Cutaneous consequences of inhibiting EGF receptor signaling in vivo: Normal hair follicle development,but retarded hair cycle induction and inhibition of adipocyte growth in EgfrWa5 mice

BackgroundThe epidermal growth factor receptor (EGFR) network is essential for proper development and homeostasis of skin and hair. However, detailed dissection of the role of the EGFR in hair follicle development and cycling have been impaired by the early mortality of EGFR knockout mice.ObjectivesWe have studied in Waved-5 mice carrying an antimorphic EGFR allele (Egfr^wa5), whose product acts as a dominant-negative receptor, whether strongly reduced EGFR signaling impacts on the hair and skin phenotype.MethodsHistomorphometry and immunohistochemistry were employed to study hair follicle morphogenesis stages and cycle induction in Waved-5 mice and control littermates during embryonic development and postnatal life.ResultsBy routine histology and quantitative histomorphometry, no significant abnormalities in the epidermis and in hair follicle morphogenesis were detected, while the initiation of hair follicle cycling was slightly, but significantly retarded. Proliferation and apoptosis of epidermal and hair matrix keratinocytes of Waved-5 mice appeared unaltered. Intriguingly, the thickness of the subcutis and the percentage of proliferating subcutaneous adipocytes were significantly reduced in Waved-5 mice around days P8.5 to P10.5. Although no differences in total body weight gain could be detected, Wa5 mice showed a significant reduction in the percentage of body fat at P8.5.ConclusionOur results suggest the presence of effective compensatory mechanisms in murine skin in vivo that ensure nearly normal epidermal and hair follicle keratinocyte function despite very low levels of EGFR-mediated signaling. Our unexpected findings of transiently reduced subcutaneous adipose tissue indicate a role for the EGFR in regulating subcutaneous fat.     

Activation of kinin B receptor triggers differentiation of cultured human keratinocytes

Background Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B₁ receptor (B₁R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. Objectives To determine the consequence of kinin B₁R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. Methods Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. Results The major proteins phosphorylated, after B₁R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170‐ and 125‐kDa proteins were identified as EGFR and p125^FAK, respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12‐myristate 13‐acetate, indicating the participation of protein kinase C. B₁R stimulation did not increase [Ca²⁺]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr⁸⁴⁵, Tyr⁹⁹² and Tyr¹⁰⁶⁸ of EGFR. B₁R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B₁R stimulation, produced an additional increase in filaggrin expression. Conclusions The kinin B₁R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.     

Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte‐specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.     

Aberrant Notch signalling contributes to hypertrophic scar formation by modulating the phenotype of keratinocytes

Hypertrophic scar (HS) is characterized by fibroblast hyperproliferation and excessive matrix deposition. Aberrant keratinocyte differentiation and their abnormal cytokine secretion are said to contribute to HS by activating fibroblasts. However, the signalling pathway causing the aberrant keratinocytes in HS has remained unidentified thus far. Given that Notch signalling is crucial in initiating keratinocyte differentiation, we hypothesized that Notch signalling contributes to HS by modulating the phenotype of keratinocytes. We found that Notch1, Notch intracellular domain, Jagged1 and Hes‐1 were overexpressed in the epidermis of patients with HS. Supernatants from recombinant‐Jagged1–treated keratinocyte cultures could accelerate dermal fibroblast proliferation and collagen production. Furthermore, Jagged1 induced keratinocyte differentiation and upregulated the expression of fibrotic factors, including transforming growth factors β₁ and β₂, insulin‐like growth factor‐1, connective tissue growth factor, vascular endothelial growth factor and epidermal growth factor, while DAPT (a Notch inhibitor) significantly suppressed these processes. In a rabbit ear model of HS, local application of DAPT downregulated the production of fibrotic factors in keratinocytes, together with ameliorated scar hyperplasia. Our findings suggest that Notch signalling contributes to HS by modulating keratinocyte phenotype. These results provide new insights into the pathogenesis of HS and indicate a potential therapeutic target.     

A novel homozygous mutation disrupting the initiation codon in the SLURP1 gene underlies mal de Meleda in a consanguineous family

Mal de Meleda (MDM) is a palmoplantar keratoderma (PPK), characterized by hyperkeratosis of the palms and soles, and keratotic skin lesions. Patients with MDM can develop perioral erythema, keratotic and lichenoid plaques over the joints (including the elbows and knees), nail abnormalities, joint contractures and stiffness, brachydactyly, sclerodactyly, pseudoainhum, and malodorous maceration. MDM is associated with mutations in the SLURP1 gene. We report a consanguineous family in which MDM was inherited in an autosomal recessive manner. Genotyping using microsatellite markers established linkage in the family to the SLURP1 gene, which has been mapped previously to chromosome 8q24.3. Sequence analysis revealed a homozygous missense mutation (c.2T>C, p.Met1Thr) in affected family members. Molecular docking studies using a ZDOCK server predicted disruption of binding of the mutant variant to its target α7‐nAChR. This study further supports the previously reported findings that homozygous mutations in the SLURP1 gene cause MDM.     

Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes

Please cite this paper as: Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes. Experimental Dermatology 2010; 19 : 730–735. Abstract: Epidermal growth factor receptor tyrosine kinase (EGFR‐TK) is a transducer of mitogenic signals, and is involved in the pathogenesis and progression of a number of cancers, including non‐small cell lung cancer (NSCLC). Gefitinib is an EGFR‐TK inhibitor that is clinically used to treat NSCLC; however, this drug frequently causes adverse effects, including skin eruptions. The mechanism underlying these skin reactions is elusive, although it is assumed that they are caused by the inhibition of EGFR‐TK signalling in epidermal and adnexal cells. In this article, we demonstrate by immunocytochemistry that the skin lesions of patients treated with oral gefitinib had higher expression of CCL2 and CCL5 compared to normal human epidermis. Further, PD153035, a gefitinib prototype, induced CCL2 and CCL5 mRNA and protein expression in HaCaT and HSC‐1 keratinocyte cell lines with or without interleukin‐1 (IL‐1) treatment in vitro. PD153035 also reduced the levels of interleukin‐1 receptor 2 (IL‐1R2), an IL‐1 decoy receptor. Moreover, we demonstrate that reduction in IL‐1R2 by RNA interference increased IL‐1‐mediated CCL2 and CCL5 mRNA and protein expression. Taken together, our data strongly suggest that IL‐1‐mediated signalling is activated to induce the high expression of CCL2 and CCL5 via reduction in IL‐1R2 in the skin lesions caused by gefitinib.     

SLURP1 mutation‐impaired T‐cell activation in a family with mal de Meleda

Background Mal de Meleda (MDM) is palmoplantar erythrokeratoderma with an autosomal recessive inheritance and is caused by a mutation in the gene encoding SLURP‐1 (lymphocyte antigen 6/urokinase‐type plasminogen activator receptor related protein‐1). SLURP‐1 is an allosteric agonist to the nicotinic acetylcholine receptor (nAchR) and it regulates epidermal homeostasis. In addition, murine studies have shown that nAchR signalling is important for the regulation of T‐cell function. Among the family members, patients with the homozygous SLURP1 (previously known as ARS component B) mutation are prone to melanoma and viral infection, which might link to defective T‐cell function as well as a derangement of epidermal homeostasis. Objectives To investigate the association of the SLURP1 gene mutation with T‐cell activation in a Taiwanese family with MDM. To test that SLURP‐1 is essential for T‐cell activation. Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from a Taiwanese MDM family bearing the G to A substitution in nucleotide 256 in the SLURP1 gene, corresponding to a glycine to arginine substitution at amino acid 86 (G86R) in the SLURP‐1 protein. PBMCs from homozygotes and wild‐type controls were stimulated with anti‐CD3/anti‐CD28 antibodies and the level of T‐cell activation was determined by the stimulation index. Results PBMCs with the heterozygous and homozygous SLURP‐1 G86R mutation had defective T‐cell activation. This was restored by the addition of 0·5 μg mL^?1 recombinant human SLURP‐1 protein. Conclusions Patients with MDM with the homozygous SLURP‐1 G86R mutation may have an impaired T‐cell activation. The presence of wild‐type SLURP‐1 is essential for normal T‐cell activation.     

Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes

Background

Skin-derived antimicrobial peptides, such as human β-defensins and cathelicidins, actively contribute to host defense by inactivating microorganisms. Catestatin, a neuroendocrine peptide that affects human autonomic functions, has recently been detected in keratinocytes upon injury/infection where it inhibits the growth of pathogens. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu, and Arg374Gln.

Objective

To investigate the effects of human catestatin and its variants on keratinocyte migration and proliferation, and to elucidate the possible signaling mechanisms involved.

Methods

The migration of normal human keratinocytes was analyzed using Boyden microchamber assay and in vitro wound closure assay. Cell proliferation was evaluated by BrdU incorporation, cell count assay and cell cycle analysis. Intracellular Ca²⁺ mobilization was measured using a fluorescent calcium assay kit. The phosphorylation of epidermal growth factor receptor (EGFR), Akt, and MAPKs was determined by Western blotting.

Results

Catestatin and its variants dose-dependently enhanced keratinocyte migration and proliferation. Moreover, catestatin peptides increased intracellular Ca²⁺ mobilization and induced the phosphorylation of EGFR, Akt, extracellular signal-regulated kinase (ERK), and p38 in keratinocytes. The induction of keratinocyte migration and proliferation by catestatin peptides involved G-proteins, phospholipase C, EGFR, PI3-kinase, ERK, and p38, as evidenced by the specific inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor), AG1478 (EGFR inhibitor), anti-EGFR antibody, wortmannin (PI3-kinase inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor), respectively.

Conclusion

Besides inhibiting the growth of skin pathogens, catestatin peptides may also contribute to cutaneous wound closure by enhancing keratinocyte migration and proliferation at the wound site.     

Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch‐wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen‐activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up‐regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.     

The role of RNase 7 in innate cutaneous defense against Pseudomonas aeruginosa

The ribonuclease RNase 7 is a major skin‐derived human antimicrobial protein expressed in keratinocytes. Here we show that the gram‐negative pathogen Pseudomonas aeruginosa secretes factor(s) that induced RNase 7 gene and protein expression in human primary keratinocytes. The metalloprotease inhibitor marimastat, the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the EGFR blocking antibody cetuximab significantly attenuated this induction, indicating an important role of the EGFR for the P. aeruginosa‐mediated RNase 7 induction. In line with this, siRNA‐mediated downregulation of ADAM17, a metalloprotease known to proteolytically mediate the release of soluble EGFR ligands, decreased the P. aeruginosa‐mediated RNase 7 induction in keratinocytes. The impact of the EGFR was also demonstrated in a human 3D skin equivalent where blockade of the EGFR diminished induction of RNase 7 by P. aeruginosa. Blockade of Toll‐like receptor 5 (TLR5), a pattern recognition receptor (PRR) known to be activated by P. aeruginosa, only moderately reduced the P. aeruginosa‐mediated RNase 7 induction in keratinocytes. The functional relevance of RNase 7 to participate in cutaneous defense against P. aeruginosa was demonstrated by antibodies that neutralized the antimicrobial activity of RNase 7. These antibodies significantly inhibited the capacity of human stratum corneum skin extracts to control growth of P. aeruginosa. Taken together, our results indicate that P. aeruginosa induces the expression of RNase 7 in keratinocytes in an EGFR‐dependent manner. Enhanced release of RNase 7 contributes to control cutaneous growth of P. aeruginosa.     

Growth-promoting Effect of Bacterial Products from Clostridium perfringens on Human Keratinocytes

Wound healing substance (WHS) from cultured Clostridium perfringens has been reported to be effective in the treatment of wounds. The effects of WHS, now named SNK-863, on proliferation and differentiation of human keratinocytes were examined. The characteristics of WHS are as follows: 1) WHS stimulates human keratinocyte growth and DNA synthesis; 2) WHS and EGF show some additive effects on human keratinocyte growth; 3) WHS does not interfere with the binding of EGF to its receptor; 4) WHS does not counteract the growth inhibitory effects of TGF-β or vitamin D₃ on human keratinocytes; 5) WHS has no significant effect on human keratinocyte differentiation. These results indicate that the growth-promoting effect of WHS on keratinocytes may contribute to the treatment of wound healing.     

Combined treatment with sodium butyrate and PD153035 enhances keratinocyte differentiation

Epidermal growth factor (EGF) receptor (EGFR) signalling is a critical determinant of keratinocyte proliferation and differentiation in both normal and diseased skin. Here we explore the effects of combined treatment with the differentiation‐promoting agent sodium butyrate (SB) and the EGFR inhibitor (EGFRI) PD153035 on terminal differentiation of normal human epidermal keratinocytes (NHEKs). Cells treated with SB showed increased expression of the levels of mRNA and protein of the differentiation markers filaggrin and transglutaminase 1. Cotreatment with EGF significantly blunted these effects of SB. Combined treatment with SB and PD153035 alleviated these inhibitory actions of EGF, resulting in improved effects of decreased cell growth and increased terminal differentiation, relative to the individual treatments. These results indicate that the combined use of a differentiation‐promoting agent and an EGFR inhibitor may offer an additional approach to the management of hyperproliferative skin diseases.     

TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation

Tumor necrosis factor (TNF)‐like weak inducer of apoptosis (TWEAK) has been reported to induce keratinocyte apoptosis in vitro by engaging its sole receptor of fibroblast growth factor‐inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the growth of psoriatic keratinocytes that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary keratinocytes were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by keratinocytes in vitro, which expressed TNF receptor 2 predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein 2 and cellular FLICE‐inhibitory protein in lesional keratinocytes. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of keratinocytes under psoriatic inflammation. The activation of nuclear factor‐κB signalling‐dependent anti‐apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in keratinocytes under psoriatic inflammation.     

Loss of FAS/FASL signalling does not reduce apoptosis in Sharpin null mice

Mice with mutations in SHANK‐associated RH domain interactor (Sharpin) develop a hypereosinophilic auto‐inflammatory disease known as chronic proliferative dermatitis. Affected mice have increased apoptosis in the keratinocytes of the skin, oesophagus and forestomach driven by extrinsic TNF receptor‐mediated apoptotic signalling pathways. FAS receptor signalling is an extrinsic apoptotic signalling mechanism frequently involved in inflammatory skin diseases. Compound mutations in Sharpin and Fas or Fasl were created to determine whether these death domain proteins influenced the cutaneous phenotype in Sharpin null mice. Both Sharpin/Fas and Sharpin/Fasl compound mutant mice developed an auto‐inflammatory phenotype similar to that seen in Sharpin null mice, indicating that initiation of apoptosis by FAS signalling is likely not involved in the pathogenesis of this disease.     

Fetuin‐A promotes primary keratinocyte migration: independent of epidermal growth factor receptor signalling

Please cite this paper as: Fetuin‐A promotes primary keratinocyte migration: independent of epidermal growth factor receptor signalling. Experimental Dermatology 2010; 19 : e289–e292. Abstract: Previously, we reported that fetuin‐A is a major component of ovine foetal skin and significantly enhances ‘wound closure’ in primary keratinocyte cultures. In this study, we found that in human newborn foreskin, a high level of fetuin‐A protein is detected throughout the dermis. However, in adult skin a low level of fetuin‐A is observed throughout the epidermal and dermal layers, except at regions surrounding hair follicles and at the epidermal‐dermal junction where the level of fetuin‐A is relatively high. Fetuin‐A significantly induces actin‐rich protrusions in human primary keratinocytes. Interestingly, blockade of epidermal growth factor (EGF) receptor signalling has a limited effect on fetuin‐A promoted ‘wound closure’ on primary human keratinocytes, but significantly inhibits fetuin‐A’s effect on HaCaT cells. These results indicate that high levels of fetuin‐A may partially contribute to less scar formation in newborn foreskin and that the effect of fetuin‐A on primary keratinocyte migration is independent of EGF receptor signalling.     

Pemphigus,a pathomechanism of acantholysis

Autoantibodies to the desmosomal proteins desmoglein 1 and 3 cause pemphigus foliaceus and pemphigus vulgaris, which are characterised by keratinocyte dissociation (acantholysis) and intraepidermal blister formation. The passive transfer of pathogenic anti‐desmoglein antibodies induces blisters in mice in vivo and the loss of keratinocyte adhesion in vitro. The pathogenetic mechanisms of acantholysis due to anti‐desmoglein autoantibodies are not fully understood. However, recent studies have revealed that signalling‐dependent and signalling‐independent pathways are operative in the loss of cell adhesion. In this review, we focus on the pathomechanism of acantholysis due to autoantibodies to desmogleins and recent therapeutic approaches.     

Keratinocyte growth factor receptors

Modulation of the number of functional growth factor receptors on the epithelial cell surface that is exposed to the action of cognate ligands represents a key strategy in cellular physiology to regulate the proliferation rate and the differentiation process. The keratinocyte growth factor receptor (KGFR) and the epidermal growth factor receptor (EGFR), among the growth factor receptors expressed on keratinocytes, are believed to play a unique crucial role in controlling epithelial proliferation. KGFR and EGFR appear to also contribute to the cell differentiation process. Modulation of KGFR and EGFR on the proliferation rate and differentiation process has been reported either in in vivo or in vitro conditions. This article reviews the architecture, the ligand binding activated-signaling pathways, and the biologic effects of KGFR and EGFR on keratinocytes.     

Gefitinib‐induced epidermal growth factor receptor‐independent keratinocyte apoptosis is mediated by the JNK activation pathway

Background Gefitinib (ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with a significant antitumour effect on various cancers. Skin toxicity induced by gefitinib is common, and has been shown to be related to the inhibition of EGFR signalling pathways. However, other mechanisms may be involved in gefitinib‐induced skin toxicity. Objectives To study the possible EGFR‐independent mechanisms of gefitinib‐induced skin toxicity. Methods The human immortalized keratinocyte cell line HaCaT and human lung adenocarcinoma cell lines (A549 and PC9) were treated with different concentrations of gefitinib for 24, 48 and 72 h. Cell viability was measured by MTT assay [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] after EGFR gene silencing. The signalling pathways were investigated by immunoblot analysis. Keratinocyte apoptosis was evaluated by nuclear condensation and flow cytometric analysis. Results Gefitinib maintained its cytotoxicity to HaCaT cells after EGFR gene silencing, indicating that an EGFR‐independent mechanism exists. Increased phosphorylation of p38 mitogen‐activated protein kinase and JNK by gefitinib was observed in a dose‐dependent manner in HaCaT cells. The JNK inhibitor, SP600125, attenuated the gefitinib‐induced cytotoxicity and apoptosis of HaCaT cells. Immunohistochemical examination of patient specimens showed an increased expression of phosphorylated JNK in lesional epidermis compared with nonlesional epidermis. Conclusions Gefitinib can induce keratinocyte apoptosis through an EGFR‐independent JNK activation pathway.