Faculty Bibliography

Keratinocytes of the upper granular layers contain unique round-to-oval granules, 100-500 nm in diameter, in their peripheral cytoplasm. These granules (known as membrane coating granules [MCG], or lamellar granules) fuse with the apical cell surface of uppermost granular cells and discharge their contents into the intercellular space, where they are believed to play a role in establishing the permeability barrier of the epidermis and possibly in regulating the orderly desquamation of terminally differentiated keratinocytes. Using two monoclonal antibodies originally prepared against hair follicle antigens, we have identified a 25K epidermal protein in association with both MCG-like granules in the peripheral cytoplasm of granular cells as well as MCG-derived intercellular material. This protein is relatively basic (pI greater than 8), largely aqueous soluble, methionine deficient, and is relatively abundant in epidermis (comprising up to approximately 0.1% of soluble proteins). Its distribution is restricted to the granular layer of keratinized (cornified) stratified squamous epithelia. The identification of this protein component opens new avenues for studying the molecular mechanisms underlying the establishment of permeability barrier and/or regulation of desquamation

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation

Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes

Hydrocortisone, administered onto the chorioallantoic membrane (CAM) of 7- to 10-day-old chick embryos, inhibits scale development, in a dose- and stage-dependent manner. The response is also region specific in that hydrocortisone treatment, at a specific dose and time, will completely block scutellate and interstitial scale development while leaving other scale types unaffected. Using histological, biochemical, and immunofluorescence techniques, we have shown that inhibition of scutellate scale morphogenesis prevents the subsequent formation of a beta stratum and alters expression of the alpha keratins. These data support the hypotheses that each avian scale type has its own distinctive temporal, morphological, and biochemical pattern of development; and in the case of scutellate scale development, hydrocortisone treatment alters keratin biosynthesis by interfering with earlier steps in morphogenesis

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogeneis, can direct tissue-specific stratification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis

In addition to containing microtubule and microfilament systems, vertebrate epithelial cells contain an elaborate keratin intermediate-filament cytoskeleton. Little is known about its structural organization or function. Using indirect immunofluorescence microscopy with an antikeratin antiserum probe, we found that destabilization of microtubules and microfilaments with cytostatic drugs induces significant alterations in the cytoskeletal organization of keratin filaments in HeLa and fetal mouse epidermal cells. Keratin filament organization was observed to undergo a rapid (1-2 h) transition from a uniform distribution to an open lattice of keratin fibers stabilized by membrane-associated focal centers. Since addition of any one drug alone did not elicit significant organizational change in the keratin cytoskeleton, we suggest that microfilaments and microtubules have a combined role in maintaining the arrangement of keratin in these cells. Vimentin filaments, the only other intermediate-sized filaments found in HeLa cells, did not co-distribute with keratin in untreated or drug-treated cells. These findings offer a new way to approach the study of the dynamics and functional roles of the keratin cytoskeleton in epithelial cells

The distribution of keratin intermediate filaments, previously considered static in organization and imperturbable by conventional drugs used to alter the structure and organization of the cytoskeleton, can be altered significantly by treatment with colchicine and cytochalasin D. The loss of microfilaments and microtubules converts the keratin cytoskeleton from a branching, even distribution to a series of starlike structures whose filaments are maintained by multiple membrane attachment sites. These findings provide a means for manipulating cytokeratin organization to investigate the role of keratins in cytoskeletal structure and function