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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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Immunofluorescent analysis  of keratins in cultured primary mouse  keratinocytes (No. 10 line). Keratinocytes were isolated from the epidermides of newborn control, K16 heterozygote, and K16 homozygote  littermates, cultured, and processed for  indirect, double-immunofluorescence  to analyze the keratin filament networks. (A–C) Keratinocytes from wild-type control (A), heterozygote (B),  and homozygote littermates (C) were  stained with the 1275 antibody to detect K16. (D–F) These same cells were  double stained with the LL001 antibody to detect K14. A low percentage  of wild-type cells express a keratin that  is recognized by the 1275 antibody (A).  Transgene expression in the heterozygote sample was detectable in all cells  (B) and colocalized with the endogenous network (E). A subset of homozygous cells (C) featured a network  in which the bulk of keratin filaments  were located near the nucleus. This included the endogenous keratins (F).  (G–I) Wild-type (G), heterozygote  (H), and homozygote (I) keratinocytes  were stained with K8.12 antibody,  which recognizes K16 in an aggregated  form (82). A small subset of heterozygote cells (H) featured punctate staining distributed throughout the cytoplasm. In contrast, many homozygous  cells as shown in I feature a much  higher density of punctate staining near  the nucleus. N, nucleus. Bar, 25 μm.
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Figure 9: Immunofluorescent analysis of keratins in cultured primary mouse keratinocytes (No. 10 line). Keratinocytes were isolated from the epidermides of newborn control, K16 heterozygote, and K16 homozygote littermates, cultured, and processed for indirect, double-immunofluorescence to analyze the keratin filament networks. (A–C) Keratinocytes from wild-type control (A), heterozygote (B), and homozygote littermates (C) were stained with the 1275 antibody to detect K16. (D–F) These same cells were double stained with the LL001 antibody to detect K14. A low percentage of wild-type cells express a keratin that is recognized by the 1275 antibody (A). Transgene expression in the heterozygote sample was detectable in all cells (B) and colocalized with the endogenous network (E). A subset of homozygous cells (C) featured a network in which the bulk of keratin filaments were located near the nucleus. This included the endogenous keratins (F). (G–I) Wild-type (G), heterozygote (H), and homozygote (I) keratinocytes were stained with K8.12 antibody, which recognizes K16 in an aggregated form (82). A small subset of heterozygote cells (H) featured punctate staining distributed throughout the cytoplasm. In contrast, many homozygous cells as shown in I feature a much higher density of punctate staining near the nucleus. N, nucleus. Bar, 25 μm.

Mentions: To visualize the entire keratin filament network in transgenic basal keratinocytes, newborn mice were killed and isolated keratinocytes were placed in culture and processed for immunofluorescence using a variety of antibodies against different keratins. When cultured cells from non-transgenic mice were stained to detect K16, ∼10% of the cells were positive for a keratin presumed to be mouse K16 (Fig. 9 A). When double stained with an antibody that recognizes the endogenous K14, the two immunofluorescence signals were found to coalign (Fig. 9 D). All cells from heterozygous transgenic mice were positive for the human K16 transgene (Fig. 9 B). The transgene colocalized with the endogenous keratin network as determined by double staining with an antibody to K14 (Fig. 9 E). No abnormalities were observed in the organization of the keratin filaments within these cells. Other keratins analyzed including K5, K6, and K17 were found to colocalize with the K16 transgene product (data not shown). No K10 staining was observed suggesting that the cells in culture are basal-like (data not shown). In contrast to wild type and heterozygotes, a subset of the homozygote keratinocytes when stained for the K16 transgene featured a severely disrupted keratin network in which the filaments had relocalized in a perinuclear fashion (Fig. 9 C). This was very reminiscent of the results obtained when K16 was transfected into Ptk2 cells (69) and when K16 was overexpressed in the suprabasal cells of trangenic mice (82). The endogenous network also featured this relocalization as determined by staining for K14 (Fig. 9 F). The type II keratins K5 and K6 also colocalized to these reorganized filaments (data not shown). The fact that this relocalization is only seen in the homozygous cells strongly argues that a certain amount of K16 is needed with respect to the other keratins to cause this effect.


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)

Immunofluorescent analysis  of keratins in cultured primary mouse  keratinocytes (No. 10 line). Keratinocytes were isolated from the epidermides of newborn control, K16 heterozygote, and K16 homozygote  littermates, cultured, and processed for  indirect, double-immunofluorescence  to analyze the keratin filament networks. (A–C) Keratinocytes from wild-type control (A), heterozygote (B),  and homozygote littermates (C) were  stained with the 1275 antibody to detect K16. (D–F) These same cells were  double stained with the LL001 antibody to detect K14. A low percentage  of wild-type cells express a keratin that  is recognized by the 1275 antibody (A).  Transgene expression in the heterozygote sample was detectable in all cells  (B) and colocalized with the endogenous network (E). A subset of homozygous cells (C) featured a network  in which the bulk of keratin filaments  were located near the nucleus. This included the endogenous keratins (F).  (G–I) Wild-type (G), heterozygote  (H), and homozygote (I) keratinocytes  were stained with K8.12 antibody,  which recognizes K16 in an aggregated  form (82). A small subset of heterozygote cells (H) featured punctate staining distributed throughout the cytoplasm. In contrast, many homozygous  cells as shown in I feature a much  higher density of punctate staining near  the nucleus. N, nucleus. Bar, 25 μm.
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Figure 9: Immunofluorescent analysis of keratins in cultured primary mouse keratinocytes (No. 10 line). Keratinocytes were isolated from the epidermides of newborn control, K16 heterozygote, and K16 homozygote littermates, cultured, and processed for indirect, double-immunofluorescence to analyze the keratin filament networks. (A–C) Keratinocytes from wild-type control (A), heterozygote (B), and homozygote littermates (C) were stained with the 1275 antibody to detect K16. (D–F) These same cells were double stained with the LL001 antibody to detect K14. A low percentage of wild-type cells express a keratin that is recognized by the 1275 antibody (A). Transgene expression in the heterozygote sample was detectable in all cells (B) and colocalized with the endogenous network (E). A subset of homozygous cells (C) featured a network in which the bulk of keratin filaments were located near the nucleus. This included the endogenous keratins (F). (G–I) Wild-type (G), heterozygote (H), and homozygote (I) keratinocytes were stained with K8.12 antibody, which recognizes K16 in an aggregated form (82). A small subset of heterozygote cells (H) featured punctate staining distributed throughout the cytoplasm. In contrast, many homozygous cells as shown in I feature a much higher density of punctate staining near the nucleus. N, nucleus. Bar, 25 μm.
Mentions: To visualize the entire keratin filament network in transgenic basal keratinocytes, newborn mice were killed and isolated keratinocytes were placed in culture and processed for immunofluorescence using a variety of antibodies against different keratins. When cultured cells from non-transgenic mice were stained to detect K16, ∼10% of the cells were positive for a keratin presumed to be mouse K16 (Fig. 9 A). When double stained with an antibody that recognizes the endogenous K14, the two immunofluorescence signals were found to coalign (Fig. 9 D). All cells from heterozygous transgenic mice were positive for the human K16 transgene (Fig. 9 B). The transgene colocalized with the endogenous keratin network as determined by double staining with an antibody to K14 (Fig. 9 E). No abnormalities were observed in the organization of the keratin filaments within these cells. Other keratins analyzed including K5, K6, and K17 were found to colocalize with the K16 transgene product (data not shown). No K10 staining was observed suggesting that the cells in culture are basal-like (data not shown). In contrast to wild type and heterozygotes, a subset of the homozygote keratinocytes when stained for the K16 transgene featured a severely disrupted keratin network in which the filaments had relocalized in a perinuclear fashion (Fig. 9 C). This was very reminiscent of the results obtained when K16 was transfected into Ptk2 cells (69) and when K16 was overexpressed in the suprabasal cells of trangenic mice (82). The endogenous network also featured this relocalization as determined by staining for K14 (Fig. 9 F). The type II keratins K5 and K6 also colocalized to these reorganized filaments (data not shown). The fact that this relocalization is only seen in the homozygous cells strongly argues that a certain amount of K16 is needed with respect to the other keratins to cause this effect.

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