Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10754-10758, November 1993 Cell Biology Overexpression of human loricrin in transgenic mice produces a normal phenotype Kozo YONEDA AND PETER M. STEINERT Skin Biology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Henry Metzger, August 25, 1993 (received for review July 7, 1993) ABSTRACT The cornified cell envelope (CE) ofterminally novel glycine loop motif which is variable in sequence and differentiating stratified squamous epithelial cells is a complex highly flexible in likely conformation (16, 17). Since the same multiprotein assembly about 15 nm thick of which loricrin is a glycine loop motifexists on the keratin intermediate filaments major component. We have produced transgenic mice bearing also expressed in terminally differentiated epithelial cells (16, the human loricrin transgene in order to study the role of 18-20), it was proposed that an interaction between the loricrin in CE assembly, structure, and function. By analyses glycine loop sequences of loricrin on the CE and on the of RNA and protein, we show that the human transgene is intracellular keratin intermediate filaments may stabilize expressed in mouse epithelial tissues in an appropriate devel- cellular structure (16). opmental manner but at an overall level about twice that of Attempts to test these hypotheses and to study the struc- endogenous mouse loricrin. Thus the 20-kbp construct used ture and function of loricrin have proven difficult for several contains all necessary regulatory elements. By immunogold reasons. First, loricrin is poorly expressed in established electron microscopy, all of the expressed protein is incorpo- epithelial cell culture systems (21). In our hands, transfec- rated into the CE. That no alternations were noted indicates tions of loricrin constructs into a wide variety of cells have that overproduction of the loricrin component of the CE does resulted in very little or no expression, apparently because it not affect the flexible structure or function of the epithelial is toxic (unpublished observations). Further, it has not been tissues. Furthermore, these data imply that loricrin may be the possible to isolate loricrin for direct biochemical experiments last protein to be deposited onto, and thus lines, the intracel- apparently because it is rapidly crosslinked after synthesis lular surface of the CE, where it may be accessible to interact (11). As an alternative, we have explored the expression of with the subjacent keratin intermediate-ifiament network. the human loricrin gene in transgenic mice. A 20-kbp genomic construct was found to direct the net overexpression of The cornified cell envelope (CE) is a 15-nm-thick layer of human loricrin that is assimilated without evident pathology. protein deposited on the intracellular surface of the plasma We interpret this to mean that it is a very late component of membrane of terminally differentiating stratified squamous CE assembly, so that the intracellular surface is layered with epithelia tissues, where its major role is to provide a barrier loricrin, an event that is consistent with the hypothesis for an against the environment (1, 2). The highly insoluble nature of interaction between loricrin and the keratin intermediate the CE can be attributed to crosslinking of its constituent filaments. proteins by both disulfide bonds and NE-(--glutamyl)lysine isodipeptide bonds formed by the action of transglutami- MATERIALS AND METHODS nases. Likely CE proteins include involucrin (3, 4), cystatin A (5, 6), members of the class of small proline-rich proteins Construction of the Human Loricrin Transgene. A plasmid (7-9), trichohyalin (10), loricrin (11, 12), and possibly other containing the human loricrin gene was constructed by stan- proteins such as filaggrin and keratin intermediate filaments dard methods in three steps. An existing human loricrin (13, 14). To date, only loricrin has been shown to be genomic clone which contained about 1.5 kbp of 5' and 9 kbp crosslinked directly to the CE by isodipeptide bonds (12). of3' flanking sequences (17) was cleaved at its unique BssHII Thus, major questions arise as to the relative contributions site in the coding sequence. By use of PCR the coding of these proteins to the structure, organization, and function sequence 3' to this site was modified by insertion of a 27-bp of the CE. It has proven difficult to ascertain the temporal fragment encoding the carboxyl-terminal part of the neu- order of expression, the abundance, and the function ofthese ropeptide substance P sequence, just prior to the termination proteins in the CE because the irreversibly crosslinked nature codon. The substance P nucleotide sequence (but not coding of the CE precludes dissection of the structure and because sequence) was modified by creation ofa unique Sal I site. The several of the proteins appear to be expressed concurrently engineered fragment was then reinserted into the genomic very late in terminal differentiation in epithelia such as the clone. Next, a 9-kbp genomic clone harboring sequence 5' to epidermis (1, 2). However, some structure-function data are the loricrin cap site was isolated by standard procedures (17) now emerging. Due to their elongated a-helical structures, and ligated to the assembly. The final construct of about 20 involucrin (15) and trichohyalin (10) have been suggested to kbp (Fig. 1) was assembled in the pGEM-3Z vector serve as "scaffold" proteins in the CE, to which other more (Promega) and contained in the following linear order, 5' to abundant proteins are later attached. Also, two types of data 3', about 9 kbp of 5' flanking sequence; the entire human suggest that loricrin is the major CE component: its mRNA loricrin gene consisting of exon I in 5' untranslated sequence, represents about 10% of the total in such tissues as the intron 1, and exon II modified with the substance P peptide; epidermis (10-13), and its amino acid composition is strik- and about 8.6 kbp of 3' noncoding sequence. Unique Sph I ingly similar to that of isolated epidermal CEs, especially andXba I sites at the 5' and 3' ends ofthe construct were used with respect to the high content ofglycine and serine (11-13). to excise the DNA from the plasmid vector. These glycine/serine-rich sequences are thought to adopt a Transgenic Mice. The construct was microinjected into fertilized mouse eggs [(C57BL6/J x SJLJ)F1, The Jackson as mice The publication costs of this article were defrayed in part by page charge Laboratory] essentially described (22). Transgenic payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CE, cell envelope. 10754 Downloaded by guest on October 4, 2021 Cell Biology: Yoneda and Steinert Proc. Natl. Acad. Sci. USA 90 (1993) 10755 8 kbp 200 bp 8 kbp ,Z.- K0~~ CAP ATG TAG MTAAA Mouse -> -1.6 ExonIl gI Exo II SphI SphM sKI Dra I BssHU1 Sal I Dra I Kpn I Xba I Human -->. i- 1.3 FIG. 1. Structure of20-kbp construct. The central 2-kbp gene was gene flanked at both the 5' and the 3' end by about 9 kbp. The consists lJ ll1 of exon I in 5' untranslated sequence, a large intron, and exon II, which contains all of the coding sequence. The coding portion was Loricrin Substance P modified by insertion of the substance P neuropeptide sequence probe probe immediately prior to the termination codon. Diagnostic control FIG. 2. Northern blot of total epidermal RNA showing expres- sequences and restriction enzyme sites are shown. CAP, mRNA sion of endogenous mouse loricrin (1.6 kb) and transgenic hL.P (1.3 start; ATG, initiation codon; TAG, termination codon; AATAAA, kb) mRNAs. Wt, wild type; Non-Tg, nontransgenic littermates; polyadenylylation signal. TgTg, homozygous transgenic mice. Similar blots of RNA from epidermis, esophagus, and stomach were scanned in a phospho- were identified by PCR using two synthetic oligonucleotides: imaging device to estimate an -1.8-fold overexpression of the hL.P plus strand in the substrance P tag, 5'-GTCGACTGTCAGT- transcript. TCTTCGG-3', and minus strand in 3' noncoding sequence, 5'-CACCTTTCCGCCAATAGAGT-3'. PCRs employed a levels of structural homology in terms of their glycine loop commercial DNA amplification kit (Perkin-Elmer/Cetus) motifs, as well as sequence homology, especially in their with an initial denaturation at 95°C (1.5 min) and 30 cycles of amino- and carboxyl-terminal glutamine-rich sequences. Ac- denaturation at 95°C (0.5 min), annealing at 54°C (1 min), and cordingly, we configured the substance P neuropeptide se- elongation at 72°C (1 min), with 1 ug of genomic DNA. quence just prior to the termination codon (Fig. 1) in order to Miscellaneous Procedures. RNA was isolated from heat- follow the expression of human loricrin (termed hL.P) in the separated epidermis or other tissues by extraction with mouse background at both the nucleic acid and protein levels. guanidine hydrochloride (23). Northern blots (12) were The 20-kbp construct used contained the entire human lori- probed with either the substance P oligonucleotide defined crin gene of 2 kbp flanked by about 9 kbp on each side (Fig. above (for specific reaction of hL.P transcripts only) or a 1). Following injection of this construct, three founder lines human loricrin cDNA coding probe (12) (which crossreacts were produced, which by PCR analysis and Southern blot with both the endogenous mouse and hL.P transcripts). RNA analysis using specific primers, had stably incorporated were done with DNase I-treated total PCR experiments five into the mouse genome (data not shown). cellular RNA (2 ng) of transgenic and nontransgenic animals about copies Such founders were then bred to produce heterozygous F1 (24).