Proliferation Of Normal Human Epidermal Keratinocytes Research Articles

Effects of Schumann resonance on the proliferation and migration of normal human epidermal keratinocytes and the expression of DEFB1 and SIRT1.

When the skin is damaged and its barrier function is disrupted, the proliferation and migration of epidermal keratinocytes are vital for repairing the damaged area. The Schumann resonance at 7.8 Hz has been reported to protect rat cardiomyocytes against oxidative stress and inhibit the proliferation of B16 mouse melanoma cells. However, its effect on the skin is unknown. In this study, we applied 7.8-Hz electromagnetic waves to normal human epidermal keratinocytes (NHEKs) and investigated its effects on cell proliferation and migration, β-defensin (DEFB1) and sirtuin 1 (SIRT1) expression. We performed cell proliferation assay, cell migrationassay and gene expression analysis of DEFB1 and SIRT1. We found that the application of 7.8-Hz electromagnetic waves caused a 2.8-fold increase in NHEK proliferation, enhanced cell migration, and increased the expression of DEFB1 and SIRT1 by 2.4-fold and 4.9-fold, respectively. These results suggest that the application of 7.8-Hz electromagnetic waves may contribute to improving the skin barrier function and skin ulcer.

Cinnamaldehyde inhibits psoriasis‑like inflammation bysuppressing proliferation and inflammatory response of keratinocytes via inhibition of NF‑κB and JNK signaling pathways.

Psoriasis is a systemic immune-mediated inflammatory disease characterized by uncontrolled keratinocyte proliferation and poor differentiation. Cinnamaldehyde (CIN) has been shown to inhibit the proliferation and inflammatory response of primary and immortalized immune cells. However, to the best of our knowledge, the role of CIN in the progression of psoriasis remains unclear. Therefore, the present study aimed to investigate the biological role of CIN in psoriasis. To mimic abnormal proliferation and differentiation in keratinocytes in vitro, normal human epidermal keratinocytes (NHEKs) were stimulated with M5 (IL-1α, IL-17A, IL-22, oncostatin M and TNF-α). The viability and proliferation of NHEKs were analyzed using Cell Counting Kit-8 and 5-Ethynyl-2′-deoxyuridine assays, respectively. Western blotting was used to analyze the expression levels of keratin 1, filaggrin and loricrin in NHEKs. The results of the present study revealed that CIN significantly inhibited the proliferation and cell cycle progression, and promoted the differentiation of M5-stimulated NHEKs. CIN also markedly attenuated the extent of oxidative stress-induced damage in M5-stimulated NHEKs. Moreover, CIN ameliorated M5-induced inflammatory injury in NHEKs, as evidenced by the decreased levels of multiple inflammatory factors. Furthermore, CIN notably downregulated the expression levels of phosphorylated (p)-inhibitor of NF-κB, p-p65 and p-JNK in M5-stimulated NHEKs. In conclusion, the present data suggested that CIN may protect NHEKs against M5-induced hyperproliferation and inflammatory injury via inhibition of NF-κB and JNK signaling pathways. These results provide a novel insight on the role of CIN in psoriasis.

Infrared-A Irradiation-induced Inhibition of Human Keratinocyte Proliferation and Potential Mechanisms.

Infrared‐A (IRA), which can penetrate deeply into the human skin, is a major component of solar radiation and is recognized to promote photoaging of human dermis. To our knowledge, however, the cellular and molecular consequences of human epidermis exposure to IRA have not been clarified. Thus, we investigated whether IRA inhibits the proliferation of normal human epidermal keratinocytes (NHEKs). IRA irradiation ed in cell cycle arrest at G1 and a dose‐dependent reduction in the proliferation of NHEKs. We found that mechanistic target of rapamycin complex 1 (mTORC1) was initially inactivated during IRA irradiation due to the formation of stress granules (SGs), and this inactivation was maintained for at least 6 h after irradiation due to Akt dephosphorylation. Furthermore, repeated exposure of human skin equivalents to IRA led to marked thinning of the epidermal cell layer. In conclusion, IRA irradiation inhibits mTORC1 activity possibly through two molecular mechanisms involving SG formation in the early‐phase and subsequent Akt dephosphorylation. This sequential mechanism seems to cause G1 cell cycle arrest and a reduction in cell proliferation, supporting the hypothesis that the decreased proliferation of basal keratinocytes that occurs during skin aging might be partly attributable to IRA radiation.

Downregulation of miR-145-5p contributes to hyperproliferation of keratinocytes and skin inflammation in psoriasis.

The extensive involvement of microRNAs (miRNAs) in the pathogenesis of psoriasis is well documented. However, little is known about the contribution of specific miRNAs to the prevalence of this disease. To explore the role of miR-145-5p in psoriasis. miRNA microarray analysis was performed in four patients with psoriasis and four controls. Quantitative reverse-transcriptase polymerase chain reaction and fluorescence insitu hybridization were used to identify the dysregulated miRNAs. Luciferase assays were performed to determine whether miR-145-5p targets mixed-lineage kinase (MLK)3. CCK-8 assay and Magnetic Luminex Assay were performed to measure cell proliferation and chemokine secretion. Western blot analysis was used to investigate the protein levels of MLK3 and its downstream effectors. Mouse models of psoriasis were established for invivo experiments. miR-145-5p was downregulated in psoriatic lesional skin. Luciferase assays showed that MLK3 is a direct target of miR-145-5p. Overexpression of miR-145-5p in normal human epidermal keratinocytes (NHEKs) suppressed cell proliferation and secretion of chemokines. In contrast, silencing miR-145-5p promoted NHEK proliferation and increased chemokine secretion. Silencing MLK3 abrogated miR-145-5p inhibitor-induced promotion of cell proliferation and chemokine expression. miR-145-5p regulates nuclear factor-κB and signal transducer and activator of transcription 3 by targeting MLK3. Delivery of agomiR-145-5p into the skin decreased epidermal hyperplasia and ameliorated psoriasis-like dermatitis. Delivery of antagomiR-145-5p led to the opposite effects. Our findings indicate that miR-145-5p negatively regulates proliferation and chemokine secretion of NHEKs by targeting MLK3, and downregulation of miR-145-5p contributes to skin inflammation in psoriasis lesions.

Cystine-thiamin-containing hair-growth formulation modulates the response to UV radiation in an in vitro model for growth-limiting conditions of human keratinocytes

BackgroundUltraviolet radiation (UVR) is known to be harmful to normal human epidermal keratinocytes (NHEKs) of the epidermal skin layer, as well as to hair-follicle-associated keratinocytes. An oral formulation containing l-cystine, thiamin, calcium d-pantothenate, medicinal yeast, keratin and p-aminobenzoic acid (Panto[vi]gar®) has demonstrated clinical efficacy for the treatment of diffuse telogen effluvium; however, its mode of action at the cellular level, and in particular whether protective mechanisms are involved, has yet to be elucidated. ObjectivesTo assess the capacity of ingredients of this oral formulation, both separately and in combination, to modulate the effects of UVR in growth-limited NHEKs in vitro. MethodsNHEKs were incubated in keratinocyte basal medium, keratinocyte basal medium lacking cystine, thiamin, calcium d-pantothenate, folic acid and biotine (minimal growth medium [MGM]) or MGM plus test compound. Test compounds comprised the following four ingredients related to the oral formulation: l-cystine, thiamin, calcium d-pantothenate and folic acid (a proposed metabolite of p-aminobenzoic acid), and a combination of these (Panto[vi]gar®-in vitro correlate; P-IC). The effect of different doses of these compounds on the metabolic activity and proliferation of NHEKs was tested, as well as their influence on the impact of UV light on NHEKs assessed by monitoring metabolic activity, cell number and apoptosis induction. ResultsCompared with basal medium, MGM reduced the proliferation of NHEKs in a time-dependent manner. Reduced proliferation is a characteristic of the multifactorial and complex phenotype associated with diffuse hair loss. l-cystine (50 μM) increased metabolic activity and proliferation 3-fold versus MGM (p < 0.05). Thiamin also had a significant effect (p < 0.05) on proliferation and metabolic activity of NHEKs, but calcium d-pantothenate and folic acid did not when tested individually in this in vitro model. In the presence of P-IC, metabolic activity increased 4-fold and proliferation 3-fold compared with MGM alone (p < 0.05 for both).Following UV irradiation, cells in MGM showed a 72% reduction in metabolic activity, while P-IC-treated cells showed only a 12–18% reduction. The observed prevention of the UV-induced reduction in metabolic activity was not simply due to filtering UVR by the P-IC components, as P-IC-mediated reduction of this effect persisted even when P-IC was washed out during UV irradiation. ConclusionThis study demonstrated that l-cystine and thiamin are essential for proliferation of epidermal keratinocytes and suggests a novel, UV-protective potential of formulations combining l-cystine and thiamin in growth-limited inter-follicular NHEKs in vitro.

Th2 cytokines enhance TrkA expression, upregulate proliferation, and downregulate differentiation of keratinocytes

BackgroundNerve growth factor (NGF), a neurotrophin that plays a critical role in developmental neurobiology, is released by proliferating keratinocytes and induces proliferation. ObjectiveThe aim of this study was to investigate the role of tyrosine kinase receptor A (TrkA), a high-affinity receptor of NGF, in human keratinocytes. MethodsExpression of TrkA and NGF in skin diseases was investigated by immunohistochemistry. Expression of TrkA in cells was examined by Western blotting and RT-PCR. Cell proliferation was assessed by BrdU assay. ResultsWe first determined the expression of TrkA and NGF in skin samples from patients with atopic dermatitis, prurigo nodularis, psoriasis vulgaris, and seborrheic keratosis. TrkA was only expressed in proliferating basal cells, and its expression was enhanced in atopic dermatitis samples. NGF expression was enhanced in atopic dermatitis and prurigo nodularis samples and in some samples from seborrheic keratosis patients. Investigation of the role of TrkA in vitro using normal human epidermal keratinocytes (NHEK) revealed that TrkA was significantly enhanced by the T helper type 2 (Th2) cytokines interleukin (IL)-4 and IL-13 but not by other inflammatory cytokines, such as IL-1β, tumor necrosis factor α, interferon γ, or epidermal growth factor. On the other hand, expression of NGF was not altered by Th2 cytokines. Notably, inhibition of TrkA significantly reversed the effects of IL-4 on proliferation and differentiation. Furthermore, overexpression of TrkA enhanced proliferation of NHEK. These results indicate that IL-4-induced TrkA expression in keratinocytes modulates proliferation and differentiation of these cells. ConclusionIncreased TrkA expression in keratinocytes in atopic dermatitis may contribute to the observed epidermal hyperproliferation in these patients.

Efficient fabrication of epidermal cell sheets using γ-secretase inhibitor

Epidermal cell sheets have been utilized for regeneration of skin when skin defects occur and prevention of esophageal stricture after endoscopic submucosal dissection. To reduce the cost of cultivation, a novel culture method to shorten a culture process needs to be developed. To shorten a culture process of epidermal cell sheets, we developed a novel culture method to accelerate the fabrication of epidermal cell sheets using γ-secretase inhibitor. Normal human epidermal keratinocytes (NHEKs) were cultured using γ-secretase inhibitor, DAPT, during expansion of the cells to confluence and culture without DAPT during stratification. The cell growth, quantitative gene expression of stem/progenitor or differentiation markers, and protein expression of these markers were analyzed to verify the effectiveness of the novel method. The proliferation of NHEKs on cell-culture inserts was promoted using DAPT. However, NHEKs were not stratified completely in the presence of DAPT. In contrast, NHEKs cultured using DAPT were stratified and differentiated by eliminating the inhibitor after the cells reached confluence. Real-time PCR analyses showed that the gene expressions of putative epithelial stem/progenitor cell markers and epidermis differentiation markers in the cell sheets fabricated using this novel method were significantly higher than those in the cell sheets fabricated without DAPT. Histological and immunofluorescence analyses revealed that it was possible to fabricate well-differentiated epidermal cell sheets efficiently by the novel culture method. The culture period was shortened to 67% of the time required for the control group. In feeder-free conditions, stratified epidermal cell sheets were also fabricated using DAPT. The novel culture method using γ-secretase inhibitor, DAPT, was found to be effective for fabricating epidermal cell sheets.

Resveratrol inhibition of human keratinocyte proliferation via SIRT1/ARNT/ERK dependent downregulation of aquaporin 3

BackgroundAquaporin 3 (AQP3) is the predominant aquaporin in the skin and is overexpressed in hyperplastic epidermal disorders. Upregulation of AQP3 contributes to keratinocyte proliferation and epidermal hyperplasia. Resveratrol, a natural polyphenol, has an anti-proliferative effect on normal human epidermal keratinocytes (NHEKs), but its exact mechanism remains largely unknown. ObjectiveTo investigate the ability and mechanism of resveratrol to affect the proliferation and the AQP3 expression in NHEKs. MethodsNHEKs treated with resveratrol were analyzed. BrdU incorporation assay, real-time PCR, Western blotting and RNA interference using small interfering RNA were employed. ResultsAt non-toxic concentrations (less than 40μM), resveratrol inhibited the proliferation of NHEKs. Resveratrol inhibited the ERK phosphorylation and the AQP3 expression with reciprocal upregulation of ARNT expression in a concentration-dependent manner. The inhibitory effects of resveratrol on the ERK phosphorylation and the AQP3 expression were canceled by transfection of siRNA for ARNT, but not by that for AhR. Furthermore, the induction effect of resveratrol on ARNT expression was canceled after SIRT1 was knocked down in NHEKs. ConclusionResveratrol inhibited NHEK proliferation by downregulating the expression of AQP3 in an SIRT1/ARNT/ERK dependent fashion. This novel mechanism may facilitate drug innovation for hyperplastic skin disorders.

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1×10−13M cortisol, whereas 1×10−5M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.

Resveratrol Induces Long-Lasting IL-8 Expression and Peculiar EGFR Activation/Distribution in Human Keratinocytes: Mechanisms and Implications for Skin Administration

Anti-inflammatory and skin tumour preventing effects of resveratrol have been extensively studied pre-clinically and resveratrol has been proposed for clinical investigations. To provide a basis or/and limitations for topical administration to human skin, molecular mechanisms underlying resveratrol effects towards normal human epidermal keratinocytes (NHEK) were evaluated. NHEK were challenged by either resveratrol alone or by its combination with TNFalpha or TGFalpha, and time-dependent molecular events were monitored. Interleukin 8 (IL-8) expression and its mRNA stability, ERK1/2, p65/RelA, and EGFR phosphorylation were determined. Intracellular distribution of EGFR/P-EGFR was measured in the membrane, cytoplasmic, and nuclear fractions. Specific DNA binding activity of NFκB (p65/RelA) and AP-1(c-Fos), NHEK proliferation, and molecular markers of apoptosis/cell cycle were detected. Resveratrol induced delayed, long-lasting and steadily growing IL-8 gene and protein over-expression as well as enhanced EGFR phosphorylation, both abrogated by the EGFR kinase inhibitor PD168393. However, resveratrol did not act as a phosphatase inhibitor. ERK phosphorylation was transiently inhibited at early time-points and activated at 6–24 h. Accordingly, c-Fos-specific DNA binding was increased by resveratrol. Cellular distribution of EGFR/P-EGFR was shifted to membrane and nucleus while cytosolic levels were reduced concomitant with enhanced degradation. Notwithstanding high nuclear levels of EGFR/P-EGFR, spontaneous and TGFalpha-triggered cell proliferation was strongly suppressed by resveratrol mainly through cell cycle arrest.Conclusions/SignificanceResveratrol synergized with TNFα in the induction of delayed, long-lasting IL-8 expression through sustained EGFR-ERK axis activation. The time course indicates that resveratrol metabolites could be implicated. Topical administration of Resv to psoriatic patients over-expressing TNFα, IL-8 and EGFR-ERK in the skin should be cautiously considered. Since high nuclear levels of EGFR correspond to increased risk of tumorigenesis, chronic resveratrol application to the skin may be potentially dangerous. Wound healing acceleration by resveratrol could not be envisaged due to its anti-proliferative effects towards normal keratinocytes.

Topical Treatment with Basic Fibroblast Growth Factor Promotes Wound Healing and Barrier Recovery Induced by Skin Abrasion

It has been reported that basic fibroblast growth factor (bFGF) promotes the healing of skin ulceration by inducing fibroblast proliferation, yet the role of bFGF on epidermal barrier function, especially from the perspective of scratch-induced skin abrasion, remains unknown. To this end, we initially developed an epidermal abrasion mouse model induced by scratching with a stainless-steel wire brush, and examined the effects of bFGF on the wound healing induced by skin abrasion. This procedure induced a significant elevation of transepidermal water loss (TEWL) in a scratch-count-dependent manner. This elevated TEWL was significantly decreased following topical application of bFGF to the skin. In addition, bFGF increased the expression of Ki67 in keratinocytes following mechanical scratching. These results suggest that bFGF enhances keratinocyte proliferation, which, in turn, repairs the skin barrier disruption and wounds caused by scratching in mice. Consistently, bFGF stimulated proliferation of normal human epidermal keratinocytes (NHEK). Intriguingly, the effect of bFGF and other growth factors on NHEK proliferation was additive. However, high cell density diminished the effect of bFGF on NHEK proliferation. This particular result can be explained by our observation that FGF receptor mRNA expression in NHEK was low under conditions of high cell density. Our findings suggest that bFGF stimulates keratinocyte proliferation, especially in a lower cell density environment, to repair skin wound in accord with skin barrier recovery.

11β-Hydroxysteroid Dehydrogenase-1 Is a Novel Regulator of Skin Homeostasis and a Candidate Target for Promoting Tissue Repair

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the interconversion of cortisone and cortisol within the endoplasmic reticulum. 11β-HSD1 is expressed widely, most notably in the liver, adipose tissue, and central nervous system. It has been studied intensely over the last 10 years because its activity is reported to be increased in visceral adipose tissue of obese people. Epidermal keratinocytes and dermal fibroblasts also express 11β-HSD1. However, the function of the enzymatic activity 11β-HSD1 in skin is not known. We found that 11β-HSD1 was expressed in human and murine epidermis, and this expression increased as keratinocytes differentiate. The expression of 11β-HSD1 by normal human epidermal keratinocytes (NHEKs) was increased by starvation or calcium-induced differentiation in vitro. A selective inhibitor of 11β-HSD1 promoted proliferation of NHEKs and normal human dermal fibroblasts, but did not alter the differentiation of NHEKs. Topical application of selective 11β-HSD1 inhibitor to the dorsal skin of hairless mice caused proliferation of keratinocytes. Taken together, these data suggest that 11β-HSD1 is involved in tissue remodeling of the skin. This hypothesis was further supported by the observation that topical application of the selective 11β-HSD1 inhibitor enhanced cutaneous wound healing in C57BL/6 mice and ob/ob mice. Collectively, we conclude that 11β-HSD1 is negatively regulating the proliferation of keratinocytes and fibroblasts, and cutaneous wound healing. Hence, 11β-HSD1 might maintain skin homeostasis by regulating the proliferation of keratinocytes and dermal fibroblasts. Thus 11β-HSD1 is a novel candidate target for the design of skin disease treatments.

Development of salmon milt DNA/salmon collagen composite for wound dressing

This study aims to develop a novel wound dressing comprising salmon milt DNA (sDNA) and salmon collagen (SC). The sDNA/SC composites were prepared by incubating a mixture of an acidic SC solution, an sDNA solution, and a collagen fibrillogenesis inducing buffer (pH 6.8) containing a crosslinking agent (water-soluble carbodiimide) for gelation, and a subsequent ventilation-drying process to give sDNA/SC films. The conjugation between sDNA and SC were confirmed by sDNA-elution assay and fluorescence microscopy. The sDNA/SC films with various doses of sDNA (sDNA/SC weight ratios of 1:5, 1:10, and 1:20) were used for in vitro cell cultures to evaluate their growth potentials of normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK). It was found that NHDF proliferation was increased by sDNA conjugation, whereas NHEK proliferation was dose-dependently inhibited. In light of the in vitro results, the appropriate dose of sDNA for in vivo study was determined to be the ratio of 1:10. For the implantation in full-thickness skin defects in rat dorsal region, the sDNA/SC films were reinforced by incorporating them on a porous SC sponge, because the sDNA/SC films exhibited early contraction and inadequate morphologic stability when implanted in vivo. The regenerated tissue in the sDNA/SC sponge group showed similar morphology to native dermis, while the SC sponge group without sDNA showed epithelial overgrowth, indicating that additional sDNA could reduce epidermal overgrowth. Furthermore, blood capillary formation was significantly enhanced in the sDNA/SC sponge group when compared to the SC sponge group. In conclusion, the results suggest that the sDNA/SC composite could be a potential wound dressing for clinical applications.

Gene expression changes associated with altered growth and differentiation in benzo[a]pyrene or arsenic exposed normal human epidermal keratinocytes

Both arsenic and benzo[a]pyrene (BaP) inhibit terminal differentiation and alter growth potential in normal human epidermal keratinocytes (NHEK) in vitro. To identify molecular alterations that may be involved in these cellular processes, microarray analysis was carried out on NHEK treated with BaP or arsenic. The gene expression microarray results measuring mRNA levels were as follows: (1) in total, the expression of 85 genes was induced and 17 genes was suppressed by 2.0 microm BaP. (2) Arsenic at an equitoxic dose (5.0 microm) induced the expression of 106 and suppressed 15 genes. Quantitative real-time RT-PCR was used subsequently to confirm microarray findings on selected genes involved in keratinocyte growth and differentiation pathways. These studies confirmed increased mRNA levels in NHEK by BaP of alpha-integrin binding protein 63 (AIBP63) (2.48-fold), retinoic acid- and interferon-inducible protein (IFIT5) (2.74-fold), interleukin-1 alpha (IL1A) (2.64-fold), interleukin-1 beta (IL1B) (2.84-fold) and Ras guanyl releasing protein 1 (RASGRP1) (3.14-fold). Real-time RT-PCR confirmed that arsenic increased mRNA levels of the following genes: retinoblastoma 1 (RB1) (5.4-fold), retinoblastoma-binding protein 1 (ARID4A) (6.8-fold), transforming growth factor beta-stimulated protein (TSC22D1) (6.84-fold), MAX binding protein (MNT) (2.44-fold), and RAD50 (4.24-fold). Collectively, these results indicate that these chemicals target different genes and molecular pathways involved in the regulatory processes controlling NHEK proliferation and differentiation. Mechanistic studies with a subset of genes may allow the correlation of alterations in these molecular markers with chemical-specific blocks to differentiation in NHEK.

Novel Function for Vascular Endothelial Growth Factor Receptor-1 on Epidermal Keratinocytes

Vascular endothelial growth factor (VEGF-A), a potent stimulus for angiogenesis, is up-regulated in the skin after wounding. Although studies have shown that VEGF is important for wound repair, it is unclear whether this is based solely on its ability to promote angiogenesis or if VEGF can also promote healing by acting directly on non-endothelial cell types. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was detected in murine keratinocytes during wound repair and in normal human epidermal keratinocytes (NHEKs). The presence of VEGF receptors on NHEKs was verified by binding studies with 125I-VEGF. In vitro, VEGF stimulated the proliferation of NHEKs, an effect that could be blocked by treatment with neutralizing VEGFR-1 antibodies. A role for VEGFR-1 in keratinocytes was also shown in vivo because treatment of excisional wounds with neutralizing VEGFR-1 antibodies delayed re-epithelialization. Treatment with anti-VEGFR-1 antibodies also reduced the number of proliferating keratinocytes at the leading edge of the wound, suggesting that VEGF sends a proliferative signal to these cells. Together, these data describe a novel role for VEGFR-1 in keratinocytes and suggest that VEGF may play several roles in cutaneous wound repair.

The effects of cyclosporin A and FK506 on proliferation and IL-8 production of cultured human keratinocytes

The present study was conducted to determine whether cyclosporin A (CsA) and FK506 could be effective in inhibiting the proliferation and cytokine secretion of normal human epidermal keratinocytes (NHEK). NHEK proliferation in the presence of CsA and FK506 at the concentrations 10 −9 to 10 −5 M at 24 and 48 h time points was measured colorimetrically by the MTS assay. CsA had inhibitory effects from 10 −6 to 10 −5 M, while FK506 had no effect, except for toxicity at the very highest concentrations (5 × 10 −6 M and higher). NHEK cells spontaneously secrete IL-8 (243.4 ± 55.5 pg/ml), and this baseline level was augmented by TNF-α alone, or synergistically by TNF-α and IFN-γ, which are thought to be secreted by T cells. Neither CsA nor FK506 had any significant effect on either spontaneous or cytokine-stimulated keratinocyte IL-8 production. Therefore, it is most likely that the two drugs indirectly inhibit the keratinocyte inflammatory response through their actions on T cells or other immunocompetent cells.

Protein Kinase C in Normal Human Epidermal Keratinocytes During Proliferation and Calcium-Induced Differentiation

Recent evidence has implicated protein kinase C (PKC) in the etiology of hyperproliferative diseases such as psoriasis and non-melanoma skin cancer. In this study, PKC activity, immunoreactive protein, and phorbol ester-binding kinetics were examined in primary cultures of normal human epidermal keratinocytes (NHEK) in order to elucidate the relationship between PKC and NHEK proliferation and differentiation. NHEK were maintained in a proliferative phase in serum-free low-calcium (0.15 mM) medium, and then were exposed to high calcium (1.6 mM) in order to stimulate growth arrest and differentiation. Staurosporine was inhibitory to Ca(++)-induced differentiation. Scatchard analysis of phorbol binding indicated that exposure to high calcium for 24 h increased the number of binding sites (Bmax) by fivefold. In correlation with the ligand-binding results, PKC activity was extremely low in proliferating (low-calcium) NHEK compared to differentiating cells (high calcium). When assayed after 24, 48, and 72 h, high calcium induced tenfold or greater increases in Ca++/phospholipid-dependent phosphotransferase activity. Immunoblot analysis of NHEK PKC using antibodies directed against the hinge region of PKC alpha/beta also indicated that exposure to high calcium resulted in higher levels of immunoreactive protein. Therefore, PKC in NHEK appears to be upregulated under conditions of Ca(++)-induced growth arrest and differentiation. In addition, NHEK and other human skin cell particulate fractions contain a protein of approximately 116 kDa that is highly immunoreactive to an antibody to PKC alpha/beta, which coelutes from DEAE-sephacel under the same buffer conditions as the 80-kDa PKC.