Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level

Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.     

All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes.

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.     

Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NR

A monoclonal antibody, designated mAb alpha(CT), was generated against a peptide of the ISP(NAP) alpha-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa. Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring. This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp. expressing the NDO inducible enzyme. The detection threshold for the ELISA assay developed in this work was 10(4) colony forming units (cfu) per ml. Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments.     

Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross-linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus-40 (SV-40)-transformed human foreskin keratinocyte line SV-K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane-associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane-associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.     

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.     

A rapid method to improve protein detection by indirect ELISA

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.     

UVA/B exposure promotes the biosynthesis of dehydroretinol in cultured human keratinocytes

Retinol and its metabolites modulate epithelial differentiation and serve as cellular UV sensors through changes in retinoid status. Of note is the dehydroretinol family which may serve functions distinct from parental retinol. This study focuses on the metabolism of this family and its potential participation in the response of normal epidermal human keratinocytes to UV irradiation. There were three findings. First, keratinocytes contain two pools of dehydroretinyl esters, one of which is shielded from UVB-, but not from UVA-induced decomposition. Second, using a novel in vitro assay we demonstrated that both UVA and UVB promote dehydroretinol biosynthesis in keratinocytes, but only UVB exposure promotes retinoid ester accretion by enhancing the activity of at least one acyl transferase. Finally, dehydroretinol sufficiency reduces UVA/B driven apoptosis more effectively than retinol sufficiency. This may in part be due to differences in the expression of Fas ligand, which we found to be upregulated by retinoic acid, but not dehydroretinoic acid. These observations implicate a role of dehydroretinol and its metabolites in UVA/B adaptation. Thus, the keratinocyte response to UV is jointly shaped by both the retinoids and dehydroretinoids.     

Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte

Plasminogen activator inhibitor 2 (PAI-2) is an enzyme inhibitor which is involved in cell differentiation, tissue growth and regeneration. In this study, immunocytochemistry, in situ hybridization and confocal laser scanning microscopy were used to investigate the expression and role of PAI-2 in differentiation of keratinocytes in vitro. The result showed that in the mono-layer differentiated keratinocytes induced by high calcium concentration, the expression of PAI-2 and its mRNA increased significantly, accompanied by expression increase of the differentiation marker keratin 10; and in the multi-layer differentiated keratinocytes induced by high calcium, PAI-2 expressed strongly mainly in the keratinocytes of middle as well as upper stratified layers, while K10 expressed in the keratinocytes of all stratified layers. Furthermore, the changes of the parameters related to keratinocyte differentiation were detected after inhibition of PAI-2 functions by its antibody, and the data showed that when treated by PAI-2 antibody, involucrin in the keratinocytes envelope expressed increasingly with an altering distribution from part to the whole envelope area. Our results indicate that during differentiation of epidermal keratinocyte, PAI-2 expresses mainly in the more differentiated keratinocytes and may protect the terminal differentiated keratinocytes from prematuration through inhibiting involucrin expression in cornified envelope.     

Effect of tumour-cell-derived or recombinant keratinocyte growth factor (KGF) on proliferation and radioresponse of human epithelial tumour cells (HNSCC) and normal keratinocytes in vitro

Purpose of this work was to test the effect of tumour-cell-derived keratinocyte growth factor (KGF) or recombinant KGF (palifermin) on cell proliferation and radiation response of human HNSCC cells and normal keratinocytes in vitro. Four tumour cell cultures derived from head and neck squamous cell carcinomas, primary keratinocytes, and immortalized keratinocytes were analysed. Fibroblasts, the natural source of KGF protein, served as controls. KGF expression was observed in primary and immortalized keratinocytes, fibroblasts, and in tumour cells, while significant KGF receptor expression was only found in keratinocytes. Recombinant KGF as well as tumour-cell-derived KGF caused a significant growth stimulation and radioprotection in keratinocytes, which was abolished by a neutralizing anti-KGF antibody. This indicates that tumour-cell-derived KGF is biologically active. In the tumour cell lines, no significant growth stimulation was induced by recombinant KGF, and the neutralizing antibody did not influence tumour cell growth or radiation response. Our results indicate that the normal, paracrine KGF regulatory mechanisms, which are based on KGF receptor expression, are lost in malignant cells, with the consequence of irresponsiveness of the tumour cells to exogenous KGF. In face of the amelioration of the radiation response of normal epithelia, demonstrated in various clinical and various preclinical animal studies, recombinant KGF represents a candidate for the selective protection of normal epithelia during radio(chemo) therapy of squamous cell carcinoma.     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

Regulation of ultraviolet B radiation-mediated activation of AP1 signaling by retinoids in primary keratinocytes

The main cause of skin cancer and photo-aging is chronic exposure to ultraviolet B (UVB) radiation. Such damage can be ameliorated by retinoid treatment. UVB-radiation-induced skin carcinogenesis is associated with the induction of activator protein 1 (AP1) signaling and factors, namely FOS and JUN family members. We investigated the effects of several retinoids, all-trans-retinoic acid (tRA), 9-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl)-retinamide (HPR), on UVB-induced damage in primary mouse keratinocytes. In addition, the interplay between UVB radiation, retinoid receptors, and AP1 signaling was assessed using Western blot analysis and ribonuclease protection and gene reporter assays. Exposure of keratinocytes to UVB radiation caused a down-regulation of the retinoid receptor protein levels in a proteasome-mediated manner. In contrast, FOS and JUN proteins were transiently induced shortly after exposure to UVB radiation. Retinoid treatment caused a dose-dependent reduction in the levels of retinoid receptor proteins. When irradiated cells were treated with retinoids, no significant effects on AP1 protein expression were noted. Interestingly, pretreatments with tRA and cRA, but not HPR, suppressed UVB-radiation-induced AP1 activity by more than 50%, whereas post-treatment failed to produce similar effects. Our findings indicate that the inhibition of AP1 activity by retinoids explains, at least in part, the chemopreventive potential of retinoids in UV-radiation-associated epidermal damage.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.     

Autocrine induction of substance P mRNA and peptide in cultured normal human keratinocytes.

In this study, we have demonstrated that normal cultured keratinocytes (KCs) could generate significant endogenous substance P (SP) in a dose- and time-dependent response to exogenous SP by sensitive ELISA assay and express preprotachinin-a mRNA by RT-PCR and Southern blotting. We performed immunohistochemical analysis to confirm the presence of SP in cultured keratinocytes. In contrast, adrenaline, acetylcholine, histamine and CGRP induced only low amount of SP from cultured normal human KCs. This is the first report that SP can be induced by skin epithelial cells in response to exogenous SP and KC derived SP might play an important role in induction and acceleration of certain cutaneous diseases.     

Considerations for in vitro retinoid experiments: importance of protein interaction

Retinoids, natural and synthetic substances structurally related to vitamin A, are important modulators of cell proliferation and differentiation, and have proven activity in cancer therapy. Experiments to reveal the mechanism of action of retinoids are routinely performed in in vitro models. As retinoids are relatively hydrophobic and unstable, we hypothesized that the composition of culture media is of critical importance for the stability and bioavailability of these compounds. Various culture media were incubated with all-trans-, 13-cis- and 9-cis-retinoic acid (RA). Without fetal calf serum (FCS) or bovine serum albumin (BSA) in the medium, the concentration of these retinoids was found to decrease to considerably low levels. This excessive loss of retinoids was due to absorption to culture plates, reaction tubes and pipet tips. Binding of retinoids to BSA was demonstrated to have attenuating effects on uptake and metabolism of all-trans-RA, as studied in oral keratinocytes and head and neck cancer cells, indicating that a balance exists between the bioavailability and the aspecific loss of retinoids. In this study we demonstrate that the type of culture medium and especially the presence of protein in the medium is of paramount importance to perform reproducible experiments with retinoids.     

Rapid isotyping of murine monoclonal antibodies by enzyme-linked immunofiltration assay

Summary As a part of the initial characterization of monoclonal antibodies, the isotype is routinely identified by enzyme-linked immunosorbent assay (ELISA). In the present study, we describe an isotyping methodology that uses the technique referred to as enzyme-linked immunofiltration assay (ELIFA) for greatly accelerating the assay compared with ELISA. In the ELIFA method, solutions are filtered through a nitrocellulose membrane using a controlled flow rate to bind proteins. By using this technology, the time required to isotype a monoclonal antibody was reduced from a minimum of 4 to 8 hr using a standard ELISA assay to 30 min with ELIFA.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.