Lujun Yang

Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

Mite allergen is a danger signal for the skin via activation of inflammasome in keratinocytes

BACKGROUND Atopic dermatitis (AD) is a chronic inflammatory skin disorder caused by multiple factors. Among them, house dust mite (HDM) allergens are important in the development of AD. In airway allergy, HDM allergens activate innate immunity. However, information regarding the activation of innate immunity by HDM allergens in the skin is limited. OBJECTIVES The inflammasome is a key regulator of pathogen recognition and inflammation. We investigated whether HDM allergens activate the inflammasome in epidermal keratinocytes. METHODS Keratinocytes were stimulated with Dermatophagoides pteronyssinus, and the activation of caspase-1 and secretion of IL-1β and IL-18 were examined. Formation of the inflammasome was studied by analyzing the subcellular distributions of inflammasome proteins. The importance of specific inflammasome proteins was studied by knocking down their expression through transfection of keratinocytes with lentiviral particles carrying short hairpin RNAs (shRNAs). RESULTS D pteronyssinus activated caspase-1 and induced caspase-1-dependent release of IL-1β and IL-18 from keratinocytes. Moreover, D pteronyssinus stimulated assembly of the inflammasome by recruiting apoptosis-associated specklike protein containing a caspase-recruitment domain (ASC), caspase-1, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin-domain containing 3 (NLRP3) to the perinuclear region. Finally, infection with lentiviral particles carrying ASC, caspase-1, or NLRP3 shRNAs suppressed the release of IL-1β and IL-18 from the keratinocytes. Activation of the NLRP3 inflammasome by D pteronyssinus was dependent on cysteine protease activity. CONCLUSION House dust mite allergens are danger signals for the skin. In addition, HDM-induced activation of the NLRP3 inflammasome may play a pivotal role in the pathogenesis of AD.

A Novel Function of Angiotensin II in Skin Wound Healing INDUCTION OF FIBROBLAST AND KERATINOCYTE MIGRATION BY ANGIOTENSIN II VIA HEPARIN-BINDING EPIDERMAL GROWTH FACTOR (EGF)-LIKE GROWTH FACTOR-MEDIATED EGF RECEPTOR TRANSACTIVATION

The role of angiotensin II (Ang II) in the control of systemic blood pressure and volume homeostasis is well known and has been extensively studied. Recently, Ang II was suggested to also have a function in skin wound healing. In the present study, the in vivo function of Ang II in skin wound healing was investigated using Ang II type 1 receptor (AT1R) knock-out mice. Wound healing in these mice was found to be markedly delayed. Keratinocytes and fibroblasts play important roles in wound healing, and thus the effect of Ang II on the migration of these cells was examined. Ang II stimulated keratinocyte and fibroblast migration in a dose-dependent manner. It has been reported that G protein-coupled receptor (GPCR) activation induces epidermal growth factor (EGF) receptor (EGFR) transactivation through the shedding of heparin-binding EGF-like growth factor (HB-EGF). As AT1R is a GPCR, it was hypothesized that Ang II-induced keratinocyte and fibroblast migration is mediated by EGFR transactivation. Ang II induced EGFR phosphorylation, which was inhibited by an AT1R antagonist, HB-EGF neutralizing antibody, and an HB-EGF antagonist in both keratinocytes and in fibroblasts. Moreover, Ang II-induced migration of keratinocytes and fibroblasts was also prevented by these inhibitors. Taken together, these findings clearly demonstrate, for the first time, that Ang II plays an important role in skin wound healing and that it functions by accelerating keratinocyte and fibroblast migration in a process mediated by HB-EGF shedding.

IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression

IL‐20 cytokine subfamily members, including IL‐19, IL‐20, and IL‐24, are highly expressed in psoriatic skin lesions. Here, we demonstrate that psoriasis mediators IL‐17 and IL‐22 synergistically induce the production of IL‐20 subfamily proteins in cultured human keratinocytes. Interestingly, expression of the IL‐22 receptor (IL‐22R) also increased in epidermal lesions versus normal skin. IL‐22R over‐expression using an adenoviral vector to mimic psoriatic conditions in cultured keratinocytes significantly enhanced IL‐17‐ and IL‐22‐induced production of IL‐20 subfamily cytokines. Furthermore, IL‐17 and IL‐22 coordinately enhanced MIP‐3α, IL‐8, and heparin‐binding EGF‐like growth factor (HB‐EGF) production, depending on the amount of IL‐22R expression. Additionally, because IL‐20 and IL‐24 share the IL‐22R with IL‐22, the function of IL‐20 and IL‐24 was also increased. IL‐20 and IL‐24 have effects similar to that of IL‐22; IL‐24 showed more potent expression than IL‐20. A combination of IL‐24 and IL‐17 increased the production of MIP‐3α, IL‐8, and HB‐EGF, as did a combination of IL‐22 and IL‐17. These data indicate that increased IL‐22R expression in epidermal keratinocytes contributes to the pathogenesis of psoriasis through enhancing the coordinated effects of IL‐22 and IL‐17, inducing the production of the IL‐20 subfamily, chemokines, and growth factors.

Transforming Growth Factor-β-activated Kinase 1 Is Essential for Differentiation and the Prevention of Apoptosis in Epidermis

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-κB (NF-κB). Given that NF-κB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7flox/flox mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7flox/flox mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis.

Living skin equivalents constructed using human amnions as a matrix

BACKGROUND Living skin equivalents (LSEs) are being used to treat burn wounds, skin defects, and chronic wounds, and today, several biomaterials are applied as dermal matrices in LSEs. The amnionic membrane (AM) is known to have useful properties as a dermal matrix and can be used to construct a LSE. OBJECTIVE To develop a new LSE with human AM as the matrix. METHODS Human AM was de-epithelialized and investigated to determine whether it supported keratinocyte adherence and proliferation, and fibroblast in-growth and proliferation. A new LSE was constructed by seeding keratinocytes on the epithelial side of fibroblast-populated, de-epithelialized AM and was investigated histologically. The LSE was transplanted onto a full-thickness wound on a nude mouse and a histological examination was conducted. RESULTS De-epithelialized AM supported the adherence and proliferation of keratinocytes and the in-growth of fibroblasts. The new LSE demonstrated good mechanical properties and revealed good morphology, with a well differentiated epidermis and well developed basement membrane. The LSE grafts survived well on nude mice, showing good morphology. CONCLUSION A LSE with amnions as a matrix exhibited good morphology, low cost, and good mechanical properties and may be useful as a skin substitute for clinical use.

IFN-α Enhances IL-22 Receptor Expression in Keratinocytes: A Possible Role in the Development of Psoriasis

TO THE EDITOR Recently, a pathological role for IFN-a in the development of psoriasis has been suggested. Clinically, exacerbation of psoriasis has often been observed after starting IFN-a therapy for other diseases. IFN-a induced by topical application of a Toll-like receptor-7 agonist can also trigger the development of psoriasis (Gilliet et al., 2004). Histopathologically, plasmacytoid dendritic cells (pDCs), the principal IFN-a–producing cells, infiltrate into the psoriatic lesions in the early phase (Wollenberg et al., 2002; Gilliet et al., 2004). In a xenograft model, in which human psoriatic skin was transplanted onto immunodeficient mice, Nestle et al. (2005) clearly demonstrated that blocking IFN-a signaling prevents the T cell–dependent development of psoriasis and that inhibiting the production of IFN-a by pDCs completely suppresses the development of a psoriatic phenotype. IFN-a may mediate the activation of T cells directly or through the induction of myeloid dendritic cell activation and/or maturation. The involvement of keratinocytes, however, remains unclear. We recently reported that IL-22 receptor (IL-22R) expression is enhanced in the epidermis of psoriasis compared with normal skin (Tohyama et al., 2009). IL-22, a member of the IL-10 family, is one of the most important cytokines in the pathogenesis of psoriasis, contributing to proliferation/ differentiation of keratinocytes and to the production of antimicrobial peptides, cytokines, and chemokines (Wolk et al., 2006; Sa et al., 2007). Receptors for IL-22 include IL-22R and IL-10 receptor 2 (IL-10R2). IL-10R2 is widely expressed on various cells and tissues. On the other hand, IL-22R expression is limited mainly to epithelial cells, including epidermal keratinocytes, where IL-22R expression levels are altered by cytokine stimulation. Increased IL-22R expression strengthens the responsiveness of keratinocytes to IL-22 stimulation (Tohyama et al., 2009). These findings suggest that an increase in IL-22R expression on epidermal keratinocytes is an important feature in psoriasis. We hypothesized that IFN-a enhances IL-22R expression on epidermal keratinocytes. To examine this, we used a living skin equivalent (LSE) model (Yang et al., 2005), as IL-22R expression of cultured keratinocyte monolayers is suppressed relative to that of stratified epidermal keratinocytes, including those in the LSE (Sa et al., 2007). To prepare the LSE, normal human epidermal keratinocytes and fibroblasts were isolated from nonlesional human skin obtained from plastic surgery and then cultured. A collagen gel containing the fibroblasts was prepared, and the keratinocytes were seeded onto the concave surface of the contracted gel. When the keratinocytes reached confluence, the LSE was raised to the air–liquid interface, and cornification medium was added. Experiments were conducted from 8 to 12 days after air-lifting. Total RNA or protein was extracted from the LSE epidermis for real-time reverse transcription-PCR analysis or western blotting, respectively. For immunofluorescence staining, frozen LSE sections (5mm) were fixed in cold acetone or methanol and reacted with primary antibodies. Sections were then incubated with donkey anti-rabbit or anti-goat antibodies labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Alternatively, LSE epidermis was treated with trypsin, and flow cytometry analysis (FCM) was performed as described previously (Tohyama et al., 2009). For each examination, results were confirmed in three independent experiments. LSE was treated with IFN-a for 4, 8, or 24 hours. IFN-a markedly enhanced IL-22R mRNA expression in the epidermis after 4 hours of stimulation (Figure 1a), and this increase was dose dependent (Figure 1b). In contrast, IL-10R2 mRNA expression level was not changed (Figure 1c). The expression of IL-20R1 and IL-20R2, receptors for IL-10 family cytokines other than IL-22, was also unaffected by IFN-a. LSE treated with or without IFN-a for 24 hours was snap-frozen, and immunofluorescence staining was performed using anti-IL-22R antibody (R&D systems, Minneapolis, MN). IL-22R expression was increased relative to controls in the LSE epidermis treated with IFN-a (Figure 1d). Increased IL-22R expression on keratinocyte cell surfaces was also observed by FCM analysis (Figure 1e). To examine whether the response to IL-22 is increased by IFN-a treatment, phosphorylation of signal transducer and activator of transcription 3 (STAT3) was examined by western blotting. Anti-phospho-STAT3 antibody was purchased from Cell Signaling Technology (Danvers, MA). As shown in Figure 2a, STAT3 was phosphorylated at 30 and 60 minutes after IL-22 stimulation in the epidermis of LSE. When LSE was pretreated with IFN-a for 24 hours, phosphorylation of STAT3 was markedly augmented. See related commentary on pg 1759

PPARγ mediates innate immunity by regulating the 1α,25-dihydroxyvitamin D3 induced hBD-3 and cathelicidin in human keratinocytes

BACKGROUND Production of antimicrobial peptides (AMPs) is the primary mechanism by which skin innate immunity protects against infection. Hormonally active vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D₃) is a vital regulator of skin innate immunity, and has been shown to increase the expression and function of AMPs. OBJECTIVE PPARγ is a ligand-activated nuclear receptor and plays a role in keratinocyte differentiation and cutaneous homeostasis. In this study, we investigate whether 1,25D₃-activated PPARγ signaling regulates AMP expression in keratinocytes. METHODS Subconfluent keratinocytes were treated with 1,25D₃ for the indicated times. The mRNA and protein levels of AMPs were detected by RT-PCR and Western blot, and the DNA binding activation of PPARγ, VDRE and AP-1 was investigated by EMSA. To examine the role of PPARγ, the recombinant adenovirus carrying a dominant-negative form of PPARγ (dn-PPARγ) was constructed and transfected into keratinocytes. RESULTS We show here that 1,25D₃ significantly enhances hBD-3 and cathelicidin expression in keratinocytes. Expression of dn-PPARγ did not affect binding to the vitamin D-responsive element (VDRE), which is crucial for cathelicidin induction by VD3; however, it did decrease 1,25D₃ induction of both hBD-3 and cathelicidin. Inhibition of the p38, ERK, and JNK signaling pathways blocked hBD-3 expression, whereas only p38 inhibition suppressed cathelicidin induction. dn-PPARγ had no effect on ERK and JNK activity, but inhibited p38 phosphorylation and suppressed 1,25D₃-induced AP-1 activation via effects on Fra1 and c-Fos proteins. CONCLUSIONS In conclusion, PPARγ regulates the 1,25D₃-induced hBD-3 and cathelicidin expression in keratinocytes through the regulation of AP-1 and p38 activity.

The NF-κB, p38 MAPK and STAT1 pathways differentially regulate the dsRNA-mediated innate immune responses of epidermal keratinocytes

The epidermis is the primary boundary between the body and the environment, and it serves as the first line of defense against microbial pathogens. Production of chemokines and cytokines is an important step in the initiation of innate immune responses to viral infections. Epidermal keratinocytes produce IFN-alpha, -beta and macrophage inflammatory protein (MIP)-1alpha in response to double-stranded RNA (dsRNA) or viral infections. We showed that human keratinocytes produced cytokines [tumor necrosis factor (TNF)-alpha, IL-1beta and IL-15] and chemokines [MIP-1beta, RANTES and liver and activation-regulated chemokine (LARC)] in response to dsRNA, with activation of the nuclear factor kappaB (NF-kappaB), p38 mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription 1 (STAT1) pathways. To study the roles of these pathways in their production, we transfected keratinocytes with adenoviral vectors (Ax) carrying a dominant-negative form of inhibitor kappaB alpha (IkappaBalpha) (IkappaBalphaM), a dominant-negative mutant form of STAT1 (STAT1F) or suppressors of cytokine signaling 1 (SOCS1). Transfection with AxIkappaBalphaM or addition of a p38 inhibitor (SB203580) significantly decreased the dsRNA-mediated production of TNF-alpha, IL-1beta and MIP-1alpha, but not of IFN-beta, IL-15, MIP-1beta, RANTES or LARC. Transfection with AxSTAT1F or AxSOCS1 inhibited the dsRNA-mediated production of TNF-alpha, IL-15, MIP-1alpha, MIP-1beta, RANTES and LARC, but not IFN-beta or IL-1beta. In conclusion, the NF-kappaB, p38 MAPK and STAT1 pathways differentially regulate dsRNA-mediated innate immune responses in epidermal keratinocytes.

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents.

BACKGROUND During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. OBJECTIVE In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. METHODS We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-β (TGF-β) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. RESULTS The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-β1 expression in the epidermis. The inhibition of TGF-β kinase completely suppressed α-SMA expression in the dermal matrix. CONCLUSIONS Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-β1, which is responsible for myofibroblast differentiation.