Isolation, sequence, and expression of a human keratin K5 gene: transcriptional regulation of keratins and insights into pairwise control.

Abstract

The mitotically active basal layers of most stratified squamous epithelia express 10 to 30% of their total protein as keratin. The two keratins specifically expressed in these cells are the type II keratin K5 (58 kilodaltons) and its corresponding partner, type I keratin K14 (50 kilodaltons), both of which are essential for the formation of 8-nm filaments. Dissecting the molecular mechanisms underlying the coordinate regulation of the two keratins is an important first step in understanding epidermal differentiation and in designing promoters that will enable delivery and expression of foreign gene products in stratified squamous epithelia, e.g., skin. Previously, we reported the sequence of the gene encoding human K14 (D. Marchuk, S. McCrohon, and E. Fuchs, Cell 39:491-498, 1984; Marchuk et al., Proc. Natl. Acad. Sci. USA 82:1609-1613, 1985. We have now isolated and characterized the gene encoding human K5. The sequence of the coding portion of this gene matched perfectly with that of a partial K5 cDNA sequence obtained from a cultured human epidermal library (R. Lersch and E. Fuchs, Mol. Cell. Biol. 8:486-493, 1988), and gene transfection studies indicated that the gene is functional. Nuclear runoff experiments demonstrated that the K5 and K14 genes were both transcribed at dramatically higher levels in cultured human epidermal cells than in fibroblasts, indicating that at least a part of the regulation of the expression of this keratin pair is at the transcriptional level. When the K5 gene was transfected transiently into NIH 3T3 fibroblasts, foreign expression of the gene caused the appearance of endogenous mouse K14 and the subsequent formation of a keratin filament array in the cells. In this case, transcriptional changes did not appear to be involved in the regulation, suggesting that there may be multiple control mechanisms underlying the pairwise expression of keratins.Only epithelial cells express keratins, a large family of proteins (molecular size, 40 to 67 kilodaltons) (kDa) that assemble into a cytoskeletal network of 8-nm intermediate filaments (IFs). There are two distinct classes of keratins: type I keratins (K9 to K19) are small (40 to 56.5 kDa) and relatively acidic (pKi, 4.5 to 5.5), whereas type II keratins (Kl to K8) are larger (53 to 67 kDa) and more basic (pKi, 5.5 to 7.5) (for reviews, see references 8, 16, 50, and 64). Both types of keratins are essential for filament assembly (12,28,41,63). Type I and type II keratins are frequently expressed as specific pairs, and different pairs are expressed in a tissue-specific, differentiation-specific, and developmentalspecific fashion (11, 66).The molecular mechanisms underlying the regulation of pairs of type I and type II keratins remain largely unknown. It is clear that self-propagating epithelial cells expressing a specific pair of keratins must necessarily cotranscribe these genes. However, coordinate expression does not restrict these genes to a common regulatory mechanism, nor does it rule out the possibility that the two genes may b...

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

“…In fact, staining for plectin revealed an almost complete colocalization with K14. The persistence of K14 is at odds with previous data showing that the stability of a keratin in cell culture is dependent on the expression of its partner keratin (Kulesh and Oshima, 1988;Kulesh et al, 1989;Lersch et al, 1989; Magin et al, 1990;Bader et al, 1991).To ascertain the expression of K14, we performed Northern and Western blots. Northern blot analysis of total RNA revealed that the K14 mRNA expression was unaffected in the absence of one wild-type allele of K5 but decreased in K5-deficient mice (Figure 2).…”

“…Previous data had shown that the stability of a keratin depended on the expression of its partner keratin (Kulesh et al, 1989;Lersch et al, 1989). Yet, the immunofluorescence analysis had revealed the persistence of K14 expression in K5 Ϫ/Ϫ mice.…”

The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14.

“…6). It was surprising that the levels of K5 were appreciable in the -/-mouse sample, since the stability of a type II keratin is known to be dependent upon its partner (Kulesh and Oshima, 1988;Lersch et al, 1989). In previous studies on the putative K14 null EBS patients, several mechanisms were proposed to account for the stabilization of K5 (Chan et al, 1994;Rugg et al, 1994).…”

“…Cell culture studies have indicated that K5 is degraded in the absence of K14 (Kulesh and Oshima, 1988;Lersch et al, 1989;Chan et al, 1994). In epidermis, however, the reduction in K5 protein was not as dramatic as might have been expected based on the paucity of filaments in the basal layer of K14 -/ -epidermis (Rugg et al, 1994; present study).…”

“…These patients still had appreciable levels of K5, which was surprising, since studies in fibroblasts had shown that in the absence of its partner, a keratin is unstable and rapidly turns over (Kulesh and Oshima, 1988;Lersch et al, 1989). Rugg et al (1994) proposed that the K5 protein might be stabilized by association with the plasma membrane.…”