Is there any relation between serum insulin and insulin‐like growth factor‐I in non‐diabetic patients with skin tag?

Background Skin tags are common benign lesion occurring mainly on the neck and major flexures as a small soft pedunculated protrusion. This study evaluate insulin and insulin‐like growth factor‐I (IGF‐I) in non‐diabetic ones. Methods and materials A case–control study was conducted in non‐diabetic persons. Comparing insulin and IGF‐I between matched cases (n = 40) and controls (n = 40) by radioimmunoassay test. Cases and controls were recruited from patients consecutively seen at an academic outpatient dermatology clinic. Results The insulin level in patients with skin tags was significantly higher than controls (P = 0.00) but IGF‐I level was not significantly different (P = 0.43). Conclusion These results show an increased insulin level in non‐diabetics ones and overall importance of insulin effect in pathogenesis of skin tags.     

Small-interfering RNA targeted at antiapoptotic mRNA increases keratinocyte sensitivity to apoptosis

Background Gene silencing RNA interference technology concentrates on downregulation of gene expression using specific double‐stranded RNA molecules (small‐interfering RNA, siRNA), which induce the degradation of complementary mRNA. SiRNA therapeutics is currently being tested in several clinical trials. Skin disorders are an attractive target for siRNA‐based technology because effective agents can be delivered by topical application. In several inflammatory skin disorders, the barrier function of the skin is markedly impaired and this may increase the delivery efficiency. Psoriasis is a very common skin disorder. The characteristics of this disorder are abnormal keratinocyte proliferation and inflammation. Keratinocytes from psoriatic epidermis express much higher levels of the antiapoptotic protein, Bcl‐xL, compared with normal keratinocytes. Insulin‐like growth factor 1 receptor (IGF‐1R) plays a major role in cell growth, differentiation and apoptosis in many cell types, including keratinocytes and IGF‐1R activation plays an important role in the pathogenesis of psoriasis. Keratinocytes from patients with psoriasis are more susceptible to IGF‐1‐stimulated proliferation compared with normal keratinocytes. IGF‐1R is expressed by proliferating basal and suprabasal keratinocytes and is more abundant in psoriatic lesions. Objectives To prove the validity of IGF‐1R and Bcl‐xL as useful targets for siRNA‐based therapeutics and to deliver siRNA selectively and efficiently to primary human keratinocytes; this is a primary essential step in the development of siRNA. Methods Primary normal human keratinocytes were transfected with various siRNA molecules, and transfection efficiency was monitored by fluorescent labelling. Cell growth and apoptosis induction were evaluated in the transfected cells. Results We were able to deliver efficiently siRNA targeting Bcl‐xL or IGF‐1R to primary human keratinocyte cultures. We also showed that siRNAs targeting Bcl‐xL and IGF‐1R induce growth inhibition, apoptosis and increased sensitivity to ultraviolet B in keratinocytes. Conclusions The present findings demonstrate that Bcl‐xL and IGF‐1R are valid, important targets for siRNA‐based technology directed at the suppression of keratinocyte hyperproliferation.     

Ultraviolet B radiation regulates cysteine‐rich protein 1 in human keratinocytes

Background: Cysteine‐rich protein 1 (CRP1) is a growth‐inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. Methods: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A‐431) by immunoblotting. UVB‐induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV‐damaged skin and squamous cell carcinoma (SCC), a UV‐induced skin cancer. Results: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis‐inducing doses of UV radiation, CRP1 was cleaved in a caspase‐dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. Conclusion: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.     

In vitro formation of organized structure between keratinocytes and dorsal‐root‐ganglion cells

We recently found that the morphology in a co‐culture system of keratinocytes and dorsal‐root ganglion‐derived cells depended on the timing of seeding of the two cell types. In skin, epidermis is formed first, followed by construction of peripheral nerve structure. Therefore, we hypothesized that formation of peripheral nerve structure in the epidermis might be driven by interaction between keratinocytes and nerve cells. In the present study, we tested this idea by incubating keratinocytes and dorsal‐root ganglion cells in a spatially separated manner and observing the morphological changes in the co‐culture system. Extension of nerve fibre‐like structures from the ganglion cells was observed, and within 3 days after seeding, many nerve fibre‐like extensions penetrated into the keratinocyte cluster, subsequently forming a network that appeared to resemble the cutaneous peripheral nervous system. Our present model may be useful for studying the formation of peripheral nerve structure in the skin.     

Vitamin D inhibits captopril‐induced cell detachment and apoptosis in keratinocytes

Background Captopril, an angiotensin I‐converting enzyme inhibitor, is a commonly prescribed antihypertensive drug. Its cutaneous side‐effects include pemphigus vulgaris acantholysis and bullous pemphigoid‐like cell–matrix detachment. This medication also triggers apoptosis in human keratinocytes. Calcitriol, the hormonally active vitamin D metabolite, protects keratinocytes from programmed cell death induced by various noxious stimuli. Objectives To examine if calcitriol protects proliferating keratinocytes from the damage inflicted by captopril. Methods Autonomously proliferating HaCaT keratinocytes, used as a model for basal layer keratinocytes, were exposed to captopril. Cell detachment was examined visually by light microscopy. Cytotoxicity was assessed by Hoechst 33342 staining and lactate dehydrogenase release. Apoptotic death was assessed by monitoring caspase 3‐like activity. Results Cells exposed to captopril detached and became round. This process was accompanied by programmed cell death. From time‐dependent monitoring of cell detachment and apoptosis, and examination of pan‐caspase inhibitor effects on cell detachment we concluded that cell death is the consequence of cell detachment from the culture plate and not vice versa. Pretreatment with calcitriol significantly attenuated these events. The effects of calcitriol were already evident at 1 nmol L^?1 concentration of the hormone. Conclusions The results of this study show that calcitriol protects keratinocytes from captopril‐induced cell detachment and apoptosis.     

Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes

Ultra‐violet B (UVB)‐induced oxidative stress crucially perturbs the epidermal homeostasis, and the skin is endowed with protective mechanisms to take action against such damage. Here, we show the possible involvement of t‐SNARE protein syntaxin3, a membrane fusion mediator of cytoplasmic vesicles, and which is released from dying keratinocytes, to play a role in this response. UVB irradiation, which generates reactive oxidative stress in cells, was shown to lead to the keratinocyte cell death accompanied by a release of cytoplasmic syntaxin3. We found that such extracellularly sourced syntaxin3 completely blocked the processing of a crucial effector for apoptotic cell death, caspase‐3, and thus facilitated the survival of keratinocytes damaged by oxidative stress. These results demonstrate the latent prosurvival function of syntaxin3 and underline the importance of intracellular molecular elements for the maintenance of homeostasis in epidermal keratinocytes.     

Altered expression of dermokine in skin disorders

Background Although dermokine‐β, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl‐terminal domain shares a high pI value with many cytokines, suggesting similar functions. Objective To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. Methods We generated an anti‐human dermokine‐β/γ monoclonal antibody cross‐reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine‐β/γ were performed with various tissue samples. Results Although human dermokine‐β/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti‐dermokine‐β/γ antibody in inflammatory skin disorders. Dermokine‐β/γ was expressed in keratoacanthoma and a part of well‐differentiated squamous cell carcinoma (SCC). However, dermokine‐β/γ was not detected in poorly differentiated SCC or tumours derived from non‐keratinocytes. In mice, dermokine‐β/γ‐expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet‐irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin‐1β, interleukin‐12, and tumour necrosis factor‐α augmented dermokine‐β/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin‐like growth factor‐I, keratinocyte growth factor and transforming growth factor‐α significantly reduced dermokine expression. Conclusion These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis.     

Expression of insulin-like growth factor I by cultured skin substitutes does not replace the physiologic requirement for insulin in vitro

Clinical efficacy of cultured skin substitutes may be increased if their carbohydrate metabolism is optimized by understanding whether endogenous insulin-like growth factor I can substitute for exogenous insulin. Cultured skin substitutes were prepared and incubated at the air-liquid interface for 4 wk in media containing 0.5 or 5 microg per ml insulin, 10 or 50 ng per ml insulin-like growth factor I, or 0 insulin and 0 insulin-like growth factor I (negative control). In situ hybridization showed that the epidermal and dermal cultured skin substitute components express insulin-like growth factor I mRNA throughout the 28 d interval. Immunohistochemistry confirmed the expression of insulin-like growth factor I protein by the human keratinocytes and fibroblasts in cultured skin substitutes. Insulin-like growth factor I at 10 or 30 ng per ml could partially replace insulin in a clonal assay of keratinocyte growth. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays showed significantly higher values in cultured skin substitutes incubated with insulin at incubation days 14 and 28 compared to negative control or the 10 ng per ml insulin-like growth factor I condition. Cultured skin substitutes incubated in 50 ng per ml insulin-like growth factor I had MTT values similar to the insulin-treated cultured skin substitutes at day 14, but were significantly lower by day 28. Light microscopy agreed with MTT data showing that cultured skin substitutes grown with insulin media had multiple layers of nucleated keratinocytes and stratum corneum at days 14 and 28. The negative control and 10 ng per ml insulin-like growth factor I exhibited poor cultured skin substitute epidermal morphology throughout the experiment. In contrast, the cultured skin substitutes in 50 ng per ml insulin-like growth factor I were similar to the insulin-treated cultured skin substitutes at day 14, but by day 28 had deteriorated to resemble the negative control. Bromodeoxyuridine incorporation at day 28 was significantly higher for 5 microg per ml insulin cultured skin substitutes versus all other treatment groups. These data suggest that medium containing 5 microg per ml insulin supports greater physiologic stability in cultured skin substitutes over time, and that expression of insulin- like growth factor I by keratinocytes and fibroblasts in cultured skin substitutes is not sufficient to fully replace the requirement for exogenous insulin in vitro.     

Insulin‐like growth factor‐I gene polymorphism in acne vulgaris

Background Acne vulgaris is a multifactorial disease of the skin. Several studies have shown that elevated levels of serum insulin‐like growth factor‐I (IGF‐I) correlate with overproduction of sebum and acne. Recently functional relationship between IGF‐I (CA) polymorphism and circulating IGF‐I levels in adults has been reported. Aims The aim of our study was to investigate for the first time whether IGF‐I (CA) polymorphism might be involved in the pathogenesis of acne or not. Methods We included 115 acne patients and 117 healthy subjects to the study. The clinical grade of acne was assessed based on the Global Acne Grading System. Participants were questioned about diabetes mellitus, PCOS and other systemic disease. We searched for the IGF‐I (CA) 19 polymorphism in this study. The IGF‐I (CA) 19 polymorphism was performed by polymerase chain reaction. Results We categorized the IGF‐I (CA) 19 polymorphism area into three groups as lower than 192 bp, 192–194 bp and higher than 194 bp. We found that the frequency of genotype IGF‐1 (CA) 19 gene was significantly different between control and acne patients (P = 0.0002). A significant association between IGF‐I (CA) genotypes and severity of acne was found (P = 0.015). No significant difference was found between male and female patients (P > 0.05). Conclusions Our results suggest that IGF‐I (CA) 19 polymorphism may contribute to a predisposition to acne in Turkish patients.     

Topical tacrolimus has a limited direct effect on ultraviolet B‐irradiated keratinocytes: implications for its photocarcinogenic potential

Background. Topical tacrolimus has shown remarkable clinical efficacy in treating many dermatoses. Combining ultraviolet (UV) B and tacrolimus is an intriguing therapeutic regimen, especially for treatment of vitiligo, for which combination therapy may show greater clinical efficacy than topical tacrolimus alone. The photocarcinogenic potential of such a regimen is unclear, and conflicting results have been reported by different investigators. Aim. To clarify this important clinical issue, we investigated the effects of tacrolimus on UVB‐irradiated cultured keratinocytes in terms of apoptosis, differentiation, cell‐cycle regulation and DNA damage. Methods. Cultured keratinocytes were treated with tacrolimus before and after UVB irradiation and the various cellular physiological changes were evaluated using trypan blue exclusion, terminal dUTP nick‐end labelling, flow cytometry and Western blotting analyses. Results. Our results showed that treatment of tacrolimus before or after UVB irradiation had no significant effects on cultured keratinocytes in terms of cell apoptosis, transglutaminase‐1, involucrin expression, cell‐cycle progression and phospho‐H₂AX compared with UVB irradiation alone. Conclusion. The direct effect of tacrolimus on UVB‐irradiated keratinocytes is small, suggesting that clinical regimens combining UVB and tacrolimus also have a limited direct effect on healthy skin compared with UVB irradiation alone.     

Redistribution of the nuclear protein IFI16 into the cytoplasm of ultraviolet B-exposed keratinocytes as a mechanism of autoantigen processing

Background The skin has long been recognized as a prominent target tissue in systemic lupus erythematosus (SLE) which plays a crucial role in the initiation and perpetuation of the autoimmune reaction cascade as a consequence of ultraviolet (UV)‐induced keratinocyte apoptosis. Antibodies against IFI16 (interferon‐inducible protein 16) have been detected in the sera of patients with SLE. Objectives To verify whether the induction of autoimmunity against IFI16 involves redistribution of this nuclear protein in keratinocytes during UVB‐induced cell death. Methods An in vitro epidermal model was developed to investigate the fate of the IFI16 protein in keratinocytes after irradiation with UVB; both keratinocyte monolayers and human skin explants were used. IFI16 expression and localization were also analysed in diseased skin sections of patients with SLE. Results We demonstrated that IFI16, normally restricted to the nucleus, can be induced to appear in the cytoplasm under conditions of UVB‐induced cell injury. This nucleus to cytoplasm translocation was also observed in skin explants exposed to UVB and in the diseased skin sections from patients with SLE. In addition, IFI16 was found in the supernatants of UVB‐exposed keratinocytes. Conclusions The finding that IFI16 is present in the cytoplasm of diseased skin cells from patients with SLE and the demonstration of IFI16 in the supernatants of UVB‐exposed keratinocytes, suggest that UVB irradiation or other stimuli may favour an abnormal IFI16 presentation to the afferent limb of the immune system and potentially an autoimmune response against the protein itself.     

UV radiation induces CXCL5 expression in human skin

CXCL5 has recently been identified as a mediator of UVB‐induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose‐dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non‐immune skin cells also contribute to UV‐induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte‐keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV‐radiated human skin and the essential role of fibroblast‐keratinocyte interaction in the regulation of epidermal CXCL5.     

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.     

Acute keratinocyte damage stimulates platelet-activating factor production

Abstract Recent evidence suggests that the phosphocholine-derived lipid mediator platelet-activating factor (PAF) is involved in keratinocyte function and cutaneous inflammation. PAF is found in various inflammatory skin diseases, and intradermal injection of PAF directly results in cutaneous inflammation. Keratinocytes also synthesize PAF and related 1-acyl species in response to ionophores, cytokines and growth factors, and in response to activation of the epidermal PAF receptor. Since keratinocytes are routinely exposed to potential damage by thermal or oxidative stressors with resultant induction of cutaneous inflammation, the objective of these studies was to assess whether exogenous thermal or oxidative damage can induce the production of PAF and related 1-acyl species. Cells of the immortalized human keratinocyte cell line HaCaT were subjected to acute heat or cold, or treatment with the pro-oxidant lipid tertiary butyl hydroperoxide, and PAF and 1-palmitoyl-2-acetyl-GPC were measured by gas chromatography/mass spectrometry. We report that these diverse toxic stimuli resulted in the accumulation of these biologically active lipids. These studies suggest that the PAF system is involved in the inflammatory response seen following acute epidermal damage. Received: 21 September 1999 / Revised: 11 January 2000 / Accepted: 14 January 2000     

Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model

BACKGROUND: Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. OBJECTIVES: To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. METHODS: We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyte-conditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo[tumour necrosis factor (TNF)-alpha] or modify collagen biochemistry [putrescine, estrone, estradiol and beta-aminopropionitrile (beta-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte/fibroblast cocultures. RESULTS: Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-alpha and beta-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. CONCLUSIONS: Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.