OBJECTIVETo observe the change of caspase-8, caspase-9 activity and apoptosis rates in the process of trichloroethylene-induced damage in keratinocytes, and explore the tentative mechanism of apoptosis.

METHODSHuman keratinocytes were exposed to 0.125, 0.250, 0.500, 1.000 and 2.000 mmol/L trichloroethylene for 4, 8, 12 and 24 h. The inhibitive groups were pretreated with 100 micromol/L Z-LEHD-FMK (a specific inhibitor of caspase-9) for 1 h, and were stimulated with 2.000 mmol/l TCE for 12 h. MTT assay was used to detect the viability of different cells; The activity of caspase were calculated according to spectrophotometry; Change of the apoptotic rates was assessed by flow cytometer (FCM) after double-stained with Annexin V-FITC and propidium iodide (PI).

RESULTS(1) The minimum effective concentration for cell viability reduction was 0.125 mmol/L at 12 h and the shortest time required to produce a change was 4 h at a concentration of 2.000 mmol/L (compared with control group, P < 0.01). Cell viability in all the groups markedly decreased from 12 h to 24 h (P < 0.05). (2) The activity of caspase-8 in the various dosage groups at different times had no statistical difference compared with the control group, P > 0.01. (3) At 8 h, 1.000 and 2.000 mmol/L TCE groups could significantly enhance caspase-9 activity (P < 0.05). The caspase-9 activity in all the groups showed differences and was significantly higher than those of control cells when time was over 12 h (P < 0.05). (4) After exposing to different dosages of TCE for 12 h, the rate of apoptosis rose to (80.43 +/- 4.21)% with the increase of dosage, compared with the control group, (9.40 +/- 2.98)%, which showed a dose-effect relationship. (5) The cells pre-treated with caspase-9 inhibitor resulted in a decrease in the caspase-9 activity and apoptosis rates (compared with 2.000 mmol/L TCE exposed group, P < 0.01). However, there was no statistical significance in comparison with the control group (P > 0.05).

CONCLUSIONCaspase-9 may be an important mediator of apoptosis in keratinocytes induced by trichloroethylene.

Apoptosis ; drug effects ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Humans ; Keratinocytes ; drug effects ; enzymology ; pathology ; Trichloroethylene ; toxicity

OBJECTIVETo investigate the toxic and DNA damaging effect of acrylamide (AA) on human keratinocytes and its mechanism.

METHODS(1) After the keratinocyte cell line HaCaT cells were exposed to AA with different concentrations for 44 hours, cell survival rate was detected by MTT method. (2) The effects of DNA damage of exposed cells were detected by comet assay. (3) After treating the cells with 2.00 mmol/L of AA plus 0.50 mmol/L of 1-aminobenzotriazole (1-ABT), an inhibitor of cytochrome P-450 enzymes (CYP-450), for 4 hours, the relationship between DNA damage and CYP-450 was studied.

RESULTS(1) Cytotoxicity measurement of AA showed that cell survival rate decreased significantly after 44-hour treatment. (2) Cytotoxicity was not detected after 4-hour AA treatment, but significant DNA damage was observed in all treatment groups, and the degree of damage increased with the concentration of AA. Moreover, the tail lengths of comet cells were in dose-effect relationship. As for cells treated by 1-ABT with 2 mmol/L AA, comet rate and tail length were 15.4% and (8.2 +/- 2.0) micro m respectively, which were decreased significantly (P < 0.01) when compared with 2 mmol/L AA treatment group [80.6% and (44.3 +/- 4.0) micro m].

CONCLUSIONSAcrylamide has significant cytotoxicity and genotoxicity on HaCaT cells. AA-induced DNA damage may be related to the oxidative metabolite(s) of AA through CYP-450.

Acrylamide ; toxicity ; Cells, Cultured ; Cytochrome P-450 Enzyme Inhibitors ; DNA Damage ; Dose-Response Relationship, Drug ; Humans ; Keratinocytes ; drug effects ; enzymology

Calpain I (mu-calpain) and II (m-calpain) are well known calcium-activated neutral cysteine proteases. Many reports have shown that activation of calpain is related to cataract formation, neuronal degeneration, blood clotting, ischemic injuries, muscular dystrophy and cornified cell envelope (CE) formation. Here, we report that insoluble CE formation was reduced after treatment with calpain I inhibitor (N-acetyl-leucyl-leucyl-norleucinal) on normal human epidermal keratinocytes (NHEK), whereas serine and thiol protease inhibitors had no effect on the reduction of CE. When NHEK cells were confluent, keratinocytes were treated with various concentrations (0.5 microM-0.5 mM) of calpain I inhibitor or serine and thiol protease inhibitors under calcium induced differentiation. Insoluble CE formation was reduced about 90% in the 50 microM calpain inhibitor I treated group by day 9 of culture, whereas insoluble CE was reduced only 10% in the same condition. Interestingly TGase activity was blocked by 90% in the 0.5 mM calpain inhibitor treated group within 72 h, whereas TGase activity was retained by 80% in the 0.5 mM serine protease inhibitor treated group at 7 day treatment. Therefore it can be suggested that cysteine protease calpains might be responsible for the activation of the TGase 1 enzyme to complete insoluble CE formation during epidermal differentiation.
Calcium/pharmacology ; Calpain/metabolism* ; Calpain/antagonists & inhibitors* ; Cell Differentiation ; Dose-Response Relationship, Drug ; Epidermis/metabolism ; Human ; In Vitro ; Keratinocytes/metabolism ; Keratinocytes/enzymology ; Protease Inhibitors/pharmacology ; Protein-Glutamine gamma-Glutamyltransferase/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/antagonists & inhibitors* ; Tissue Culture

OBJECTIVETo investigate the effects of arecoline and nicotine on the expression of human telomerase reverse transcriptase (hTERT) mRNA and protein in cultured normal human oral keratinocytes (KC).

METHODSThe experiments were divided into arecoline group, arecoline/nicotine group and control group. The hTERT mRNA and protein expression of KC was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot.

RESULTSArecoline could induce the hTERT mRNA and protein expression of KC in a dose dependent manner, the hTERT mRNA and protein expression of KC was higher in 0.030, 0.060, 0.090 g/L arecoline group than control group (P < 0.001). Nicotine (0.025 g/L) increased hTERT mRNA and protein expression of KC induced by arecoline.

CONCLUSIONSArecoline could increase the expression of hTERT mRNA and protein in oral keratinocytes. Nicotine had a synergistic effect on arecoline. hTERT over-expression induced by arecoline and nicotine may play an important role in the malignant transformation of oral submucous fibrosis.

Arecoline ; pharmacology ; Cells, Cultured ; Cholinergic Agonists ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Ganglionic Stimulants ; pharmacology ; Humans ; Keratinocytes ; drug effects ; enzymology ; Mouth Mucosa ; enzymology ; pathology ; Nicotine ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics ; metabolism

Transglutaminase (TGase) isoenzymes are involved in the process of the differentiation and cornification of keratinocytes in the epidermis. This study investigates the presence and localization of three TGase isoenzymes to elucidate the nature and differentiation status of the squamous epithelium in human aural cholesteatoma. Twenty cholesteatoma specimens were used. The presence and localization of three TGase isoenzymes were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. mRNA expression of three TGase isoenzymes were detected in the tested cholesteatomas with variable levels. The immunohistochemical staining patterns of three TGase isoenzymes showed variations within specimens, relating to keratinizing activity. TGase K is the most abundant among three isoenzymes. Keratinizing epithelium of cholesteatoma have similar expression profiles of TGase isoenzymes with those of epidermis of the skin. Other areas, particularly those showing non-keratinizing epithelium, showed weak immunostaining of TGase E and C, suggesting its different maturation status from keratinizing epithelium. The results of this study indicate that epithelium of cholesteatoma undergoes same direction of maturation and differentiation characteristics as the epidermis of skin, evidenced by similar expressions of TGases both in mRNA level and immunohistochemistry.
Cell Differentiation ; Cholesteatoma, Middle Ear/genetics ; Cholesteatoma, Middle Ear/enzymology* ; Comparative Study ; Diagnosis, Differential ; Epidermis/enzymology ; Epithelial Cells/enzymology ; Human ; Immunohistochemistry ; Isoenzymes/metabolism ; Isoenzymes/genetics ; Keratinocytes/enzymology ; Protein-Glutamine gamma-Glutamyltransferase/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/genetics ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium- induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis.
Acute-Phase Proteins/*biosynthesis ; Calcium/*metabolism ; Cell Differentiation ; Culture Media ; Culture Media, Conditioned ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Homeostasis ; Humans ; Keratinocytes/enzymology ; Lipocalins/*biosynthesis ; Models, Biological ; Proto-Oncogene Proteins/*biosynthesis ; Psoriasis/enzymology ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/*metabolism ; Skin Neoplasms/enzymology

OBJECTIVETo evaluate the effects of sodium arsenite on the activity, the mRNA and the protein expression of CAT in human keratinocyte cell line (HaCaT).

METHODSThe activity of catalase (CAT) was detected by ultraviolet direct velocity assay. RT-PCR was used to detect the mRNA expression of CAT and Western blotting was conducted to detect the protein expression of CAT.

RESULTSIf the cells were treated with higher than 5.0 micromol/L sodium arsenite, the activity, mRNA and protein expression of CAT were decreased significantly and in a dosage dependent fashion (P < 0.05).

CONCLUSIONCAT is inhibited by sodium arsenite in the transcription, translation and activity levels.

Arsenites ; toxicity ; Blotting, Western ; Catalase ; biosynthesis ; genetics ; Cell Line ; Dose-Response Relationship, Drug ; Humans ; Keratinocytes ; drug effects ; enzymology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Compounds ; toxicity

Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced HO production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.
Antioxidants ; pharmacology ; Benzopyrans ; pharmacology ; Caesalpinia ; chemistry ; Humans ; Hydrogen Peroxide ; toxicity ; Keratinocytes ; cytology ; drug effects ; enzymology ; radiation effects ; Oxidative Stress ; drug effects ; radiation effects ; Peroxidases ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Ultraviolet Rays

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKC alpha isoform. When PKC alpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKC alphakinase active mutant (PKC alpha- CAT) in the undifferentiated keratinocyte, but not PKC beta-CAT, also increased differentiation marker expressions. On the other hand, PKC alphadominant negative mutant (PKC beta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKC beta-KR did not, suggesting that PKC alphais responsible for keratinocyte differentiation. When downstream pathway of PKC alphain Ca2+ - mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+- mediated differentiation, and that only ERK1/2 pathway is specific for PKCa-mediated differentiation in mouse keratinocytes.
Animals ; Calcium/pharmacology/physiology ; Cell Differentiation/physiology ; Intermediate Filament Proteins/analysis/metabolism ; Keratinocytes/cytology/*enzymology ; Membrane Proteins/analysis/metabolism ; Mice ; Mitogen-Activated Protein Kinase 1/*metabolism ; Mitogen-Activated Protein Kinase 3/*metabolism ; Phosphorylation ; Protein Kinase C/genetics/*physiology ; Research Support, Non-U.S. Gov't ; p38 Mitogen-Activated Protein Kinases/metabolism

BACKGROUNDSolar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.

METHODSFollowing UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCulture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts.

CONCLUSIONSUVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

Cell Line ; Enzyme Activation ; Fibroblasts ; enzymology ; radiation effects ; Humans ; Interleukin-1 ; pharmacology ; Keratinocytes ; physiology ; radiation effects ; Matrix Metalloproteinase 1 ; biosynthesis ; Mitogen-Activated Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; RNA, Messenger ; analysis ; Skin ; radiation effects ; Skin Aging ; Transcription Factor AP-1 ; metabolism ; Ultraviolet Rays