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Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation.

Paladini RD, Coulombe PA - J. Cell Biol. (1998)

Bottom Line: Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative.Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion.We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We previously hypothesized that the type I keratin 16 (K16) plays a role in the process of keratinocyte activation that occurs in response to skin injury (Paladini, R.D., K. Takahashi, N.S. Bravo, and P.A. Coulombe. 1996. J. Cell Biol. 132:381-397). To further examine its properties in vivo, the human K16 cDNA was constitutively expressed in the progenitor basal layer of transgenic mouse skin using the K14 gene promoter. Mice that express approximately as much K16 protein as endogenous K14 display a dramatic postnatal phenotype that consists of skin that is hyperkeratotic, scaly, and essentially devoid of fur. Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative. Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion. The phenotype normalizes at approximately 5 wk after birth. In contrast, control mice expressing a K16-K14 chimeric protein to comparable levels are normal. The character and temporal evolution of the phenotype in the K16 transgenic mice are reminiscent of the activated EGF receptor- mediated signaling pathway in skin. In fact, tyrosine phosphorylation of the EGF receptor is increased in the newborn skin of K16 transgenic mice. We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

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Generation of transgenic mice. (A) Schematic representation of the DNA constructs used. The human K16 and K16-C14 cDNAs were subcloned into a modified version of the K14  expression cassette (75). The cassette features 0.6 kb of human  K14 polyA sequence, the rabbit β-globin intron, and 2 kb of human K14 promoter sequence (88). Arrow, direction and initiation  site of transcription. (B) Phenotype of the K16 transgenic mice at  9 d after birth (No. 21 line). Wild-type (top) and homozygous  transgenic (bottom) littermates are pictured. The smaller phenotypic mouse shows an absence of fur coat and a flaky, wrinkled,  and thickened skin surface. Also, the ears have not yet completely erupted. (C) Phenotype at 3 wk (No. 10 line). Wild-type  (top) and homozygous transgenic (bottom) littermates are pictured. The first hair cycle in mice is complete at ∼3 wk (5, 20) and  the phenotypic littermate is essentially lacking the first hair coat.  The epidermis, however, is no longer flaky and wrinkled in appearance. In contrast, the K16-C14 transgenic mice have a wild-type appearance (data not shown).
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Figure 1: Generation of transgenic mice. (A) Schematic representation of the DNA constructs used. The human K16 and K16-C14 cDNAs were subcloned into a modified version of the K14 expression cassette (75). The cassette features 0.6 kb of human K14 polyA sequence, the rabbit β-globin intron, and 2 kb of human K14 promoter sequence (88). Arrow, direction and initiation site of transcription. (B) Phenotype of the K16 transgenic mice at 9 d after birth (No. 21 line). Wild-type (top) and homozygous transgenic (bottom) littermates are pictured. The smaller phenotypic mouse shows an absence of fur coat and a flaky, wrinkled, and thickened skin surface. Also, the ears have not yet completely erupted. (C) Phenotype at 3 wk (No. 10 line). Wild-type (top) and homozygous transgenic (bottom) littermates are pictured. The first hair cycle in mice is complete at ∼3 wk (5, 20) and the phenotypic littermate is essentially lacking the first hair coat. The epidermis, however, is no longer flaky and wrinkled in appearance. In contrast, the K16-C14 transgenic mice have a wild-type appearance (data not shown).

Mentions: As the first step in creating mice that express human K16 in the absence of K14, transgenic mice were generated to express either the K16 or K16-C14 cDNA in the basal layer of stratified squamous epithelia. The cDNAs were subcloned into a modified version of the K14 expression cassette (88, 75) as shown in Fig. 1 A. The two constructs were transiently transfected in PTK2 cells and in both cases the transgene was expressed and incorporated into the endogenous keratin network as detected by indirect immunofluorescence (data not shown). The K16-C14 chimeric cDNA encodes the head and rod of K16 fused to the tail of K14. The two keratins share 91% amino acid sequence identity in their head domains and 86% identity in their rod domains. The identity is only 38% in the tail domain. As previously shown (69), K16, when transiently expressed in PtK2 cells, can cause the reorganization of the endogenous keratin filament network whereas K14 doesn't have the same ability. When the chimera was transfected into PtK2 cells, the results were similar to those obtained with K14 (data not shown), suggesting that the tail domains are responsible for the differences seen between the two keratins. Therefore, the chimeric construct was chosen as a control and was predicted to behave in a K14-like fashion.


Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation.

Paladini RD, Coulombe PA - J. Cell Biol. (1998)

Generation of transgenic mice. (A) Schematic representation of the DNA constructs used. The human K16 and K16-C14 cDNAs were subcloned into a modified version of the K14  expression cassette (75). The cassette features 0.6 kb of human  K14 polyA sequence, the rabbit β-globin intron, and 2 kb of human K14 promoter sequence (88). Arrow, direction and initiation  site of transcription. (B) Phenotype of the K16 transgenic mice at  9 d after birth (No. 21 line). Wild-type (top) and homozygous  transgenic (bottom) littermates are pictured. The smaller phenotypic mouse shows an absence of fur coat and a flaky, wrinkled,  and thickened skin surface. Also, the ears have not yet completely erupted. (C) Phenotype at 3 wk (No. 10 line). Wild-type  (top) and homozygous transgenic (bottom) littermates are pictured. The first hair cycle in mice is complete at ∼3 wk (5, 20) and  the phenotypic littermate is essentially lacking the first hair coat.  The epidermis, however, is no longer flaky and wrinkled in appearance. In contrast, the K16-C14 transgenic mice have a wild-type appearance (data not shown).
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Related In: Results  -  Collection

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Figure 1: Generation of transgenic mice. (A) Schematic representation of the DNA constructs used. The human K16 and K16-C14 cDNAs were subcloned into a modified version of the K14 expression cassette (75). The cassette features 0.6 kb of human K14 polyA sequence, the rabbit β-globin intron, and 2 kb of human K14 promoter sequence (88). Arrow, direction and initiation site of transcription. (B) Phenotype of the K16 transgenic mice at 9 d after birth (No. 21 line). Wild-type (top) and homozygous transgenic (bottom) littermates are pictured. The smaller phenotypic mouse shows an absence of fur coat and a flaky, wrinkled, and thickened skin surface. Also, the ears have not yet completely erupted. (C) Phenotype at 3 wk (No. 10 line). Wild-type (top) and homozygous transgenic (bottom) littermates are pictured. The first hair cycle in mice is complete at ∼3 wk (5, 20) and the phenotypic littermate is essentially lacking the first hair coat. The epidermis, however, is no longer flaky and wrinkled in appearance. In contrast, the K16-C14 transgenic mice have a wild-type appearance (data not shown).
Mentions: As the first step in creating mice that express human K16 in the absence of K14, transgenic mice were generated to express either the K16 or K16-C14 cDNA in the basal layer of stratified squamous epithelia. The cDNAs were subcloned into a modified version of the K14 expression cassette (88, 75) as shown in Fig. 1 A. The two constructs were transiently transfected in PTK2 cells and in both cases the transgene was expressed and incorporated into the endogenous keratin network as detected by indirect immunofluorescence (data not shown). The K16-C14 chimeric cDNA encodes the head and rod of K16 fused to the tail of K14. The two keratins share 91% amino acid sequence identity in their head domains and 86% identity in their rod domains. The identity is only 38% in the tail domain. As previously shown (69), K16, when transiently expressed in PtK2 cells, can cause the reorganization of the endogenous keratin filament network whereas K14 doesn't have the same ability. When the chimera was transfected into PtK2 cells, the results were similar to those obtained with K14 (data not shown), suggesting that the tail domains are responsible for the differences seen between the two keratins. Therefore, the chimeric construct was chosen as a control and was predicted to behave in a K14-like fashion.

Bottom Line: Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative.Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion.We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We previously hypothesized that the type I keratin 16 (K16) plays a role in the process of keratinocyte activation that occurs in response to skin injury (Paladini, R.D., K. Takahashi, N.S. Bravo, and P.A. Coulombe. 1996. J. Cell Biol. 132:381-397). To further examine its properties in vivo, the human K16 cDNA was constitutively expressed in the progenitor basal layer of transgenic mouse skin using the K14 gene promoter. Mice that express approximately as much K16 protein as endogenous K14 display a dramatic postnatal phenotype that consists of skin that is hyperkeratotic, scaly, and essentially devoid of fur. Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative. Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion. The phenotype normalizes at approximately 5 wk after birth. In contrast, control mice expressing a K16-K14 chimeric protein to comparable levels are normal. The character and temporal evolution of the phenotype in the K16 transgenic mice are reminiscent of the activated EGF receptor- mediated signaling pathway in skin. In fact, tyrosine phosphorylation of the EGF receptor is increased in the newborn skin of K16 transgenic mice. We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

Show MeSH
Related in: MedlinePlus