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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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Determination of the level of transgene expression in the epidermis. Urea-soluble protein extracts were obtained from the  epidermides of 7-d-old control, heterozygous, and homozygous littermates from the various transgene lines. Equivalent amounts of total  urea-extractable protein (20 μg) were electrophoresed via SDS-PAGE and transferred to nitrocellulose for subsequent Western blot  analysis. (A) Western blot analysis of the K16 transgenics. The indicated amounts of purified human recombinant K14 and K16 were  used to establish a standard curve for densitometric analysis. The LL001 antibody (71) was used to detect K14 and the 1275 antibody  (82) to detect K16. (B) Western blot analysis of the K16-C14 chimera transgenics. The LL001 antibody was used to detect both mouse  K14 and the K16-C14 transgene protein. Their position of migration of each protein is indicated by the arrows on the right. Purified recombinant K16-C14 was used to determine the migration position of the transgene product. (C) The three No. 21 line samples were subjected to Western blot analysis using the K8.12 antibody (this antibody is known to react with at least K13, K15, and K16). No bands of  molecular weight ⪝40 kD were detected in any of the three samples. Note that a minor degradation product of the purified K16 (see D)  reacts with the antibody. The lower arrow indicates the bottom of the gel. (D) The same samples were subjected to SDS-PAGE and  stained with Coomassie blue. The are no significant protein products below the type I keratin cluster (⪝40 kD) and no significant differences in the total amount of keratin proteins among the three samples.
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Figure 2: Determination of the level of transgene expression in the epidermis. Urea-soluble protein extracts were obtained from the epidermides of 7-d-old control, heterozygous, and homozygous littermates from the various transgene lines. Equivalent amounts of total urea-extractable protein (20 μg) were electrophoresed via SDS-PAGE and transferred to nitrocellulose for subsequent Western blot analysis. (A) Western blot analysis of the K16 transgenics. The indicated amounts of purified human recombinant K14 and K16 were used to establish a standard curve for densitometric analysis. The LL001 antibody (71) was used to detect K14 and the 1275 antibody (82) to detect K16. (B) Western blot analysis of the K16-C14 chimera transgenics. The LL001 antibody was used to detect both mouse K14 and the K16-C14 transgene protein. Their position of migration of each protein is indicated by the arrows on the right. Purified recombinant K16-C14 was used to determine the migration position of the transgene product. (C) The three No. 21 line samples were subjected to Western blot analysis using the K8.12 antibody (this antibody is known to react with at least K13, K15, and K16). No bands of molecular weight ⪝40 kD were detected in any of the three samples. Note that a minor degradation product of the purified K16 (see D) reacts with the antibody. The lower arrow indicates the bottom of the gel. (D) The same samples were subjected to SDS-PAGE and stained with Coomassie blue. The are no significant protein products below the type I keratin cluster (⪝40 kD) and no significant differences in the total amount of keratin proteins among the three samples.

Mentions: In Fig. 2 A the results are shown for the phenotypic K16 transgenic line Nos. 10, 21, and 6 and in Fig. 2 B are the results for the K16-C14 chimera line Nos. A2, B1, and C1. Rabbit polyclonal antibody No. 1275, which specifically reacts with the tail of K16 (human and mouse; see references 18, 55) was used to detect the K16 transgene. Mouse mAb LL001 (71), which recognizes a tail epitope that is identical between human and mouse K14 was used to detect mouse K14 and the human K16-C14 transgene. The amount of mouse K14 in the extracts was equivalent regardless of the genotype, phenotype, or transgene line from which they were derived as determined by densitometry of the K14 blot (Fig. 2 A). From this result, mouse K14 was used as an internal reference to allow for quantitation of transgene protein.


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)

Determination of the level of transgene expression in the epidermis. Urea-soluble protein extracts were obtained from the  epidermides of 7-d-old control, heterozygous, and homozygous littermates from the various transgene lines. Equivalent amounts of total  urea-extractable protein (20 μg) were electrophoresed via SDS-PAGE and transferred to nitrocellulose for subsequent Western blot  analysis. (A) Western blot analysis of the K16 transgenics. The indicated amounts of purified human recombinant K14 and K16 were  used to establish a standard curve for densitometric analysis. The LL001 antibody (71) was used to detect K14 and the 1275 antibody  (82) to detect K16. (B) Western blot analysis of the K16-C14 chimera transgenics. The LL001 antibody was used to detect both mouse  K14 and the K16-C14 transgene protein. Their position of migration of each protein is indicated by the arrows on the right. Purified recombinant K16-C14 was used to determine the migration position of the transgene product. (C) The three No. 21 line samples were subjected to Western blot analysis using the K8.12 antibody (this antibody is known to react with at least K13, K15, and K16). No bands of  molecular weight ⪝40 kD were detected in any of the three samples. Note that a minor degradation product of the purified K16 (see D)  reacts with the antibody. The lower arrow indicates the bottom of the gel. (D) The same samples were subjected to SDS-PAGE and  stained with Coomassie blue. The are no significant protein products below the type I keratin cluster (⪝40 kD) and no significant differences in the total amount of keratin proteins among the three samples.
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Figure 2: Determination of the level of transgene expression in the epidermis. Urea-soluble protein extracts were obtained from the epidermides of 7-d-old control, heterozygous, and homozygous littermates from the various transgene lines. Equivalent amounts of total urea-extractable protein (20 μg) were electrophoresed via SDS-PAGE and transferred to nitrocellulose for subsequent Western blot analysis. (A) Western blot analysis of the K16 transgenics. The indicated amounts of purified human recombinant K14 and K16 were used to establish a standard curve for densitometric analysis. The LL001 antibody (71) was used to detect K14 and the 1275 antibody (82) to detect K16. (B) Western blot analysis of the K16-C14 chimera transgenics. The LL001 antibody was used to detect both mouse K14 and the K16-C14 transgene protein. Their position of migration of each protein is indicated by the arrows on the right. Purified recombinant K16-C14 was used to determine the migration position of the transgene product. (C) The three No. 21 line samples were subjected to Western blot analysis using the K8.12 antibody (this antibody is known to react with at least K13, K15, and K16). No bands of molecular weight ⪝40 kD were detected in any of the three samples. Note that a minor degradation product of the purified K16 (see D) reacts with the antibody. The lower arrow indicates the bottom of the gel. (D) The same samples were subjected to SDS-PAGE and stained with Coomassie blue. The are no significant protein products below the type I keratin cluster (⪝40 kD) and no significant differences in the total amount of keratin proteins among the three samples.
Mentions: In Fig. 2 A the results are shown for the phenotypic K16 transgenic line Nos. 10, 21, and 6 and in Fig. 2 B are the results for the K16-C14 chimera line Nos. A2, B1, and C1. Rabbit polyclonal antibody No. 1275, which specifically reacts with the tail of K16 (human and mouse; see references 18, 55) was used to detect the K16 transgene. Mouse mAb LL001 (71), which recognizes a tail epitope that is identical between human and mouse K14 was used to detect mouse K14 and the human K16-C14 transgene. The amount of mouse K14 in the extracts was equivalent regardless of the genotype, phenotype, or transgene line from which they were derived as determined by densitometry of the K14 blot (Fig. 2 A). From this result, mouse K14 was used as an internal reference to allow for quantitation of transgene protein.

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