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© The Rockefeller University Press,
0021-9525/2000//381 $5.00
The Journal of Cell Biology, Volume 151, Number 2,
, 2000 381-388
Original Article |
Targeted Ablation of the Murine Involucrin Gene
Involucrin is synthesized in abundance during terminal differentiation of keratinocytes. Involucrin is a substrate for transglutaminase and one of the precursors of the cross-linked envelopes present in the corneocytes of the epidermis and other stratified squamous epithelia. These envelopes make an important contribution to the physical resistance of the epidermis. We have generated mice lacking involucrin from embryonic stem cells whose involucrin gene had been ablated by homologous recombination. These mice developed normally, possessed apparently normal epidermis and hair follicles, and made cornified envelopes that could not be distinguished from those of wild-type mice. No compensatory increase of mRNA for other envelope precursors was observed.
Key Words: embryonic stem cells • envelopes • skin morphology • skin resistance • involucrin
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
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(
-glutamyl)lysine cross-links formed under the action of transglutaminase 1, an enzyme specific to keratinocytes (Rice and Green 1978; Simon and Green 1985; Thacher and Rice 1985). The protein envelope is covered with a monomolecular layer of N-(
-hydroxyacyl)sphingosine bound to protein by ester bonds (Swartzendruber et al. 1987; Wertz and Downing 1987; Marekov and Steinert 1998). Involucrin is synthesized in the outer living layers of terminally differentiating keratinocytes of all stratified squamous epithelia (Banks-Schlegel and Green 1981). Human involucrin has been shown to be a preferred substrate of keratinocyte transglutaminase and a precursor of the cross-linked envelope (Rice and Green 1979; Eckert et al. 1993; Yaffe et al. 1993). Expression of human involucrin in transgenic mice results in abnormalities of the skin and hair (Crish et al. 1993).
The nucleotide sequence of the involucrin gene has been determined in a large number of anthropoid and nonanthropoid mammals. In all species examined, the coding region of the gene contains a segment of short tandem repeats, which accounts for one half to two thirds of the coding region. The entire involucrin molecule, and particularly its segment of repeats, contains numerous glutamine residues (Eckert and Green 1986), clearly relating to the function of the protein as an amine acceptor in transglutaminase-catalyzed cross-linking (Tseng and Green 1988, Tseng and Green 1990; Phillips et al. 1990, Phillips et al. 1997; Djian and Green 1991; Djian et al. 1993).
Other precursors of the cross-linked envelope are loricrin (Mehrel et al. 1990), the small proline-rich proteins (SPRRs) (Kartasova and van de Putte 1988), and two membrane-bound proteins, periplakin and envoplakin (Simon and Green 1984; Ruhrberg et al. 1996, Ruhrberg et al. 1997). Recently, additional envelope constituents including S100 proteins have been described (Robinson et al. 1997). Whereas the role of transglutaminase 1 in the synthesis of the cornified envelope is clear (Jeon et al. 1998), the relative importance of the various substrates has not been established. Therefore, we decided to generate mice lacking involucrin by targeted ablation of the gene. Surprisingly, the absence of involucrin had no obvious effect on the cornified envelope, the morphology of the epidermis, or the hair follicles.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FIXII vector (Stratagene). Screening of 106 phage DNA plaques (Sambrook et al. 1989) with a 1.2-kb HindIII fragment containing most of the involucrin coding region identified two positive clones, of which clone 17B was used to prepare the targeting vector. This clone contained the coding region flanked by 1.5 kb of upstream sequence and 11.5 kb of downstream sequence. The entire insert of 17B was excised with Not1 (whose site is present in the FIXII vector, on both sides of the insert) and subcloned into pBluescript. The resulting plasmid (psv6) was cut with Not1, Xba1, and Pst1 to excise most of the coding region and to subclone the flanking sequences. These sequences were isolated as a 1.7-kb Not1–Pst1 fragment (upstream) and a 10.6-kb Xba1–Not1 fragment (downstream), thus generating psv18 and psv26, respectively. Since psv18 retained 95 codons, including the initiator ATG, and since we were not certain that the encoded peptide could not act as a substrate in transglutaminase-catalyzed cross-linking, we deleted those codons with BAL31. The resulting clones were sequenced and one clone (psv23) was used in subsequent experiments; it lacked the entire coding region, the 3' splice site, and a small 3' part of the intron (see Fig. 1). The neomycin phosphotransferase gene under the control of the phosphoglucokinase promoter and followed by the phosphoglucokinase 3' end, including the polyA addition site, was excised from pGKneo (a gift from Dr. Fred Alt, Children's Hospital, Boston, MA) with EcoR1 and HindIII. An Xho1 site was added immediately 3' of the HindIII site by subcloning the EcoR1–HindIII fragment into pBluescript and reexcising it with EcoR1 and XhoI. The resulting fragment was cloned directly into the linker of psv23, in the right orientation and downstream of the involucrin 5' sequence. The insert of the resulting plasmid was excised with Not1 and Xho1, blunt-ended with the Klenow fragment, and cloned with Xba1 linkers upstream of the Xba1–Not1 insert of psv26. The sequence flanking the Xba site of the linkers was such as to regenerate the Not1 site that had been partially abolished by treatment with the Klenow enzyme. The orientation of the Xba insert in the clones obtained was determined, and one of the constructs, psv29, was used to transfect ES cells. To facilitate homologous recombination, we excised the insert with Not1 and removed the pieces of linker on both ends by a brief treatment with BAL31, before transfection of the ES cells.
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Southern Blot Analysis
DNA was prepared from ES cells or mouse tails by lysis in the presence of 100 mM Tris-HCl, pH 8.5, 200 mM NaCl, 5 mM EDTA, 0.2% SDS, and 0.1 mg/ml proteinase K. The lysate was incubated at 55°C for at least 6 h, and the DNA was precipitated in the presence of isopropanol, spooled out, and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (Laird et al. 1991). Southern blots were performed as described in Tseng and Green 1988. The probe was a 1.0-kb PstI–HindIII fragment located immediately 5' to the genomic fragment used to prepare the targeting vector (see Fig. 1). To clone the probe, we first constructed a library from size-fractionated genomic DNA. After digestion with Pst1 and electrophoresis of the digest, fragments of 
2.6 kb were excised from the agarose gel and ligated to pBluescript. The ampicillin-resistant bacteria were divided into 22 pools, each containing 7,000 primary transformants. Screening by Southern blotting with the 1.25-kb Not1–Pst1 fragment located immediately upstream of the involucrin coding region revealed two positive pools. The fragment of interest was amplified by PCR, using an upstream primer located in the plasmid, and a downstream primer corresponding to codons 2–8 of the involucrin gene. The PCR product was cut with Pst1 and Hind III and the mixture of fragments was ligated directly into pGEM3z cut with PstI and HindIII. All recombinants obtained contained the fragment of interest (psv35).
Northern Blot Analysis
For preparation of total skin RNA, mice were first anesthetized by intraperitoneal injection of tribromoethanol (Sigma-Aldrich). Hairy mice were depilated with a commercial preparation containing thioglycolic acid. The skin was then removed, frozen immediately in liquid nitrogen, pulverized in a mortar, resuspended in Tripure isolation reagent, and purified according to the manufacturer's recommendation (Boehringer). Total RNA was analyzed by electrophoresis in agarose gels containing formaldehyde, according to Sambrook et al. 1989, and RNA was transferred to nylon without prior treatment of the gel. Prehybridization, hybridization, and washing conditions were exactly as those described for Southern blotting. The probes consisted of a 1.2-kb fragment containing most of the involucrin coding region (Djian et al. 1993), a 0.9-kb BamHI–PstI fragment containing the 3' half of the loricrin coding region (Mehrel et al. 1990), and a 0.7-kb EcoR1 fragment derived from the SPRR1 cDNA (Kartasova et al. 1996).
Western Blot Analysis
Protein extracts containing involucrin were prepared from mouse skin. Pulverized skin was prepared as described above for RNA. Approximately 400 mg of tissue was resuspended in 2 ml of a buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 0.1 mM leupeptin, 0.01 mM pepstatin, and 1 mM PMSF. The tissue was homogenized (Potter Elvekjem), clarified by two centrifugations at 120 g for 15 min and 48,000 g for 30 min, and the supernatant was collected. The protein concentration of the supernatant was determined with a protein assay kit using IgG as standards (Bio-Rad Laboratories). Protein extracts were stored at 4°C in the presence of 1% SDS. Partially purified mouse involucrin was prepared according to Etoh et al. 1986.
The proteins were resolved by SDS-PAGE, using 4 and 6% acrylamide plus bisacrylamide for the stacking and resolving gels, respectively (a ratio of 29:1 for both stacking and resolving gel). Immunoblot analysis was carried out according to Kahlem et al. 1998. The antiinvolucrin (Djian et al. 1993) and the secondary antibody were both added at a 1:4,000 dilution.
Histological Analysis and Preparation of Cornified Envelopes
For sections stained with hematoxylin and eosin, the skin samples were first fixed in 10% buffered formalin and embedded in paraffin then stained. For immunofluorescence studies of involucrin, skin samples were snap frozen in OCT (Miles) and isopentane and cut with a cryomicrotome at 5 mm. Sections were fixed in acetone/methanol (1:1) at –20°C and stained with rabbit anti–mouse involucrin (Covance), then were detected with goat antiserum to rabbit IgG coupled to Alexa 488 (Molecular Probes). DNA was stained using Vectashield mounting medium containing DAPI (Vector Laboratories).
For preparation of envelopes, the tip of the ear was cut, placed in water containing 25 mM DTT and 2% SDS, heated to 100°C for 15 min, and centrifuged. The pellet was resuspended in 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. Envelopes were examined in a hemocytometer under phase microscopy.
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Results |
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The Epidermis and Hair Follicles of Mice Lacking Involucrin Appear Histologically Normal
Histological sections of the back skin of adult wild-type and mice lacking involucrin were compared. No differences were observed either in the epidermis or in the hair follicles (Fig. 4A and Fig. B). As mouse epidermis of the trunk is very thin and contains only two to three layers, we also examined epidermis of the tail, whose thickness is comparable with that of the human epidermis, and which possesses clearly distinguishable basal, spinous, granular, and cornified layers. Neither in the spinous and granular layers, where free involucrin is found, nor in the stratum corneum, where the involucrin is cross-linked in the envelopes, was there a perceptible difference between wild-type (Fig. 4C and Fig. E) and inv–/– (Fig. 4D and Fig. F) littermates. The healing time of the wounds resulting from the skin biopsies used for histological analysis was compared in mice lacking involucrin and normal mice and was not found to be significantly longer in the former. The gross appearance of the esophageal, conjunctival, and corneal epithelia, which also contain involucrin, appeared unchanged in its absence.
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Absence of Involucrin Is Not Compensated by Increased Synthesis of Other Envelope Precursors
In view of the lack of any detectable phenotype produced by the absence of involucrin, we thought that increasing levels of other envelope precursor proteins might have compensated for the absence of involucrin. The levels of the mRNAs encoding loricrin (Mehrel et al. 1990) and SPRR1 (Kartasova and van de Putte 1988; Kartasova et al. 1996) were found to be identical in the mice lacking involucrin and in the wild-type mice (Fig. 6). Therefore, the absence of involucrin is not compensated by increased levels of either loricrin or SPRR1 mRNAs.
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Discussion |
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A large body of data supported the earlier conclusion that involucrin was an essential component of the cross-linked envelope. The synthesis of involucrin is tightly regulated: it is restricted to terminally differentiated keratinocytes of stratified squamous epithelia. Human involucrin is a preferred substrate of transglutaminase 1, since after incubation of a crude extract of cultured epidermal cells in the presence of labeled putrescine and transglutaminase 1, involucrin was labeled at least 80 times more intensely than the average of the other cytosolic protein (Simon and Green 1985). Involucrin was also shown to promote the cross-linking of the particulate fraction containing membrane proteins. In other respects, involucrin is thought suitable as a protein able to cross-link other proteins (Yaffe et al. 1992). Involucrin is rich in glutamine residues (Djian et al. 1993; Eckert and Green 1986), a property in keeping with its function as an amine acceptor in transglutaminase-catalyzed cross-linking; other envelope precursors, such as loricrin, envoplakin, periplakin, or the small proline-rich proteins, are not glutamine-rich. For all of these reasons, it is surprising that envelopes should form normally in mice lacking involucrin. The presence of these cross-linked envelopes in inv–/– mice clearly establishes that contrary to earlier suggestions (Steinert and Marekov 1997), whatever properties might be conferred on envelopes by involucrin, this protein is not essential for the assembly of envelopes, which in its absence consist exclusively of other proteins.
It remains to be seen whether more detailed examination of the skin of inv–/– mice will reveal a detectable difference from that of wild-type mice. We have provided the inv–/– mice to Dr. Peter Elias (University of California at San Francisco, San Francisco, CA) and to Drs. M. Jensen and E. Proksch (University of Kiel, Kiel, Germany) for detailed examination of the water vapor barrier and ultrastructural studies of the envelopes.
The disruption of the loricrin gene, which encodes another envelope precursor (Mehrel et al. 1990), also does not prevent envelope assembly, although it results in transitory redness of skin immediately after birth (Koch et al. 2000, this issue). It seems that whereas the transglutaminase is required in order to make the cross-linked envelope, neither involucrin nor loricrin is individually required. Mutations in the COOH-terminal part of human loricrin cause Vohwinkel syndrome, but the mutated alleles are dominant and the disease is likely to result from a gain of function (Maestrini et al. 1996).
The involucrin gene of the mouse is very polymorphic and this in itself would be compatible with the absence of strong selective pressure. However, this polymorphism includes the addition of numerous repeats that increase the size of the protein from 448 to 695 residues (Delhomme and Djian 2000). The involucrins of the BALB/c and C57BL/6 strains used in this study contain 448 and 468 residues, respectively. It is therefore necessary to suppose either that the increases in size occur in the absence of any demonstrated necessity for the protein, or that evolutionary development of a larger involucrin will eventually render the protein indispensable.
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Acknowledgments |
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We are indebted for research support from the National Cancer Institute, the Centre National de la Recherche Scientifique, and the European Community (Biomed 2).
Submitted: 4 May 2000
Revised: 18 July 2000
Accepted: 27 July 2000
Abbreviations used in this paper: ES, embryonic stem; SPRR, small proline-rich protein.
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