Expression of Unusually Large Keratins during Terminal Differentiation: Balance of Type Type II Keratins Is Not Disrupted land

When a basal epidermal cell undergoes a commitment to terminally differentiate, it ceases to divide and begins to migrate outward towards the surface of the skin. Dramatic changes in its cytoskeletal architecture take place, accompanied by numerous changes in the expression of keratins, a family of related polypeptides that form 8-nm filaments in these cells. We show here that a shift to the synthesis of unusually large keratins occurs that does not seem to disrupt the ratio of two distinct subfamilies of keratins. Preliminary studies indicate that this differentiation-specific shift may be at the level of transcriptional rather than post-trancriptional regulation. The striking similarities between these large keratins and the type I and type II keratins of basal epidermal cells suggests the important role that both classes of large keratin sequences must play in the assembly of the intermediate filaments within the differentiating keratinocyte. Several laboratories have reported differences between the keratins of stratum corneum and those of the living layers of epidermis (1--4). During the course of terminal differentiation, changes occur in the expression of the keratin polypeptides that comprise the 8-nm tonofilaments (5-10). For the human, keratins of size 46, 48.5, 50, 52, 56, and 58 kd are synthesized by the basal epidermal cells (Fig. 1, lane 1), whereas additional keratins of size 67, 65.5, and 56.5 kd are produced by the terminally differentiating epidermis (lane 3). As the cells pass through the granular layer to the stratum corneum, a slight reduction in the size of the keratins takes place. As judged by in vitro translation of mRNAs isolated from basal (lane 2) and differentiating (lane 4) keratinocytes, the changes in keratin pattern that occur early in the course of terminal differentiation are clearly at the level of mRNA biosynthesis (5). Recently, it was demonstrated that the keratins produced by basal epidermal cells can be divided into two distinct groups based on the ability of their mRNAs to cross-hybridize with two different cloned keratin cDNAs (11). Other epithelia express different subsets of keratins, but most if not all of these seem to be similar to one or the other of the two epidermal keratin subfamilies (12). The small (40-52 kd) and relatively acidic keratins have been named the type I class, and the large (53-58 kd) and more basic keratins have been named the type II class (13, 14). At least one member of each of the two keratin types seem to be expressed in all cells at all times, suggesting their combined importance in filament assembly (14). Sequence analyses (13, 15-18) have shown that while the two types of keratins share only low (<30%) homology, their predicted secondary structures are strikingly similar and are compatible with their playing an essential role in forming the coiled-coil backbone of the protofilament of the 8-nm keratin filament (13, 18). The keratins typical of terminally differentiating keratinocytes seem to be unusually large and are not found in other epithelial cells (for review, see reference 19). Whether these keratins are members of the same two subfamilies of sequences already described for basal epidermal cells and other epithelial cells has not yet been determined. In this paper, we explore the relation of the differentiation-specific keratins to other epithelial keratins and we investigate the balance of the ratio of type I and type II keratins during terminal differentiation in human epidermis. MATERIALS AND METHODS Extraction of Keratins and Poly(A)+ RNAs from Cultured Human Basal Epidermal Cells and from Human Skin: Human epidermal cell strains were derived from newborn foreskin and used in their second to fourth subculture. They were grown according to the procedure of Rheinwald and Green (20, 21). When vitamin A is present in the cell culture medium, terminal differentiation is largely inhibited, and the cells resemble basal epidermal ceils (22). Whole human epidermis was obtained fresh as discarded material from surgical operations and used immediately. After the subcutaneous fat and dermis were clipped away, the epidermis was minced in the presence of vanadyi ribonucleoside complex and then frozen in liquid nitrogen prior to mRNA and THE JOURNAL OF CELL BIOLOGY . VOLUME 99 NOVEMBER 1984 1872-1877 1872 © The Rockefeller University Press 0021-9525/84/11/1872106 $1.00 on July 1, 2017 jcb.rress.org D ow nladed fom Fic:ure 1 Synthesis of human epidermal keratins from Poly(A)+ RNA of cultured human basal epidermal cells and differentiating human epidermis. Poly(A)+ RNAs from cultured basal epidermal cells and from adolescent human foreskin epidermis were isolated and translated in vitro and the synthesized products were immunoprecipitated (5). The keratins were separated electrophoretically and the gel was fluorographed. Lane 1, [3SS]methionine-labeled keratin extract from cultured cells; lane 2, radiolabeled immunoprecipitated translation products from cultured cell RNA; lane 3, radiolabeled keratin extract from epidermis; lane 4, radiolabeled immunoprecipitated translation products from epidermis RNA. Molecular weight values are in kilodaltons. A, actin. 285 bp Alul/Pstl fragment beginning at 235 nucleotide residues 3' downstream from the TAA stop codon and ending at the polyA addition site was subeloned. Isolation and Characterization of the Cene Encoding the Type I (50 kd) Human Keratin: Purification of the Bgll Fragment Containing the 3' Noncoding Portion of the Gene: A Haelll/Alul human genomic library (Ed Fritsch, Genetics Institute, Boston, MA) has been screened with the human epidermal keratin eDNA probes KAI and KB-2 (11), and the genes encoding the type I and type II kerafins have been isolated and partially characterized. The gene encoding the 50-kd type I keratin has been identified, and its sequence and characterization will be reported elsewhere. The 3' noncoding region of the 50-kd keratin mRNA hybridized with a single 800 bp BglIfrngment of the gene. The 5' end of this BglI fragment contains the 130 bp noncoding portion of the 50 kd keratin raRNA, while the remainder of the sequence includes a 670 bp stretch that is 3' downstream from the polyadenylation site. This fragment was isolated and purified according to the procedure of Birnboim and Doily (25), and then radiolabeled as described previously (l 1). Immunoblot Analysis: Duplicate samples containing 5 ~g of total protein were resolved by polyacrylamide gel electrophoresis, and unstained gels were then transferred electrophoretically to nitrocellulose paper. Each blot was first incubated in 10 ml of BSA-saline solution containing 100/A of antiserum specific for either the type I or the type II keratin~ and later placed in a solution containing l0 s Clara per ml of ~2SI-labeled Staphylococcus aureus protein A (26). Antisera were prepared by injecting male New Zealand white rabbits with gelpurified 50 (type I) or 56 kd (type II) basal epidermal kerafins. These antisera were shown to cross-react with a number of other type I and type II keratins,