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The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 phenotype by keratin 16.

Paladini RD, Coulombe PA - J. Cell Biol. (1999)

Bottom Line: Degenstein, E.Fuchs. 1995.Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Chemistry and Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The type I epidermal keratins K14 and K16 are remarkably similar at the primary sequence level. While a structural function has been clearly defined for K14, we have proposed that a function of K16 may be to play a role in the process of keratinocyte activation that occurs after acute injury to stratified epithelia. To compare directly the functions of the two keratins we have targeted the expression of the human K16 cDNA to the progenitor basal layer of the epidermis of K14 mice. Mice for K14 blister extensively and die approximately 2 d after birth (Lloyd, C., Q.C. Yu, J. Cheng, K. Turksen, L. Degenstein, E. Hutton, and E. Fuchs. 1995. J. Cell Biol. 129:1329-1344). The skin of mice expressing K16 in the absence of K14 developed normally without evidence of blistering. However, as the mice aged they featured extensive alopecia, chronic epidermal ulcers in areas of frequent physical contact, and alterations in other stratified epithelia. Mice expressing a control K16-C14 cDNA also rescue the blistering phenotype of the K14 mice with only a small percentage exhibiting minor alopecia. While K16 is capable of rescuing the blistering, phenotypic complementation in the resulting skin is incomplete due to the multiple age dependent anomalies. Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

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Immunofluorescence analysis of keratins in cultured primary keratinocytes from replacement mice. Keratinocytes were isolated from the epidermides of newborn K16 and K16-C14 replacement mice, cultured, and analyzed by indirect double immunofluorescence to examine the keratin filament networks. Keratinocytes from K16 replacement mice (A–C) were stained with the 1275 antibody to detect K16. Some K16 replacement keratinocytes had fragmented keratin networks and cytoplasmic areas devoid of filaments (B and C). Others exhibited large bundling of filaments randomly distributed throughout the cytoplasm (A). Keratinocytes from K16-C14 replacement mice (D) were stained with the LL001 antibody to detect K16-C14. These keratinocytes appeared identical to wild-type keratinocytes (data not shown) in all morphological aspects as there was no evidence of keratin reorganization, fragmentation, or loss. Bar, 25 μm.
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Figure 3: Immunofluorescence analysis of keratins in cultured primary keratinocytes from replacement mice. Keratinocytes were isolated from the epidermides of newborn K16 and K16-C14 replacement mice, cultured, and analyzed by indirect double immunofluorescence to examine the keratin filament networks. Keratinocytes from K16 replacement mice (A–C) were stained with the 1275 antibody to detect K16. Some K16 replacement keratinocytes had fragmented keratin networks and cytoplasmic areas devoid of filaments (B and C). Others exhibited large bundling of filaments randomly distributed throughout the cytoplasm (A). Keratinocytes from K16-C14 replacement mice (D) were stained with the LL001 antibody to detect K16-C14. These keratinocytes appeared identical to wild-type keratinocytes (data not shown) in all morphological aspects as there was no evidence of keratin reorganization, fragmentation, or loss. Bar, 25 μm.

Mentions: The electron microscopy results prompted the examination of the replacement keratinocytes in culture in order to analyze the global organization of the keratin filaments within the context of an intact keratinocyte. Primary cultures of newborn keratinocytes from K16 and K16-C14 replacement mice were established and analyzed by immunofluorescence to further determine if there were any possible abnormalities in the keratin networks of the K16 replacement mice. K16 replacement keratinocytes appeared normal after one day in culture (data not shown). All cells were positive for K5, K6, human K16, and K17, and negative for K14 (data not shown). As the cells remained in culture, however, they began to display time-dependent changes in their keratin filament networks. In a subset of cells (<50%), the keratin filament networks began to appear fragmented and even absent in some areas of the cytoplasm (Fig. 3B and Fig. C). There were also large bundles of filaments that were distributed throughout the cell (Fig. 3 A) rather than preferentially located adjacent to the nucleus as previously noted for the K16 ectopic keratinocytes 39. There was no evidence of keratin reorganization, fragmentation, or loss in any of the K16-C14 replacement keratinocytes regardless of the time spent in culture. These keratinocytes featured filament networks that were indistinguishable from wild-type keratinocytes (Fig. 3 D). These data suggest that in the absence of K14, K16 is not able to support a keratin filament network and that over time this fragile network is susceptible to fragmentation and loss. Furthermore, they provide further evidence that the carboxy-terminal ∼105 amino acids of K16 are responsible for these differences in filament organization.


The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 phenotype by keratin 16.

Paladini RD, Coulombe PA - J. Cell Biol. (1999)

Immunofluorescence analysis of keratins in cultured primary keratinocytes from replacement mice. Keratinocytes were isolated from the epidermides of newborn K16 and K16-C14 replacement mice, cultured, and analyzed by indirect double immunofluorescence to examine the keratin filament networks. Keratinocytes from K16 replacement mice (A–C) were stained with the 1275 antibody to detect K16. Some K16 replacement keratinocytes had fragmented keratin networks and cytoplasmic areas devoid of filaments (B and C). Others exhibited large bundling of filaments randomly distributed throughout the cytoplasm (A). Keratinocytes from K16-C14 replacement mice (D) were stained with the LL001 antibody to detect K16-C14. These keratinocytes appeared identical to wild-type keratinocytes (data not shown) in all morphological aspects as there was no evidence of keratin reorganization, fragmentation, or loss. Bar, 25 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169494&req=5

Figure 3: Immunofluorescence analysis of keratins in cultured primary keratinocytes from replacement mice. Keratinocytes were isolated from the epidermides of newborn K16 and K16-C14 replacement mice, cultured, and analyzed by indirect double immunofluorescence to examine the keratin filament networks. Keratinocytes from K16 replacement mice (A–C) were stained with the 1275 antibody to detect K16. Some K16 replacement keratinocytes had fragmented keratin networks and cytoplasmic areas devoid of filaments (B and C). Others exhibited large bundling of filaments randomly distributed throughout the cytoplasm (A). Keratinocytes from K16-C14 replacement mice (D) were stained with the LL001 antibody to detect K16-C14. These keratinocytes appeared identical to wild-type keratinocytes (data not shown) in all morphological aspects as there was no evidence of keratin reorganization, fragmentation, or loss. Bar, 25 μm.
Mentions: The electron microscopy results prompted the examination of the replacement keratinocytes in culture in order to analyze the global organization of the keratin filaments within the context of an intact keratinocyte. Primary cultures of newborn keratinocytes from K16 and K16-C14 replacement mice were established and analyzed by immunofluorescence to further determine if there were any possible abnormalities in the keratin networks of the K16 replacement mice. K16 replacement keratinocytes appeared normal after one day in culture (data not shown). All cells were positive for K5, K6, human K16, and K17, and negative for K14 (data not shown). As the cells remained in culture, however, they began to display time-dependent changes in their keratin filament networks. In a subset of cells (<50%), the keratin filament networks began to appear fragmented and even absent in some areas of the cytoplasm (Fig. 3B and Fig. C). There were also large bundles of filaments that were distributed throughout the cell (Fig. 3 A) rather than preferentially located adjacent to the nucleus as previously noted for the K16 ectopic keratinocytes 39. There was no evidence of keratin reorganization, fragmentation, or loss in any of the K16-C14 replacement keratinocytes regardless of the time spent in culture. These keratinocytes featured filament networks that were indistinguishable from wild-type keratinocytes (Fig. 3 D). These data suggest that in the absence of K14, K16 is not able to support a keratin filament network and that over time this fragile network is susceptible to fragmentation and loss. Furthermore, they provide further evidence that the carboxy-terminal ∼105 amino acids of K16 are responsible for these differences in filament organization.

Bottom Line: Degenstein, E.Fuchs. 1995.Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biological Chemistry and Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
The type I epidermal keratins K14 and K16 are remarkably similar at the primary sequence level. While a structural function has been clearly defined for K14, we have proposed that a function of K16 may be to play a role in the process of keratinocyte activation that occurs after acute injury to stratified epithelia. To compare directly the functions of the two keratins we have targeted the expression of the human K16 cDNA to the progenitor basal layer of the epidermis of K14 mice. Mice for K14 blister extensively and die approximately 2 d after birth (Lloyd, C., Q.C. Yu, J. Cheng, K. Turksen, L. Degenstein, E. Hutton, and E. Fuchs. 1995. J. Cell Biol. 129:1329-1344). The skin of mice expressing K16 in the absence of K14 developed normally without evidence of blistering. However, as the mice aged they featured extensive alopecia, chronic epidermal ulcers in areas of frequent physical contact, and alterations in other stratified epithelia. Mice expressing a control K16-C14 cDNA also rescue the blistering phenotype of the K14 mice with only a small percentage exhibiting minor alopecia. While K16 is capable of rescuing the blistering, phenotypic complementation in the resulting skin is incomplete due to the multiple age dependent anomalies. Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

Show MeSH
Related in: MedlinePlus