Rainer Schmidt

The Cornified Envelope: A Model of Cell Death in the Skin

The epidermis functions as a barrier against the environment by means of several layers of terminally differentiated, dead keratinocytes — the cornified layer, which forms the endpoint of epidermal differentiation and death. The cornified envelope replaces the plasma membrane of differentiating keratinocytes and consists of keratins that are enclosed within an insoluble amalgam of proteins, which are crosslinked by transglutaminases and surrounded by a lipid envelope. New insights into the molecular mechanisms and the physiological endpoints of cornification are increasing our understanding of the pathological defects of this unique form of programmed cell death, which is associated with barrier malfunctions and ichthyosis.

Refined Characterization of Corneodesmosin Proteolysis during Terminal Differentiation of Human Epidermis and Its Relationship to Desquamation

Corneodesmosin is a putative adhesion glycoprotein located in the extracellular part of the desmosomes in the upper layers of the epidermis. Synthesized by granular keratinocytes as a 52–56-kDa protein, corneodesmosin is progressively proteolysed during corneocyte maturation. This processing is a prerequisite for desquamation. Two glycine- and serine-rich domains of the protein might take on the conformation of adhesive secondary structures similar to glycine loops. Corneodesmosin proteolysis was further characterized. Deglycosylation experiments and reactivity with lectins demonstrated that the corneodesmosin carbohydrate moiety does not prevent the proteolysis. Immunoblotting, immunohistochemistry, and immunoelectron microscopy experiments using affinity-purified anti-peptide antibodies raised to four of the five structural domains of corneodesmosin and a monoclonal antibody against its fifth central domain showed that the first step in corneodesmosin processing is the cleavage of its extremities and probably occurs before its incorporation into desmosomes. Then the glycine loop-related domains are cleaved, first the N-terminal and then part of the C-terminal domain. At the epidermis surface, the multistep proteolytic cleavage leaves intact only the central domain, which was detected on exfoliated corneocytes and probably lacks adhesive properties. Importantly, corneodesmosin was demonstrated to be a preferred substrate of two serine proteases involved in desquamation, the stratum corneum tryptic and chymotryptic enzymes.

Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes.

When confluent cultures of the transformed human keratinocyte line SV‐K14 are shifted to serum‐free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross‐linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane‐bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.

Reconstructed skin from cultured human keratinocytes and fibroblasts on a collagen-glycosaminoglycan biopolymer substrate.

A lattice prepared from biopolymer substrate bovine skin collagen and chondroitin-6-sulfate (glycosaminoglycan) served as a support for normal human keratinocytes and fibroblasts. Air exposure of the lattice on an agarose block gave rise to reconstructed epidermis, the histological features of which are very similar to normal human epidermis. Indirect immunofluorescence staining of the plasma membrane-associated transglutaminase, the enzyme responsible for the synthesis of the cornified envelope, revealed the same tissue distribution as observed in vivo. Cell cycle analysis showed a large shift of the normal human keratinocyte population into S-phase and cell division during the 1st week postinoculation. Furthermore, the effects of two modulators of differentiation (25-hydroxycholesterol and sodium butyrate) on the reconstruction of the epidermis were evaluated.

The use of reconstructed human skin to evaluate UV-induced modifications and sunscreen efficacy.

Abstract Biological and clinical effects of sun exposures are characterized by short‐term reactions, i.e. sunburn reaction and suntan, as well as long‐term consequences corresponding to photoaging and photocancers. We have developed several human in vitro three‐dimensional models in order to assess both the photodamage and the photoprotection afforded by sunscreens. Using a full thickness reconstructed skin comprising a differentiated epidermis and a living dermal equivalent, UVB‐ and UVA‐induced biological markers could be found at both the keratinocyte and the fibroblast level. Typical markers of the sunburn reaction could be reproduced in that model as well as dermal damages related to the photoaging process. Another model of reconstructed epidermis, comprising keratinocytes but also melanocytes and Langerhans cells, has been developed. The study of the UV‐induced pigmentation as possible using the pigmented reconstructed epidermis and allowing to reproduce the epidermal melanin unit. The assessment of cellular parameters related to UV‐induced immunosuppression could be performed using the reconstructed epidermis containing Langerhans cells. Exposure to solar‐simulated radiation provokes morphological alterations and the reduction in numbers of Langerhans cells within the exposed epidermis. Using all these models, the efficiency of sunscreens could be envisaged after topical application. The results showed that appropriate sunscreens could efficiently prevent the damage described above.

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.

Determination of retinoid activity by an enzyme-linked immunosorbent assay

In normal human keratinocytes, retinoic acid suppresses the expression of the plasma membrane associated enzyme transglutaminase (TGm) at the pretranslational level. This finding led us to develop an enzyme-linked immunosorbent assay (ELISA) for the evaluation of the biological activity of retinoids, i.e., natural and synthetic derivatives of vitamin A. In this assay, keratinocytes are cultured in a 96-well cluster in the presence of different retinoid concentrations. The expression of TGm is then quantified, without any extraction or purification step, using a TGm-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. The dose-response curves obtained show this ELISA to be a sensitive and reproducible assay to determine the potency of retinoids.

Incomplete epidermal differentiation of A431 epidermoid carcinoma cells

SummaryA431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10−3M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth.

Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis

Abstract:u2002 Using fluorescein isothiocyanate (FITC)‐labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid‐rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pHu20035.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model.

Plasma membrane transglutaminase and cytosolic transglutaminase form distinct envelope-like structures in transformed human keratinocytes.

Cross‐linked envelope formation in the transformed human keratinocyte line SV‐K14 requires treatment of the cells with a Ca2+ ionophore. Depending on the culture conditions, different extracellular Ca2+ concentrations are necessary to trigger the process which is catalyzed by the enzyme transglutaminase. Confluent cells grown in the presence of serum express only the cytosoluble form of the enzyme and need 5 mM Ca2+ for optimum protein cross‐linking, whereas serum‐starved cells which additionally contain the plasma membrane associated form of the enzyme require only 1 mM Ca2+. The envelope‐like structures thus synthesized are morphologically and biochemically distinct.