitor (NSC 74859) and the AKT inhibitor (MK-2206) significantly decreased the expression of involucrin, keratin1, and keratin 5 (Figure 4D,E).

| DISCUSSION
LMO4 is a transcription factor that regulates the proliferation and differentiation of various cells such as neurons, retinal cells, and mammary epithelial cells. 13,40,41MO4 interacts with LDB1 to regulate normal epithelial cell differentiation and proliferation. 42,43However, in the mammary epithelium, the dysregulation of LMO4 causes mammary neoplasia by altering cell proliferation, differentiation, and invasion. 42,446][47] We demonstrated that LMO4 overexpression in psoriatic lesions is involved in the abnormal differentiation and hyperplasia of epidermal keratinocytes. 16onsistent with LMO4 expression, IL-23 expression significantly increased in psoriatic lesions.IL-23 is a key cytokine in the pathogenesis of psoriasis and mainly involved in Th17 cell development. 20,48,49However, IL-23induced LMO4 expression plays a critical role in regulating keratinocyte proliferation and differentiation.Furthermore, LMO4 overexpression in psoriatic keratinocytes is required for IL-23 to activate the AKT/STAT3 signaling pathway. 16L-6 is another important member of the cytokine network that regulates keratinocyte proliferation and differentiation in psoriasis. 20,35,50Although there is no correlation between IL-6 level and disease activity, studies have confirmed that patients with psoriasis have significantly increased serum IL-6 levels and overexpression in psoriatic lesions versus healthy controls. 51,52In this study, we found that plasma IL-6 levels were higher in patients with psoriasis than in healthy populations and were abnormally expressed in psoriatic lesions and imiquimod-induced psoriasis-like lesions.Interestingly, IL-6 expression was significantly increased in psoriatic lesions consistent with LMO4 overexpression in the present study.We investigated the association between LMO4 overexpression and elevated levels of IL-6.We found that IL-6 induced LMO4 expression in HaCaT cells in a time-dependent manner.The IHC analysis indicated that the AKT/STAT3 signaling pathway is activated in both human psoriatic lesions and mouse psoriasis-like skin.STAT3, an important regulator of inflammation, is recognized as the main mediator of IL-6 function and is activated in psoriatic skin. 5,53We previously identified that p-STAT3 regulates LMO4 expression by binding to a specific motif in the promotion of LMO4. 16Our in vitro experiments further demonstrated that IL-6 induced LMO4 overexpression in HaCaT cells through the AKT/STAT3 signaling pathway activation.
IL-23-induced LMO4 highexpression plays a critical roles in regulating keratinocyte proliferation and differentiation, here we show that IL-6 has a function similar to IL-23.IL-6-induced LMO4 overexpression accelerated cell proliferation and increased expression of keratin-1, keratin-5, keratin-10, and involucrin in HaCaT cells.Remarkably, both cell growth and differentiation were weakened when the IL-6-activated AKT/STAT3 pathway was inhibited.Thus, high IL-6 levels activate the AKT/ STAT3 signaling pathway to increase LMO4 expression and contribute to psoriatic keratinocyte growth and differentiation.The abnormal proliferation and differentiation of epidermal keratinocytes is an important link in the development of psoriasis.
Together, we found that in addition to IL-23, IL-6 also induces LMO4 overexpression in epidermal keratinocytes.IL-6-activated AKT/STAT3 signaling induces LMO4 expression, leading to keratinocyte hyperproliferation and mis-differentiation.This study provides new insight into the role of LMO4 in the development of psoriasis.

F
I G U R E 1 IL-6 upregulates LMO4 expression in psoriatic keratinocytes.(A) Plasma IL-6 levels in patients with psoriasis versus healthy controls using a chemiluminescence immunoassay system.(B) Hematoxylin and eosin staining of human and mouse skin tissue sections.(C) Histopathological staining of IL-6 in skin sections from healthy controls and psoriatic lesions of human and mouse.(D) Immunohistochemical analysis of LMO4 in human and mouse skin sections.(E) Immunoblotting analysis of LMO4 expression induced by IL-6 (15 ng/mL) in epidermal keratinocytes HaCaT cells.Scale bars = 20 μm.H, human skin tissues from a healthy control versus a patient with psoriasis; M, tissue sections of the imiquimod-induced psoriasis-like dermatitis and normal mouse skin; LMO4, LIM domain only 4; IL-6, interleukin (IL)-6.F I G U R E 2 IL-6-induced LMO4 expression via AKT/STAT3 signaling pathway activation.(A) Immunohistochemical staining of p-AKT and p-STAT3 in human normal epidermis, perilesional tissue, and psoriatic epidermis.(B) Histopathological staining of IL-6, p-AKT, and p-STAT3 in mouse normal epidermis, perilesional tissue, and imiquimode-induced psoriasis-like dermatitis.Scale bars = 20 μM.Detection of LMO4 expression induced by IL-6 or after inhibition AKT and STAT3 using western blot analysis (c-f).(C) The LMO4 expression induced by IL-6 (15 ng/mL) was consistent with the increased p-AKT and p-STAT3 expression in HaCaT cells.(D) LMO4 expression was decreased after inhibition of AKT and STAT activation in HaCaT cells exposed to IL-6 and JAK2 inhibitor (AG490).(E) The AKT inhibitor MK-2206 dramatically reduced p-STAT3, STAT3, and LMO4 expressions.(F) The STAT3 inhibitor NSC 74859 effectively inhibited STAT3 expression and activation.AKT, Protein kinase B; p-AKT, phospho-AKT; STAT3, Signal transducer and activator of transcription 3; p-STAT3, phospho-STAT3; JAK2, Janus kinase 2.