Peter M. Steinert

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Bricks and mortar of the epidermal barrier

A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(ε)-(γ-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of ω-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.

The molecular biology of intermediate filaments

From the hairs on our head to the soles of our feet, our bodies are composed of cells rich in intracellular fibrous proteins called intermediate filaments (IF). Immunological and biochemical data established the existence of five subclasses of IF proteins that can form IF in our various tissues: a complex group of 30 or so keratins of 40-70 kd in epithelia; a single protein desmin of 52 kd in muscle; a single protein vimentin of 53 kd in cells of mesenchymal origin; glial fibrillary acidic protein (GFAP), a single protein of 50 kd in astroglia; and a triplet of neurofilament proteins, NF-L (about 65 kd), NF-M (about 105 kd), and NF-H (about 135 kd) in neuronal cells (Lazarides, 1980; 1982; Steinert, 1981; Zackroff et al., 1981; Steinert et al., 1984a; Weber and Geisler, 1984). The recent plethora of amino acid sequence information now allows a more rigorous classification of these proteins into three (or more) distinct sequence Types (see below). There is growing evidence that our invertebrate passengers and all of their relatives also contain most of these subclasses and sequence Types (Gilbert et al., 1975; Koury and Eckert, 1984; Walter and Biessmann, 1984). Having passed beyond an infancy of phenomenology, the field of IF has now matured to the point where it is being explored by the various approaches generically termed “molecular biology.” This work has largely been directed toward finding answers to fundamental questions about the structure, complexity, origins, expression, and functions of IF. At this time, there are no complete answers to any of these questions, although much progress has been made. In this short review, we examine the most recent data that impinge on these questions.

Identification of a major keratinocyte cell envelope protein, loricrin

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

Self-assembly of bovine epidermal keratin filaments in vitro.

The seven α-keratin polypeptides which comprise the subunits of the in situ epidermal keratin filaments of bovine epidermis polymerize in vitro in dilute salt solution into filaments that have the same structure as the in situ keratin filaments. Most combinations of two or three of the purified polypeptides also polymerize into filaments of the same general structure and these contain the polypeptides in the precise molar ratios of 1:2 or 1:1:1. On the basis of these polypeptide stoichiometries and the characteristic α-type X-ray diffraction pattern by all filaments, it is concluded that the keratin filaments polymerized in vitro and thus the in situ epidermal keratin filaments are comprised of a three-chained unit structure. Turbidometric measurements of the kinetics of filament assembly revealed a biphasic mechanism, involving the initial rate-limiting formation of a hexamer nuclear particle, followed by a more rapid rate of polymerization to filaments. The nuclear particle probably consists of a pair of triple-chained units.

Intermediate filament structure

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.

A leucine→proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis is an autosomal dominant disorder affecting the structural integrity of the suprabasal layers of human epidermis. We have recently documented in one family linkage of the disease phenotype to the cluster of type II keratins. We have now identified a leucine----proline amino acid substitution in the conserved H1 subdomain of keratin 1 that is present only in affected family members. Using a quantitative assay and electron microscopy with synthetic peptides, we show that, whereas the wild-type H1 peptide rapidly disassembles preformed keratin filaments in vitro, the mutant peptide does this far less efficiently. Therefore the mutation in keratin 1 is likely to cause defective keratin filaments and hence a defective cytoskeleton in the epidermal cells in vivo.

Differential expression of multiple transglutaminases in human brain. Increased expression and cross-linking by transglutaminases 1 and 2 in Alzheimer's disease.

The transglutaminase (TGase) family of enzymes, of which seven different members are known in the human genome, participate in many biological processes involving cross-linking proteins into large macromolecular assemblies. The TGase 2 enzyme is known to be present in neuronal tissues and may play a role in neuronal degenerative diseases such as Alzheimers disease (AD) by aberrantly cross-linking proteins. In this paper, we demonstrate by reverse transcriptase-polymerase chain reaction and immunological methods with specific antibodies that in fact three members, the TGase 1, TGase 2, and TGase 3 enzymes, and are differentially expressed in various regions of normal human brain tissues. Interestingly, the TGase 1 and 3 enzymes and their proteolytically processed forms are involved in terminal differentiation programs of epithelial cell development and barrier function. In addition, we found that the levels of expression and activity of the TGase 1 and 2 enzymes were both increased in the cortex and cerebellum of AD patients. Furthermore, whereas normal brain tissues contain ≈1 residue of cross-link/10,000 residues, AD patient cortex and cerebellum tissues contain 30–50 residues of cross-link/10,000 residues. Together, these findings suggest that multiple TGase enzymes are involved in normal neuronal structure and function, but their elevated expression and cross-linking activity may also contribute to neuronal degenerative disease.

Direct Evidence That Involucrin Is a Major Early Isopeptide Cross-linked Component of the Keratinocyte Cornified Cell Envelope

Involucrin was the first protein to be identified as a likely constituent of the insoluble cornified cell envelope (CE) of stratified squamous epithelia. However, to date, direct isolation from CEs of involucrin cross-linked by way of the transglutaminase-induced isopeptide bond has not been reported. We have treated human foreskin CEs with methanol/KOH (saponification) to hydrolyze off much of the lipids. By immunogold electron microscopy, this exposed large amounts of involucrin epitopes as well as of desmoplakin, a desmosomal structural protein. About 20% of the total CE protein could be solubilized by proteolytic digestion after saponification, of which involucrin was the most abundant. Subsequent amino acid sequencing revealed many peptides involving involucrin cross-linked either to itself or to a variety of other known CE protein components, including cystatin α, desmoplakin, elafin, keratins, members of the small proline-rich superfamily, loricrin, and unknown proteins related to the desmoplakin family. Specific glutamines or lysines of involucrin were used to cross-link the different proteins, such as glutamines 495 and 496 to desmoplakin, glutamine 288 to keratins, and lysines 468, 485, and 508 and glutamines 465 and 489 for interchain involucrin cross-links. Many identical peptides were obtained from immature CEs isolated from the inner living cell layers of foreskin epidermis. The multiple cross-linked partners of involucrin provide experimental confirmation that involucrin is an important early scaffold protein in the CE. Further, these data suggest that there is significant redundancy in the structural organization of the CE.

Ceramides Are Bound to Structural Proteins of the Human Foreskin Epidermal Cornified Cell Envelope

An important component of barrier function in human epidermis is contributed by ceramides that are bound by ester linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of masses ranging from about 690 to 890 atomic mass units, of which six quantitatively major species were common. The array of ceramides was similar to that obtained from pig skin, the composition of which is known, thereby providing strong indirect data for their fatty acid and sphingosine compositions. The recovered peptides accounted for about 20% of the total foreskin CE ceramides. By amino acid sequencing, about 35% of the peptides were derived from ancestral glutamine-glutamate-rich regions of involucrin, an important CE structural protein. Another 18% derived from rod domain sequences of periplakin and envoplakin, which are also known or suspected CE proteins. Other peptides were too short for unequivocal identification. Together, these data indicate that involucrin, envoplakin, periplakin, and possibly other structural proteins serve as substrates for the attachment of ceramides by ester linkages to the CE for barrier function in human epidermis.