Limits...
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.

Show MeSH

Related in: MedlinePlus

Determination of  EGFR levels and tyrosine  phosphorylation in the K16  transgenic mice (No. 6 line).  Equivalent amounts (∼30  μg) of newborn skin lysates  from control, heterozygous,  and homozygous K16 transgenic littermates were electrophoresed via SDS-PAGE  and transferred to nitrocellulose for Western blot analysis (in duplicate). Top blot, the four samples were probed with an antibody  specific for the EGFR. Approximately equivalent amounts of the  receptor were present in each of the samples as determined by  densitometry. Bottom blot, the samples were probed with an antibody specific for phosphotyrosine. The control + EGF sample  featured a strong band migrating at the same apparent molecular  weight as the EGFR. The control and heterozygous samples featured faint reactivity. The homozygous sample is increased twofold with respect to the non-stimulated controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132878&req=5

Figure 10: Determination of EGFR levels and tyrosine phosphorylation in the K16 transgenic mice (No. 6 line). Equivalent amounts (∼30 μg) of newborn skin lysates from control, heterozygous, and homozygous K16 transgenic littermates were electrophoresed via SDS-PAGE and transferred to nitrocellulose for Western blot analysis (in duplicate). Top blot, the four samples were probed with an antibody specific for the EGFR. Approximately equivalent amounts of the receptor were present in each of the samples as determined by densitometry. Bottom blot, the samples were probed with an antibody specific for phosphotyrosine. The control + EGF sample featured a strong band migrating at the same apparent molecular weight as the EGFR. The control and heterozygous samples featured faint reactivity. The homozygous sample is increased twofold with respect to the non-stimulated controls.

Mentions: The general morphology of the skin and the temporal nature of the phenotype in the K16 transgenic mice suggested that an activated EGF receptor might be involved in generating the phenotype (see Discussion for details). To test this, skin from newborn wild-type, heterozygous, and homozygous littermates was obtained and the amount and the tyrosine phosphorylation status of the receptor was determined by Western blot analysis. EGF was subcutaneously injected into a control littermate to generate tyrosine-phosphorylated receptor to serve as a reference (Fig. 10, control + EGF).


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  EGFR levels and tyrosine  phosphorylation in the K16  transgenic mice (No. 6 line).  Equivalent amounts (∼30  μg) of newborn skin lysates  from control, heterozygous,  and homozygous K16 transgenic littermates were electrophoresed via SDS-PAGE  and transferred to nitrocellulose for Western blot analysis (in duplicate). Top blot, the four samples were probed with an antibody  specific for the EGFR. Approximately equivalent amounts of the  receptor were present in each of the samples as determined by  densitometry. Bottom blot, the samples were probed with an antibody specific for phosphotyrosine. The control + EGF sample  featured a strong band migrating at the same apparent molecular  weight as the EGFR. The control and heterozygous samples featured faint reactivity. The homozygous sample is increased twofold with respect to the non-stimulated controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132878&req=5

Figure 10: Determination of EGFR levels and tyrosine phosphorylation in the K16 transgenic mice (No. 6 line). Equivalent amounts (∼30 μg) of newborn skin lysates from control, heterozygous, and homozygous K16 transgenic littermates were electrophoresed via SDS-PAGE and transferred to nitrocellulose for Western blot analysis (in duplicate). Top blot, the four samples were probed with an antibody specific for the EGFR. Approximately equivalent amounts of the receptor were present in each of the samples as determined by densitometry. Bottom blot, the samples were probed with an antibody specific for phosphotyrosine. The control + EGF sample featured a strong band migrating at the same apparent molecular weight as the EGFR. The control and heterozygous samples featured faint reactivity. The homozygous sample is increased twofold with respect to the non-stimulated controls.
Mentions: The general morphology of the skin and the temporal nature of the phenotype in the K16 transgenic mice suggested that an activated EGF receptor might be involved in generating the phenotype (see Discussion for details). To test this, skin from newborn wild-type, heterozygous, and homozygous littermates was obtained and the amount and the tyrosine phosphorylation status of the receptor was determined by Western blot analysis. EGF was subcutaneously injected into a control littermate to generate tyrosine-phosphorylated receptor to serve as a reference (Fig. 10, control + EGF).

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