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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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Keratin solubility in the skin. Skins from 7-d-old ectopic and replacement mice (no. 6 line) were homogenized in 1% Triton-X in PBS with 5 mM EDTA to obtain the soluble fraction of keratins. The remaining insoluble cytoskeletal fraction was solubilized in buffer A to obtain the insoluble fraction of keratins. 100 μg of total protein from the soluble fractions and 15 μg of total protein from the insoluble fractions were electrophoresed via SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis. Keratins analyzed included K14, K15, mouse K16, and human K16. No major differences were noted in the partitioning of any of the keratins between the soluble and insoluble fractions.
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Figure 6: Keratin solubility in the skin. Skins from 7-d-old ectopic and replacement mice (no. 6 line) were homogenized in 1% Triton-X in PBS with 5 mM EDTA to obtain the soluble fraction of keratins. The remaining insoluble cytoskeletal fraction was solubilized in buffer A to obtain the insoluble fraction of keratins. 100 μg of total protein from the soluble fractions and 15 μg of total protein from the insoluble fractions were electrophoresed via SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis. Keratins analyzed included K14, K15, mouse K16, and human K16. No major differences were noted in the partitioning of any of the keratins between the soluble and insoluble fractions.

Mentions: We have previously shown that a single proline residue in the 1B rod domain of human K16 (residue 188) completely accounts for the reduced stability of K16-containing heterotetramers under denaturing buffer conditions in vitro 65. The reduced stability of these tetramers correlated with the diminished ability of K16 to form 10-nm filaments efficiently in vitro. This led to the possibility that the solubility and partitioning of K16 between the soluble and insoluble keratin pools in the epidermis may be an important factor in determining its effect on keratinocytes 65. To determine if the partitioning of K16 was different among the various types of mice, skin from 7-d-old mice was lysed and the soluble and insoluble (cytoskeletal) fractions were isolated and subjected to Western blot analysis using various keratin antibodies (Fig. 6).


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)

Keratin solubility in the skin. Skins from 7-d-old ectopic and replacement mice (no. 6 line) were homogenized in 1% Triton-X in PBS with 5 mM EDTA to obtain the soluble fraction of keratins. The remaining insoluble cytoskeletal fraction was solubilized in buffer A to obtain the insoluble fraction of keratins. 100 μg of total protein from the soluble fractions and 15 μg of total protein from the insoluble fractions were electrophoresed via SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis. Keratins analyzed included K14, K15, mouse K16, and human K16. No major differences were noted in the partitioning of any of the keratins between the soluble and insoluble fractions.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169494&req=5

Figure 6: Keratin solubility in the skin. Skins from 7-d-old ectopic and replacement mice (no. 6 line) were homogenized in 1% Triton-X in PBS with 5 mM EDTA to obtain the soluble fraction of keratins. The remaining insoluble cytoskeletal fraction was solubilized in buffer A to obtain the insoluble fraction of keratins. 100 μg of total protein from the soluble fractions and 15 μg of total protein from the insoluble fractions were electrophoresed via SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis. Keratins analyzed included K14, K15, mouse K16, and human K16. No major differences were noted in the partitioning of any of the keratins between the soluble and insoluble fractions.
Mentions: We have previously shown that a single proline residue in the 1B rod domain of human K16 (residue 188) completely accounts for the reduced stability of K16-containing heterotetramers under denaturing buffer conditions in vitro 65. The reduced stability of these tetramers correlated with the diminished ability of K16 to form 10-nm filaments efficiently in vitro. This led to the possibility that the solubility and partitioning of K16 between the soluble and insoluble keratin pools in the epidermis may be an important factor in determining its effect on keratinocytes 65. To determine if the partitioning of K16 was different among the various types of mice, skin from 7-d-old mice was lysed and the soluble and insoluble (cytoskeletal) fractions were isolated and subjected to Western blot analysis using various keratin antibodies (Fig. 6).

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