Transglutaminases in normal and transformed human keratinocytes in culture

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.     

Periderm cells form cornified cell envelope in their regression process during human epidermal development.

Terminally differentiated stratified squamous epithelium forms a lining of the plasma membrane called the cornified cell envelope, a thick layer of several covalently cross-linked precursor proteins including involucrin, small proline-rich proteins, and loricrin. Their cross-linking isodipeptide bonds are formed by epidermal transglutaminases 1-3. Material from lamellar granules is attached on the extracellular surface of corneocytes during the keratinization process. The formation of cornified cell envelope and sequential expression of major cornified cell envelope precursor proteins, transglutaminases, and 25 kDa lamellar granule-associated protein were studied in human embryonic and fetal skin. Ultrastructurally, membrane thickening has already started in periderm cells of the two-layered epidermis and an electron-dense, thickened cell envelope similar to cornified cell envelope in adult epidermis is observed in periderm cells at the three-layered and later stages of skin development. In the two-layered epidermis (49-65 d estimated gestational age), immunoreactivities of involucrin, small proline-rich proteins, all the transglutaminases, and lamellar granule-associated protein were present only in the periderm. In the three-layered epidermis and thereafter (66-160 d estimated gestational age), loricrin became positive in the periderm cells, transglutaminases extended to the entire epidermis, and lamellar granule-associated protein was detected in intermediate cells as well as periderm cells. Immunoelectron microscopy demonstrated that both major cornified cell envelope precursor proteins, involucrin and loricrin, were restricted to the cornified cell envelope in periderm cells at this stage of development. After 160 d estimated gestational age, the periderm had disappeared and cornified cell envelope proteins and lamellar granule-associated proteins were expressed in the spinous, granular, and cornified cells and transglutaminases were detected in the entire epidermis. These findings indicate that cornified cell envelope precursor proteins, transglutaminases, and lamellar granule-associated proteins are expressed in coordination in periderm cells during human epidermal development and suggest that periderm cells form cornified cell envelope in the process of regression.     

Effect of 1,24R-dihydroxyvitamin D3 on the growth of human keratinocytes

The effect of 1,24R-dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic analogue of a biologically active form of vitamin D3 (1,25-dihydroxyvitamin D3, 1,25(OH)2D3), on the growth of human keratinocytes cultured in serum-free medium was investigated. The growth of cultured normal human keratinocytes was inhibited by 65% by 10(-8)M 1,24R(OH)2D3 and by 90% by 10(-7)M 1,24(OH)2D3. It inhibited cell growth almost completely at 10(-6)M. The DNA synthesis of keratinocytes was also inhibited with 1,24R(OH)2D3 by 27% at 10(-8)M, 59% at 10(-7)M, and 92% at 10(-6)M. The inhibition of cell growth and DNA synthesis were more remarkable by 1,24R(OH)2D3 than by 1,25(OH)2D3. 1,24R(OH)2D3 also inhibited the growth of keratinocytes derived from patients with psoriasis vulgaris; the growth inhibitory effect was again more remarkable with 1,24R(OH)2D3 than with 1,25(OH)2D3. The viability and protein synthesis of keratinocytes were not affected by 1,24R(OH)2D3, suggesting that the growth inhibitory effect is due to its biological activity, not to cytotoxicity. The binding of [3H]-labeled 1,25(OH)2D3 to its receptor in the cytosolic fraction of cultured keratinocytes was competitively substituted by unlabeled 1,24R(OH)2D3 as well as 1,25(OH)2D3, suggesting that 1,24R(OH)2D3 binds to the 1,25(OH)2D3 receptor. It was found that the affinity of 1,24R(OH)2D3 for the receptor was slightly higher than that of 1,25(OH)2D3. These results demonstrate that 1,24R(OH)2D3 functions as a potent growth inhibitor in vitro in human keratinocytes from both normal and psoriatic epidermis, and it possesses a higher affinity for the 1,25(OH)2D3 receptor in cultured human keratinocytes. The difference in affinity of 1,24R(OH)2D3 for the 1,25(OH)2D3 receptor correlates with its greater inhibition of keratinocyte growth than 1,25(OH)2D3. 1,24R(OH)2D3 may be useful in the treatment of psoriasis.     

Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal and psoriatic keratinocytes

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth and DNA synthesis of cultured human keratinocytes obtained from involved and uninvolved psoriatic epidermis and normal epidermis were studied. Treatment with 10(-8) M and 10(-7) M of 1,25(OH)2D3 inhibited cell growth as follows: 58.5 +/- 19.3% and 21.3 +/- 13.6% in normal keratinocytes (n = 6); 43.8 +/- 22.8% and 17.8 +/- 12.3% in psoriatic uninvolved keratinocytes (n = 4); 51.7 +/- 18.2% and 13.2 +/- 6.4% in psoriatic involved keratinocytes (n = 6). Inhibition was virtually complete at 10(-6) M. DNA synthesis was also inhibited by 10(-8) M, 10(-7) M and 10(-6) M of 1,25(OH)2D3 as follows: 70.0 +/- 8.3%, 59.0 +/- 6.8% and 16.7 +/- 4.0%, respectively, in normal keratinocytes (n = 3); 78.5 +/- 13.5%, 51.5 +/- 25.5% and 24.5 +/- 21.5%, respectively, in psoriatic uninvolved keratinocytes (n = 2); and 69.3 +/- 14.5%, 41.3 +/- 19.1% and 14.8 +/- 11.2%, respectively, in psoriatic involved keratinocytes (n = 4). These results indicate that 1,25(OH)2D3 functions as a growth inhibitor for cultured human keratinocytes derived from both normal and psoriatic skin.     

Keratinocyte differentiation is induced by cell-permeant ceramides and its proliferation is promoted by sphingosine

Ceramide and sphingosine have been suggested to be intracellular modulators of cell growth and differentiation. The effects of these sphingolipids on the growth and differentiation of keratinocytes were examined using cultured human keratinocytes (the squamous cell carcinoma cell line, DJM-1). The synthetic short-chain cell-permeant analogues of ceramides, N-acetylsphingosine, N-hexanoylsphingosine and N-octanoylsphingosine, significantly promoted differentiation as confirmed by upregulation of cornified envelope formation, synthesis of involucrin and increased transglutaminase activity, and inhibited proliferation as shown by a reduction in cell numbers, DNA amount and thymidine incorporation. Generally, these activities were greater the longer the N-acyl carbon chain. On the other hand, sphingosine at an appropriate concentration modestly stimulated the proliferation of cultured cells. Our results suggest the possibility that the growth and differentiation of keratinocytes are at least partially regulated by ceramide and sphingosine.     

Interleukin-15 is not a constitutive cytokine in the epidermis, but is inducible in culture or inflammatory conditions

The regulation in the skin of interleukin-15 (IL-15), a potent modulator of T-cell-mediated immune responses, is not fully understood. We investigated the levels of IL-15 and its mRNA produced by epidermal and cultured keratinocytes and found that normal keratinocytes did not constitutively express IL-15 in the epidermis, but in culture began to produce the cytokine. Some epidermal keratinocytes expressed IL-15 in inflammatory conditions associated with infiltration of neutrophils and eosinophils. IL-15 was detected only in the cell lysates, not in the supernatants of cultured keratinocytes. Dexamethasone (10(-5)-10(-6) M) markedly inhibited IL-15 mRNA expression by normal and transformed keratinocytes in a range of pharmacological concentrations. IFN-gamma (200 and 400 U/ml) slightly increased the IL-15 message level in a squamous cell carcinoma cell line, HSC-5, in a dose-dependent fashion, whereas no significant change was observed in cultured normal human keratinocytes. Our data indicate that IL-15 is not a constitutive cytokine in epidermal keratinocytes but is inducible.     

Effects of calcium concentration on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes

BACKGROUND/PURPOSE: Cellular differentiation due to the extracellular calcium (Ca(2+)) concentration affects the level of several antioxidant enzymes in cultured human keratinocytes. Because the epidermis includes well- and un-differentiated keratinocytes, we expected that keratinocytes possess different antioxidant capacity and sensitivity to damaging effects of ultraviolet-B (UVB) depending on the differentiation. We examined the effects of Ca(2+) concentration of culture medium (DMEM (Dulbecco's modified Eagle's medium)) on the superoxide dismutase (SOD) activity and UVB-induced cytotoxicity in cultured human keratinocytes in order to investigate the relationship between cell differentiation and antioxidant defense. METHODS: Human keratinocytes (HaCaT cells) were incubated in high Ca(2+) (>1 mM) or low Ca(2+) (<0.1 mM) concentration DMEM for 24 h at 37 degrees C in 5% CO(2). Then, we measured total SOD activity and also individual Cu,Zn- and Mn-SOD activities in keratinocytes. Furthermore, after incubation in high or low Ca(2+) concentration DMEM, human keratinocytes were irradiated with 10, 20 or 30 mJ/cm(2) UVB. The quantity of lactate dehydrogenase (LDH) leaked in the supernatant from damaged keratinocytes, cell viability and TdT-mediated dUTP nick end labelings (TUNEL) positive keratinocytes were measured at 24 h after UVB irradiation. RESULTS: Total SOD activity and Cu,Zn-SOD activity in human keratinocytes cultured in low Ca(2+) were significantly lower than in keratinocytes cultured in high Ca(2+) concentration DMEM. In contrast, Mn-SOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca(2+) concentration was significantly higher than that from keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. The cell viability of keratinocytes cultured in low Ca(2+) concentration DMEM was significantly decreased compared to that of keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. Furthermore, UVB-induced apoptosis was increased in keratinocytes cultured in low Ca(2+) concentration DMEM by the TUNEL method. CONCLUSIONS: These results suggest that cellular differentiation due to the change of Ca(2+) concentration of culture medium affects the Cu,Zn-SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.     

Effects of cyclosporin and ultraviolet radiation on growth and ornithine decarboxylase activity in cultured human epidermal keratinocytes

Both cyclosporin (CyA) and ultraviolet radiation are effective in the treatment of psoriasis, but their precise mechanisms of action are uncertain. We investigated their effects on ornithine decarboxylase (ODC) activity, ODC gene expression, and cellular proliferation stimulated by epidermal growth factor (EGF), in cultured normal human epidermal keratinocytes. CyA (5 μg/ml) inhibited ODC activity, ODC mRNA level, and cell growth induced by 50ng/ml EGF. Ultraviolet B (10 mJ/cm²) irradiation suppressed the induction of ODC, ODC mRNA, and cell proliferation stimulated by EGF, but ultraviolet A (0-15 J/cm²) irradiation inhibited neither EGF-stimulated ODC activity nor cell proliferation. These findings indicate that reduction of ODC activity in CyA- or ultraviolet B-treated human keratinocytes may contribute to the antiproliferative mechanism of these agents. These results also suggest that the regulation of ODC activity by ultraviolet B and A irradiation may be mediated by different signal transduction pathways.     

Formation and removal of 8-MOP-DNA photoadducts in keratinocytes: effects of calcium concentration and retinoids

8-methoxypsoralen (8-MOP)-DNA photoadducts were quantified in freshly isolated human and murine keratinocytes and cultured keratinocyte cell lines after in vitro treatment with 8-MOP (1-200 ng/ml) and ultraviolet A (UVA; 0.2-24.0 J/cm2). Greater doses of 8-MOP and UVA led to proportionately greater numbers of photoadducts, with a dose reciprocity relationship between the amounts of 8-MOP and UVA. No significant difference in photoadduct formation was observed between basal and differentiated cells. However, the transformed keratinocyte cell lines showed fewer photoadducts than did normal keratinocytes, which appeared to be correlated with the finding that the adduct formation was inhibited in normal keratinocytes cultured with phorbol 12-myristate 13-acetate, because this agent leads to epidermal hyperproliferation. In viable keratinocytes that were treated with a sublethal dose of 8-MOP and UVA (15 ng/ml and 1 J/cm2, respectively), 54% of photoadducts formed were removed over a 20-h period. Adduct removal depended on the calcium concentration in the media; cells cultured in standard high calcium levels showed a higher removal rate than those cultured in low-calcium media. The addition of retinoids (etretinate, acitretin, and 13-cis retinoic acid) to the culture induced 55 to 80% of suppression of the adduct removal. The calcium ionophore A23187 partially restored the suppression of photoadduct removal induced by retinoids. The present studies suggest that calcium performs an important role in the photoadduct removal and raise the possibility that the synergism of systemic retinoids and psoralen plus UVA photochemotherapy relates to the former's inhibition of repair of 8-MOP photoadducts in DNA.     

Binding of 125I-gamma interferon to cultured human keratinocytes

Gamma interferon (IFN-gamma), a product of activated lymphocytes, influences keratinocyte proliferation and differentiation. Recombinant gamma interferon (r-IFN-gamma) was radioiodinated using the Bolton-Hunter reagent and retained 90% of its biologic activity as determined by induction of HLA-DR expression. The biochemical ligand-binding properties of iodinated IFN-gamma to cultured human keratinocytes revealed a plateau of binding at 150 min at 4 degrees C, and a single class of specific high affinity receptors (kD = 1.3 X 10(-10) M; 2200 sites/cell). The binding of human IFN-gamma, to keratinocytes was inhibited by human r-IFN-gamma, but not by either murine recombinant gamma interferon or human recombinant beta interferon (r-IFN-beta). The presence of high affinity receptor sites on human keratinocytes assures the reception of appropriate immunologic signals in lymphocyte-keratinocyte interactions.     

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker. When freshly isolated mouse and human epidermal cells were incubated with LDL-gold complexes, only keratinocytes with the morphologic characteristics of basal cells showed binding and uptake of LDL-gold. No LDL receptor activity was found on Langerhans cells, melanocytes or highly differentiated keratinocytes. Since cell separation techniques can destroy receptors, the staphylococcal epidermolytic toxin was utilized to produce intercellular and intra-epithelial splitting of the epidermis. In preparations of both normal mouse and human epidermis, LDL-gold binding was restricted to basal cells and a few suprabasal keratinocytes. In contrast, in psoriatic epidermis, and to a lesser extent, essential fatty acid-deficient mouse epidermis, cells in the stratum spinosum showed abundant LDL-gold binding. Thus LDL-gold may be a useful marker for epidermal differentiation.     

Analysis of epidermal-type transglutaminase (transglutaminase 3) in human stratified epithelia and cultured keratinocytes using monoclonal antibodies

BACKGROUND: Epidermal-type transglutaminase (TGase 3) is involved in the cross-linking of structural proteins in the epidermis, which results in the formation of the cornified envelope. TGase 3 is activated by limited proteolysis of a 77 kDa zymogen during keratinocyte differentiation. OBJECTIVE: To characterize the expression of TGase 3 in human epidermis and cultured keratinocytes, we established specific monoclonal antibodies against the TGase 3. METHODS: Recombinant proteins for human TGase 3 produced in bacteria and baculovirus-infected insect cells were purified as an antigen. Hybridomas are established and used for characterization of expression in epidermis and keratinocytes. RESULTS: Four antibodies were generated against recombinant human TGase 3, which reacted with the 77 kDa zymogen and in some cases either the 47 or 30 kDa active proteolytic fragments. In human epidermis and cultured keratinocytes, only the zymogen form of TGase 3 was detected. Immunohistochemical analysis of the skin revealed that the enzyme is present in the cells of the granular and cornified layers consistent with its role in cornified envelope formation. In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions. CONCLUSION: Using monoclonal antibody against human TGase 3, we showed the expression of TGase 3 in upper layers of epidermis. TGase 3 displayed a diffuse cytoplasmic distribution in vitro consistent with its proposed role in the early phase of cornified cell envelope assembly in the cytoplasm.     

CO2激光碳化尖锐湿疣残存HPVDNA活性的实验研究

对20例尖锐湿疣（CA）患者，局部皮损经CO2激光治疗后，碳末DNA进行PCR扩增，并与正常角质形成细胞（KC）培养，观察KC增殖情况，观察其是否具有活性。结果发现：碳末DNAPCR扩增，40％阳性，碳末DNA对KC有明显抑制作用。从而推断碳末DNA仍有活性，可能是引起该病复发的原因之一。     

The wound-healing effect of a glycoprotein fraction isolated from aloe vera

BACKGROUND: Aloe vera has been used as a family medicine for promoting wound healing, but it is not known which component of the plant is effective for this purpose. OBJECTIVES: To isolate and characterize the component effective in wound healing. METHODS: Chromatography, electrophoresis and spectroscopic methods were used. The cell-proliferation activity of each component isolated was measured by a [3H]thymidine uptake assay. The cell-proliferation activity of the effective component was tested on a three-dimensional raft culture (cell culture technique by which artificial epidermis is made from keratinocytes). The effect of the active component on cell migration and wound healing was observed on a monolayer of human keratinocytes and in hairless mice. RESULTS: A glycoprotein fraction was isolated and named G1G1M1DI2. It showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, with an apparent molecular weight of about 5.5 kDa. It exhibited significant [3H]thymidine uptake in squamous cell carcinoma cells. The effect of G1G1M1DI2 on cell migration was confirmed by accelerated wound healing on a monolayer of human keratinocytes. When this fraction was tested on a raft culture, it stimulated the formation of epidermal tissue. Furthermore, proliferation markers (epidermal growth factor receptor, fibronectin receptor, fibronectin, keratin 5/14 and keratin 1/10) were markedly expressed at the immunohistochemical level. The glycoprotein fraction enhanced wound healing in hairless mice by day 8 after injury, with significant cell proliferation. CONCLUSIONS: It is considered that this glycoprotein fraction is involved in the wound-healing effect of aloe vera via cell proliferation and migration.     

Calcium entry into keratinocytes induces exocytosis of lysosomes

During the final steps of epidermal differentiation, extracellular calcium ions enter keratinocytes and induce transglutaminase activity and cornified envelope formation. In other cell types, entry of calcium mediated by ionophores has been reported to induce exocytosis of lysosomes. In this study, we investigated whether lysosomes of keratinocytes might exhibit a similar behaviour. Ionomycin treatment induced cornified envelope formation in keratinocytes, but also morphological changes including plasma membrane blebbing, although no immediate alteration in cell viability could be detected. The activity of the soluble lysosomal enzymes cathepsin C and -galactosidase in the culture medium was increased upon ionomycin treatment. Cell leakage did not seem to be responsible for this phenomenon, as suggested by measurements of the cytosolic enzymes adenylate kinase and dipeptidylpeptidase III in the culture medium. Metabolic labelling followed by immunoprecipitation showed that ionomycin induced release of cathepsin D into the culture medium. Simultaneously, lysosome-associated membrane proteins (Lamps) 1 and 2 were detected at the cell surface of ionomycin-treated keratinocytes by biochemical and morphological approaches. These results suggest that upon ionomycin treatment, calcium entry stimulates exocytosis of lysosomes in keratinocytes.Abbreviations AK Adenylate kinase - BSA Bovine serum albumin - DMSO Dimethylsulphoxide - DPPIII Dipeptidylpeptidase III - DTT Dithiothreitol - EDTA Ethylene diaminotetraacetic acid - EGTA Ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N,N-tetraacetic acid - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - SDS Sodium dodecylsulphate     

Sphingomyelinase in pig and human epidermis.

The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine.