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M Darmon

Publications and source records attributed to M Darmon.

At least 37 records · Page 2Linked to original sources
PubMed1995

Retinoic acid provokes metaplasia of epithelium formed in vitro by adult human epidermal keratinocytes.

A striking effect of retinoids is their ability to alter cell fate during development. The mucous metaplasia produced by treating chick embryo skin in organ culture with retinoic acid is a classical example of this property. It has been impossible so far to demonstrate that retinoids are able to provoke metaplasia of adult keratinocytes grown in vitro, although these agents have been shown to block terminal epidermal differentiation, to induce increased synthesis of mucopolysaccharides, and to induce the ectopic expression of K19 and K13 keratins. Our previous work showed that adult human epidermal keratinocytes grown on dermal equivalents at the surface of a culture medium containing physiological amounts of retinoids form a normal keratinized epidermis, while when excess retinoic acid is added to the culture medium, keratinization is prevented but stratification is not. Here we show that the distribution of tissue- and differentiation-stage-specific markers in retinoic acid-treated epithelium is similar to that of the oral mucosa. Moreover, when the excess retinoic acid is removed, a new epithelium is formed beneath the "old" one and this epithelium displays an epidermal orthokeratinized phenotype, whereas the "old" epithelium remains unchanged. This phenomenon of "partial reversibility", as well as the mutually exclusive distribution of the markers of the two alternative routes of differentiation, demonstrate that retinoic acid is indeed able to provoke metaplasia of adult epidermal keratinocytes.

Adult↗
PubMed1995

Immunolabeling of central serotonin 5-HT1D beta receptors in the rat, mouse, and guinea pig with a specific anti-peptide antiserum.

A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT1B/1D beta receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT1B/1D beta receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin-O-carboxymethylglycyl[125I]iodotyrosinamide ([125I]GTI; a selective 5-HT1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT1B receptors. The remaining 40% of [125I]GTI binding sites were shown to be 5-HT1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT1B/1D beta, but not the 5-HT1D/1D alpha, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT1D beta receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [125I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT1D beta component of [125I]GTI specific receptor binding sites.

Amino Acid Sequence↗
PubMed1994

Biologic activities of retinoic acid and 3,4-didehydroretinoic acid in human keratinocytes are similar and correlate with receptor affinities and transactivation properties.

The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.

Cells, Cultured↗
PubMed1994

Computer analysis of two-dimensional electrophoresis gels as a tool in cell biology: study of the protein expression of human keratinocytes from normal to tumor cells.

The usefulness of computer analysis of two-dimensional electrophoresis gels has been investigated on the example of human keratinocytes transformation. For this purpose, the protein expression of various keratinocytes strains from normal to tumor cells has been analysed by two-dimensional electrophoresis. The resulting gels have been submitted to computer analysis, including various data analysis techniques allowing to select spots on the gels or to classify the gels themselves. The latter techniques appeared very useful, since they demonstrated that the major transition in words of variation of the protein expression lies at the normal cell/transformed cell transition rather than at the transformed cell/tumorigenic cell transition.

Animals↗
PubMed1993

Coordinated posttranscriptional control of gene expression by modular elements including Alu-like repetitive sequences.

We previously reported that in rat fibroblasts, accumulation of a set of mRNAs ("pIL genes") was modulated as a function of cell growth and transformation, at a posttranscriptional stage, and by a mechanism that depends on a short nucleotide sequence containing an ID repetitive element. In mouse fibroblasts, hybridization with rat pIL probes identified mRNAs with the same pattern of expression, which did not contain ID sequences but contained a different regulatory element, encompassing a repetitive sequence of the B1 family. Expression in mouse cells of a reporter beta-globin gene carrying this element inserted in its 3' noncoding region was growth- and transformation-dependent. The nucleotide sequences of two murine and of three rat pIL cDNAs showed clear similarities in the region immediately adjacent to the ID and B1 repeats. Both the repeat and the flanking sequence were required to confer on beta-globin constructs the pattern of expression characteristic of the pIL genes. The hypothesis is presented that repetitive sequences in the eukaryotic genome might be modular parts of complex regulatory elements ensuring the coordinated expression of various mRNA species.

3T3 Cells↗
PubMed1993

Epidermis reconstructed from the outer root sheath of human hair follicle. Effect of retinoic acid.

In the present paper, we show that a multilayered and well-differentiated epidermis can easily and rapidly be generated in vitro from the outer root sheath of human hair follicles deposited on de-epidermized demis. Histologically, this epidermis presented characteristic features of normal human epidermis in vivo. Moreover, markers specific for interfollicular keratinocyte terminal differentiation, such as the K10 keratin, involucrin, membrane-bound transglutaminase, filaggrin and loricrin, were expressed in the reconstructed tissue. By in situ hybridization, keratin K5 and K10 mRNAs were detected in the basal and suprabasal cells, respectively, as in normal human epidermis. The differentiation pattern achieved in this reconstructed epidermis confirms the already reported phenotypical shift from outer root sheath cells to interfollicular keratinocytes and shows that this transition takes place in the absence of living fibroblasts. The differentiation of the reconstructed epidermis thus obtained was modulated by retinoic acid in a dose-dependent manner. This culture system on dead dermis is easier to handle than similar cultures on collagen-fibroblast lattices because of the resistance of dermis to mechanical forces and to collagenolysis. It could represent a valuable wound-healing model and a promising tool for pharmacological studies on in vitro reconstructed skin.

Cell Differentiation↗
PubMed1992

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.

Animals↗
PubMed1992

Delayed onset of epidermal differentiation in psoriasis.

In normal epidermis, as previously reported, the first signs of differentiation occur within the basal layer in a subpopulation of keratinocytes that start to express K1 and K10 "supra-basal" keratin transcripts (20-30% of the basal cells) and proteins (5-10% of the basal cells). We found that in psoriatic lesions, the basal layer was devoid of cells expressing these early differentiation markers. This was already the case at the periphery of the lesions, where epidermis, although slightly acanthotic, still completes the keratinization process. In the center of the lesions, not only the basal layer, but also several rows of suprabasal cells, were negative for keratin K10 transcripts or protein. Moreover, the upper nucleated layers of involved epidermis were also devoid of K10 keratin transcripts or proteins. In normal epidermis, as previously reported, transcripts for the "basal" K5 keratin were mainly restricted to the basal layer, whereas the protein persisted in a few suprabasal layers. We found that in psoriatic epidermis, K5 keratin transcripts persisted in several suprabasal layers up to the level where K10 keratin transcripts appeared. These data, although not contradictory with previous reports showing a reduction of K1-K10 keratins and other differentiation markers in psoriasis, demonstrate that these quantitative changes are in fact the result of major qualitative differences in the distribution of these markers in psoriatic versus normal skin. Our results indicating that the onset of differentiation is delayed in psoriasis show that, contrary to conclusions accepted so far, not only the suprabasal compartment, but also the basal one, is abnormal in psoriatic epidermis.

Cell Differentiation↗
PubMed1992

Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.

Adapalene↗
PubMed1992

Expression of loricrin is negatively controlled by retinoic acid in human epidermis reconstructed in vitro.

In epidermis, the last steps of keratinocyte differentiation are characterized by the covalent cross-linking of cornified envelope precursors such as involucrin and loricrin, a hydrophobic protein recently described in mouse and human epidermis. In situ hybridization of normal human skin sections with a human loricrin cRNA probe and immunolabeling with an antiserum directed against a synthetic peptide corresponding to the carboxyterminus of human loricrin revealed the presence of loricrin transcripts and protein in the granular layers of epidermis. In human epidermis reconstructed in vitro by growing keratinocytes on dermal equivalents, loricrin and loricrin mRNAs were also restricted to granular cells, but their amounts seemed higher than in epidermis from skin biopsies. The reactivities for both loricrin and loricrin mRNAs were abolished by a treatment of the cultures with a retinoic acid concentration (10(-6) M) provoking a complete inhibition of terminal epidermal differentiation (parakeratosis). Thus, the regulation of loricrin synthesis is different from that of another envelope precursor, involucrin, which does not seem to be significantly modulated by retinoic acid. Together with the well-documented inhibition of epidermal transglutaminase by retinoic acid, our results provide a molecular basis for the inhibition of cornified envelope formation by retinoic acid.

Amino Acid Sequence↗
PubMed1992

A new marker of epidermal differentiation associated with the membrane coating granules: characterization and applications to pathology.

A murine monoclonal antibody, BC12, was obtained after immunization against suprabasal human keratinocytes. In the epidermis of normal human skin, the antigen recognized by BC12 (BC12 antigen) is located at the apex of keratinocytes in the upper stratum spinosum and stratum granulosum but is absent in other layers. The BC12 antigen is also present in hair follicles. Immunoblotting performed on keratinocyte subpopulations confirmed the presence of the BC12 antigen in differentiated keratinocytes only. Two-dimensional immunoblotting showed that the BC12 antigen corresponds to a set of polypeptides with an apparent molecular weight of approximately 33kD. In keratinocyte cultures, the antigen is present only in stratified areas. The distribution of the BC12 antigen, as studied by indirect immunofluorescence and immunoelectron microscopy, and its presence in certain subcellular fractions of epidermal cells suggest that it is a component of membrane coating granules (MCGs) or that it is associated with these structures. Strikingly, in psoriasis, eczema and many other diseases, the BC12 antibody does not label the epidermis, but vessels in dermal papillae. The BC12 antibody may thus be a useful tool in the study of keratinocyte differentiation and MCG physiology, and, also, in pathology.

Antibodies, Monoclonal↗
PubMed1992

Expression of loricrin in skin disorders.

Loricrin, the major component of the cornified envelope, is normally expressed in the granular layer of epidermis during the last steps of keratinocyte differentiation. Using an antiloricrin antiserum (A8-73), an increased expression of this envelope precursor was found in some disorders of hyperorthokeratosis (ichthyosiform erythroderma; lichen ruber), but not in others (keratodermia ichthyosis vulgaris). In disorders accompanied by parakeratosis, a sign of incomplete differentiation (psoriasis, prurigo nodularis) loricrin was not detected, whereas the tissue expressed filaggrin. Treatment of normal skin with retinoic acid, increasing epidermal thickness in some subjects, led to an increased expression of loricrin. Loricrin might be a useful indicator of the extent of terminal epidermal differentiation in skin disorders.

Biopsy↗
PubMed1991

The respecification of limb pattern by new synthetic retinoids and their interaction with cellular retinoic acid-binding protein.

We describe here experiments to examine the role of cellular retinoic-acid-binding protein (CRABP) during the induction of limb duplication in the chick limb bud and regenerating axolotl limb by retinoids. A newly synthesised class of retinoic acid analogues have been used because among them, some have been specifically designed with the property of binding to the retinoic acid receptors, but not to CRABP. We can thus test whether binding to CRABP is an obligatory step during limb respecification. The binding of four of these compounds to chick limb bud and axolotl CRABP was tested in sucrose density gradient assays and then their potencies at inducing limb duplications tested. Two of the four compounds do not bind to limb CRABP and yet are able to induce limb duplications, suggesting that an interaction with CRABP is not an obligatory step in the process. However, the two compounds which do bind to CRABP are more potent than the two which do not, suggesting that an interaction with CRABP may, nevertheless, increase the potency of a retinoid.

Ambystoma mexicanum↗
PubMed1991

1,25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes.

Human keratinocytes grown on deepidermized dermis (DED) are able to reconstruct a morphologically normal stratified and keratinized epidermis. This culture system is suitable for studying in vitro the effects of various hormones and factors on epidermal differentiation, and the goal of the present work was to study the effect of vitamin D. We found that the hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3, produced very specific alterations in epidermal architecture in a dose-dependent manner, consisting of significant reduction of the nucleated layers of the epithelium, but not of the stratum corneum, which was instead slightly thickened. The study of stage-specific differentiation markers showed that the two extreme layers of epidermis, i.e. the basal layer and the stratum corneum, were unaffected by the hormone, but that the reduction involved specifically the intermediate differentiation compartment, i.e. the spinous and granular layers. It was shown that the reduction of the intermediate compartment provoked by 1,25-dihydroxyvitamin D3 is not due to a block in the proliferation of basal cells or to inhibition of their differentiation into suprabasal cells, but to stimulation of the terminal differentiation of suprabasal cells into corneocytes.

Calcitriol↗
PubMed1991

Selective high affinity retinoic acid receptor alpha or beta-gamma ligands.

Biological effects of retinoic acid (RA) are mediated through its binding to three closely related nuclear receptors (RAR alpha, RAR beta, and RAR gamma) belonging to the steroid-thyroid nuclear receptor family. RARs are able to modulate the transcription of specific genes by binding to responsive elements located in the promoter-enhancer region of these genes. As demonstrated by in situ hybridization, the distribution of each RAR type in the developing embryo, as well as in the adult, is not uniform. In this context, synthetic retinoids that would behave as selective ligands would be invaluable for studying the respective roles of each RAR type in cultured cells, whole animals, and embryos. Moreover, from a pharmacological point of view, such selective compounds may possess a higher therapeutic index and a lower teratogenic risk, because they might affect specific tissues and spare some others. As an approach to this problem, we have set up two complementary assays, (i) an in vitro binding assay to determine the Kd values of retinoids for RAR alpha, RAR beta, and RAR gamma and (ii) a functional assay in cultured cells to evaluate the potential of retinoids to transactivate, through their binding to one type of RAR, a reporter gene. The binding assay uses nuclear extracts of COS-7 cells transfected with vectors expressing RAR alpha, RAR beta, or RAR gamma. The functional assay is a measure of chloramphenicol acetyltransferase (CAT) activity in HeLa cells co-transfected with the expression vectors used in the binding assay and the reporter gene TRE-tk-CAT. Selective agonists for RAR alpha (Am80 and Am580) and RAR beta-RAR gamma (CD495 and CD564) were identified. However, compounds with pure RAR beta or RAR gamma selectivity have not yet been identified.

Carrier Proteins↗
PubMed1990

Deficiency in catalase activity correlates with the appearance of tumor phenotype in human keratinocytes.

Biopsies isolated from various human stratified epitheliomas and cultures of the associated fibroblasts exhibit a breakdown in catalase activity (70 to 95%). We report here that primary human cultures of keratinocytes and various immortalized keratinocyte cell lines differ from one other in their catalase activity. Moreover, deficient catalase activity appears to be related to the intensity of tumor phenotype expression. Since catalase activity in cell lines transformed in vitro is not as weak as activity observed in cell lines isolated from tumors, catalase deficiency may follow the successive steps of tumor progression.

Acatalasia↗
PubMed1990

Isolation of a GC-rich cDNA identifying mRNA present in human epidermis and modulated by calcium and retinoic acid in cultured keratinocytes. Homology with murine loricrin mRNA.

Differential screening of a human epidermal cDNA library led to the isolation of cDNA clones homologous to mRNAs specifically expressed in epidermis but weakly or not expressed in the undifferentiated squamous carcinoma cell line TR146. One of these 'differentiation-specific' cDNA clones, A8, hybridized with a 1.7 kb transcript among RNAs isolated from normal human epidermis, but with several transcripts ranging from 1.4 to 2.1 kb when mRNAs were isolated from cultured keratinocytes. We examined the effects of modulators of epidermal differentiation such as calcium and retinoic acid on the production of these transcripts. Their amount was found to increase in the presence of high calcium concentration, but to decrease in the presence of retinoic acid. These results strongly suggest that A8 messages are up-regulated during epidermal differentiation. The sequence of the 1371 bp of A8 cDNA shows a very high GC content. Because of its homology with the murine loricrin mRNA, A8 is likely to correspond either to the human loricrin or to a related protein.

Animals↗