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Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, Kartasova T, Jarnik M, Steven AC, Roop DR - J. Cell Biol. (2000)

Bottom Line: The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes.Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls.Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.

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Targeting strategy and confirmation of gene targeting event. (A) Partial restriction enzyme maps and schematic representation of the strategy used to ablate loricrin expression in mice. The targeting vector (pDV140, top, arrows indicate the orientation of transcription units), the loricrin gene of 129Sv mice (129-wt, middle) and the recombinant loricrin locus (129-mut, bottom) are shown. Solid lines represent loricrin sequences included in the targeting vector. Thin lines represent sequences excluded from the targeting vector. Zigzag lines symbolize plasmid sequences. The location of the 5′ (SacI–SphI) and 3′ (SacI–BamHI) probes used to identify the recombinant allele in Southern blots are indicated above the 129-wt gene. The dotted lines below the middle and bottom schematics indicate DNA fragments that hybridize to the 3′ and 5′ probe, respectively. In the gene targeting vector, a Pst I fragment containing the entire coding sequence of the loricrin gene (middle, solid box) was deleted and replaced by a neomycin-resistance minigene (PGKneobpA). To facilitate the identification of recombinant ES cell clones, we introduced an HSV-tk into the targeting vector, as well. Note that recombinantion between the targeting vector and the wild-type loricrin gene locus deleted a BamHI site (middle, solid box) and introduced a new SacI site (in PGKneobpA cassette, bottom). (B) Example of a Southern blot analysis. Tail DNA from the offspring of a lor+/− intercross was cut with BamHI and hybridized to the 3′ probe. The genotype of the mice analyzed is indicated above the lanes. Arrows indicate the position of mutant (mut) and wild-type (wt) DNA fragments.
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Figure 1: Targeting strategy and confirmation of gene targeting event. (A) Partial restriction enzyme maps and schematic representation of the strategy used to ablate loricrin expression in mice. The targeting vector (pDV140, top, arrows indicate the orientation of transcription units), the loricrin gene of 129Sv mice (129-wt, middle) and the recombinant loricrin locus (129-mut, bottom) are shown. Solid lines represent loricrin sequences included in the targeting vector. Thin lines represent sequences excluded from the targeting vector. Zigzag lines symbolize plasmid sequences. The location of the 5′ (SacI–SphI) and 3′ (SacI–BamHI) probes used to identify the recombinant allele in Southern blots are indicated above the 129-wt gene. The dotted lines below the middle and bottom schematics indicate DNA fragments that hybridize to the 3′ and 5′ probe, respectively. In the gene targeting vector, a Pst I fragment containing the entire coding sequence of the loricrin gene (middle, solid box) was deleted and replaced by a neomycin-resistance minigene (PGKneobpA). To facilitate the identification of recombinant ES cell clones, we introduced an HSV-tk into the targeting vector, as well. Note that recombinantion between the targeting vector and the wild-type loricrin gene locus deleted a BamHI site (middle, solid box) and introduced a new SacI site (in PGKneobpA cassette, bottom). (B) Example of a Southern blot analysis. Tail DNA from the offspring of a lor+/− intercross was cut with BamHI and hybridized to the 3′ probe. The genotype of the mice analyzed is indicated above the lanes. Arrows indicate the position of mutant (mut) and wild-type (wt) DNA fragments.

Mentions: We have described previously the isolation and characterization of the loricrin gene of the BALB/c mouse strain (Rothnagel et al. 1994). To achieve a high frequency of recombination with the genome of mouse embryonic stem (ES) cells, gene targeting/replacement vectors were constructed using gene sequences from the 129/Sv mouse strain (Capecchi 1989; Deng and Capecchi 1992; Ramirez-Solis et al. 1993; Horie et al. 1994). We have isolated the loricrin gene from this mouse strain. In our targeting vector (Fig. 1 A), a 2.457-kb DNA fragment encompassing the entire loricrin coding sequence is deleted and replaced by a neomycin-resistance minigene (see Fig. 1 A, PGKneobpA) (provided by Allan Bradley, Baylor College of Medicine). The minigene is flanked by DNA sequences derived from the loricrin gene locus (5.8-kb 5′ flanking sequence; 1.5-kb 3′ flanking sequence). To facilitate the isolation of targeted ES cell clones, we inserted a herpes simplex virus type 1 thymidine-kinase minigene (HSV-tk, Fig. 1 A) (Mansour et al. 1988) into the targeting vector (provided by John Lydon, Baylor College of Medicine). Both minigenes are under the transcriptional control of the phosphoglycerate kinase I promotor.


Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, Kartasova T, Jarnik M, Steven AC, Roop DR - J. Cell Biol. (2000)

Targeting strategy and confirmation of gene targeting event. (A) Partial restriction enzyme maps and schematic representation of the strategy used to ablate loricrin expression in mice. The targeting vector (pDV140, top, arrows indicate the orientation of transcription units), the loricrin gene of 129Sv mice (129-wt, middle) and the recombinant loricrin locus (129-mut, bottom) are shown. Solid lines represent loricrin sequences included in the targeting vector. Thin lines represent sequences excluded from the targeting vector. Zigzag lines symbolize plasmid sequences. The location of the 5′ (SacI–SphI) and 3′ (SacI–BamHI) probes used to identify the recombinant allele in Southern blots are indicated above the 129-wt gene. The dotted lines below the middle and bottom schematics indicate DNA fragments that hybridize to the 3′ and 5′ probe, respectively. In the gene targeting vector, a Pst I fragment containing the entire coding sequence of the loricrin gene (middle, solid box) was deleted and replaced by a neomycin-resistance minigene (PGKneobpA). To facilitate the identification of recombinant ES cell clones, we introduced an HSV-tk into the targeting vector, as well. Note that recombinantion between the targeting vector and the wild-type loricrin gene locus deleted a BamHI site (middle, solid box) and introduced a new SacI site (in PGKneobpA cassette, bottom). (B) Example of a Southern blot analysis. Tail DNA from the offspring of a lor+/− intercross was cut with BamHI and hybridized to the 3′ probe. The genotype of the mice analyzed is indicated above the lanes. Arrows indicate the position of mutant (mut) and wild-type (wt) DNA fragments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192642&req=5

Figure 1: Targeting strategy and confirmation of gene targeting event. (A) Partial restriction enzyme maps and schematic representation of the strategy used to ablate loricrin expression in mice. The targeting vector (pDV140, top, arrows indicate the orientation of transcription units), the loricrin gene of 129Sv mice (129-wt, middle) and the recombinant loricrin locus (129-mut, bottom) are shown. Solid lines represent loricrin sequences included in the targeting vector. Thin lines represent sequences excluded from the targeting vector. Zigzag lines symbolize plasmid sequences. The location of the 5′ (SacI–SphI) and 3′ (SacI–BamHI) probes used to identify the recombinant allele in Southern blots are indicated above the 129-wt gene. The dotted lines below the middle and bottom schematics indicate DNA fragments that hybridize to the 3′ and 5′ probe, respectively. In the gene targeting vector, a Pst I fragment containing the entire coding sequence of the loricrin gene (middle, solid box) was deleted and replaced by a neomycin-resistance minigene (PGKneobpA). To facilitate the identification of recombinant ES cell clones, we introduced an HSV-tk into the targeting vector, as well. Note that recombinantion between the targeting vector and the wild-type loricrin gene locus deleted a BamHI site (middle, solid box) and introduced a new SacI site (in PGKneobpA cassette, bottom). (B) Example of a Southern blot analysis. Tail DNA from the offspring of a lor+/− intercross was cut with BamHI and hybridized to the 3′ probe. The genotype of the mice analyzed is indicated above the lanes. Arrows indicate the position of mutant (mut) and wild-type (wt) DNA fragments.
Mentions: We have described previously the isolation and characterization of the loricrin gene of the BALB/c mouse strain (Rothnagel et al. 1994). To achieve a high frequency of recombination with the genome of mouse embryonic stem (ES) cells, gene targeting/replacement vectors were constructed using gene sequences from the 129/Sv mouse strain (Capecchi 1989; Deng and Capecchi 1992; Ramirez-Solis et al. 1993; Horie et al. 1994). We have isolated the loricrin gene from this mouse strain. In our targeting vector (Fig. 1 A), a 2.457-kb DNA fragment encompassing the entire loricrin coding sequence is deleted and replaced by a neomycin-resistance minigene (see Fig. 1 A, PGKneobpA) (provided by Allan Bradley, Baylor College of Medicine). The minigene is flanked by DNA sequences derived from the loricrin gene locus (5.8-kb 5′ flanking sequence; 1.5-kb 3′ flanking sequence). To facilitate the isolation of targeted ES cell clones, we inserted a herpes simplex virus type 1 thymidine-kinase minigene (HSV-tk, Fig. 1 A) (Mansour et al. 1988) into the targeting vector (provided by John Lydon, Baylor College of Medicine). Both minigenes are under the transcriptional control of the phosphoglycerate kinase I promotor.

Bottom Line: The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes.Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls.Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.

Show MeSH
Related in: MedlinePlus