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Cited by 109Selenium in Biological SystemsOrganoselenium and organotellurium chemistryHair Growth and Disorders

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Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development

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Sengupta, Aniruddha, Lichti, Ulrike F., Carlson, Bradley A., Ryscavage, Andrew O., Gladyshev, Vadim N., Yuspa, Stuart H., Hatfield, Dolph L.·2010·PLoS ONE
DOI 10.1371/journal.pone.0012249PMC PMC2923614
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Abstract

Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

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Figures

Figure 1 Neonatal mortality and runt phenotype in keratin-14 expressing epithelial cells encoding a Trsp deletion. (A) Kaplan-Meier survival plot of Trsp knockout (– – –, n = 42) and control mice (——, n = 51) (p<0.0001). (B) Growth curve of knockout (– – –) and control (——) littermates (p<0.01). (C–D) Phenotype of knockout (KO) mice showing fragile, flaky and wrinkled skin with sparse hair compared to control (CT) littermates. (E) Picture showing short and oddly angled vibrissae of whisker pads in knockout mouse compared to a control littermate at day 9. (F) Photograph showing that knockout mice are smaller and exhibit alopecia in comparison to control littermates of same age.
Figure 1 Neonatal mortality and runt phenotype in keratin-14 expressing epithelial cells encoding a Trsp deletion. (A) Kaplan-Meier survival plot of Trsp knockout (– – –, n = 42) and control mice (——, n = 51) (p<0.0001). (B) Growth curve of knockout (– – –) and control (——) littermates (p<0.01). (C–D) Phenotype of knockout (KO) mice showing fragile, flaky and wrinkled skin with sparse hair compared to control (CT) littermates. (E) Picture showing short and oddly angled vibrissae of whisker pads in knockout mouse compared to a control littermate at day 9. (F) Photograph showing that knockout mice are smaller and exhibit alopecia in comparison to control littermates of same age.CC0 · PubMed Central
Figure 2 Tissue-specific detection of Trsp and histological examination of affected tissues. (A) Genotyping PCR of various tissues from knockout mice (KO) and control (CT) littermates is shown. Trspfl/fl (upper band), Trsp+/+ (center band) and ΔTrsp (lower band) products in the upper panel were 1100, 900 and 450 bp, respectively, and the amplified product from cre was 700 bp (lower panel). (B) H&E staining of skin, oral cavity and tongue from 8 day old knockout mice shows abnormalities in skin, oral cavity and tongue in comparison to control mice. Scale bars: 100 µm.
Figure 2 Tissue-specific detection of Trsp and histological examination of affected tissues. (A) Genotyping PCR of various tissues from knockout mice (KO) and control (CT) littermates is shown. Trspfl/fl (upper band), Trsp+/+ (center band) and ΔTrsp (lower band) products in the upper panel were 1100, 900 and 450 bp, respectively, and the amplified product from cre was 700 bp (lower panel). (B) H&E staining of skin, oral cavity and tongue from 8 day old knockout mice shows abnormalities in skin, oral cavity and tongue in comparison to control mice. Scale bars: 100 µm.CC0 · PubMed Central
Figure 3 K14-mediated Trsp deletion ablates selenoproteins in epidermis of knockout mice. Loss of selenoproteins in freshly isolated keratinocytes from new born mice was detected by western blotting with antibodies against GPX1, SELT, TR1 and SEP15.
Figure 3 K14-mediated Trsp deletion ablates selenoproteins in epidermis of knockout mice. Loss of selenoproteins in freshly isolated keratinocytes from new born mice was detected by western blotting with antibodies against GPX1, SELT, TR1 and SEP15.CC0 · PubMed Central
Figure 4 Progressive epithelial dysfunction and aberrant follicular morphogenesis in knockout mice. (A–P) Back skin sections from control (A, C, E, G, I, K, M, O) and knockout mice (B, D, F, H, J, L, N) were stained with H&E for histological examination. Thickened cornified layer (arrow), hyperplastic epidermis (asterisk) and detachment along dermal–epidermal junction (arrowhead) along with decrease in fat cells (triangle) is evident in knockout mice. Defective hair follicle morphogenesis in mutant skin is characterized by irregularly spaced malformed hair follicles. Scale bars: 100 µm.
Figure 4 Progressive epithelial dysfunction and aberrant follicular morphogenesis in knockout mice. (A–P) Back skin sections from control (A, C, E, G, I, K, M, O) and knockout mice (B, D, F, H, J, L, N) were stained with H&E for histological examination. Thickened cornified layer (arrow), hyperplastic epidermis (asterisk) and detachment along dermal–epidermal junction (arrowhead) along with decrease in fat cells (triangle) is evident in knockout mice. Defective hair follicle morphogenesis in mutant skin is characterized by irregularly spaced malformed hair follicles. Scale bars: 100 µm.CC0 · PubMed Central
Figure 5 Mice lacking selenoproteins in keratin-14 expressing cells show reduced proliferation of hair matrix cells. (A–B) BrdU was administered intraperitoneally in 9 day old control and knockout mice and immunodetected for incorporation in back skin sections. (C–D) Ki67 staining for proliferating cells in skin sections of 10 day old control and knockout mice. Proliferating cells were noted in the basal layer of epidermis (arrowhead), hair follicle bulb (thin arrow) and outer root sheath of hair follicle (broad arrow). Scale bar: 100 µm.
Figure 5 Mice lacking selenoproteins in keratin-14 expressing cells show reduced proliferation of hair matrix cells. (A–B) BrdU was administered intraperitoneally in 9 day old control and knockout mice and immunodetected for incorporation in back skin sections. (C–D) Ki67 staining for proliferating cells in skin sections of 10 day old control and knockout mice. Proliferating cells were noted in the basal layer of epidermis (arrowhead), hair follicle bulb (thin arrow) and outer root sheath of hair follicle (broad arrow). Scale bar: 100 µm.CC0 · PubMed Central
Figure 6 Abnormalities in keratinocyte morphology and differentiation. (A–B) H&E staining of back skin section of 10 day old control and knockout mice. Arrows indicate basal keratinocytes and arrowheads indicate blistering between epidermis and dermis. (C–J) Back skin sections of 10 day old control and knockout mice were stained for keratinocyte differentiation marker proteins. (C–D) keratin-1; (E–F) keratin-14; (G–H) loricrin; (I–J) keratin-6. (K–L) Barrier function of skin was tested using a hematoxylin dye penetration assay, showing hematoxylin staining only in stratum corneum (broad arrow). Scale bar: 100 µm.
Figure 6 Abnormalities in keratinocyte morphology and differentiation. (A–B) H&E staining of back skin section of 10 day old control and knockout mice. Arrows indicate basal keratinocytes and arrowheads indicate blistering between epidermis and dermis. (C–J) Back skin sections of 10 day old control and knockout mice were stained for keratinocyte differentiation marker proteins. (C–D) keratin-1; (E–F) keratin-14; (G–H) loricrin; (I–J) keratin-6. (K–L) Barrier function of skin was tested using a hematoxylin dye penetration assay, showing hematoxylin staining only in stratum corneum (broad arrow). Scale bar: 100 µm.CC0 · PubMed Central
Figure 7 Selenoprotein deficiency alters adhesion and proliferation of keratinocytes in culture. (A) Attachment and proliferation of keratinocytes from knockout mice following 3 days of culture in ColI/FN coated plates is improved in vitamin E supplemented media. (B) Expression of selenoproteins in keratinocyte cultures from control and knockout mice. Left panel, incorporation of 75Se in proteins; right panel, Coomassie blue staining. (C) Adhesion assay for keratinocytes from control (—•—) and knockout mice (– –○– –) following 30, 60 and 120 min of attachment ± S.D for each time point. (D) Growth of keratinocytes from control (—⧫—) and knockout mice (– –⋄– –) following attachment for 2 h. The unattached cells were removed and media changed following attachment, with replenishment after 14 h of attachment (arrow). The curve represents the mean values ± S.D. for each time point. (E–F) TUNEL assay for determining cell death by apoptosis in keratinocyte cultures from control (CT) and knockout (KO) mice. The abundance of apoptotic cells (depicted in black) is plotted as percentage of the total population. Scale bar: 20 µm.
Figure 7 Selenoprotein deficiency alters adhesion and proliferation of keratinocytes in culture. (A) Attachment and proliferation of keratinocytes from knockout mice following 3 days of culture in ColI/FN coated plates is improved in vitamin E supplemented media. (B) Expression of selenoproteins in keratinocyte cultures from control and knockout mice. Left panel, incorporation of 75Se in proteins; right panel, Coomassie blue staining. (C) Adhesion assay for keratinocytes from control (—•—) and knockout mice (– –○– –) following 30, 60 and 120 min of attachment ± S.D for each time point. (D) Growth of keratinocytes from control (—⧫—) and knockout mice (– –⋄– –) following attachment for 2 h. The unattached cells were removed and media changed following attachment, with replenishment after 14 h of attachment (arrow). The curve represents the mean values ± S.D. for each time point. (E–F) TUNEL assay for determining cell death by apoptosis in keratinocyte cultures from control (CT) and knockout (KO) mice. The abundance of apoptotic cells (depicted in black) is plotted as percentage of the total population. Scale bar: 20 µm.CC0 · PubMed Central
Figure 8 Loss of selenoproteins induces infiltration of inflammatory molecules and alters cell-adhesion in knockout mice skin. (A–B) Immunostaining for infiltrating macrophages with macrophage/monocyte specific antigen F4/80 in 10 day old mouse back skin section. (C–D) Detection of granulocytes with myeloperoxidase staining in back skin section of 10 day old mice. Scale bar: 100 µm.
Figure 8 Loss of selenoproteins induces infiltration of inflammatory molecules and alters cell-adhesion in knockout mice skin. (A–B) Immunostaining for infiltrating macrophages with macrophage/monocyte specific antigen F4/80 in 10 day old mouse back skin section. (C–D) Detection of granulocytes with myeloperoxidase staining in back skin section of 10 day old mice. Scale bar: 100 µm.CC0 · PubMed Central
Figure 9 Selenoprotein ablation generates ROS and induces lipid peroxidation in knockout mice skin. (A) Immunofluorescent detection of DCFH-DA to measure ROS in 14 day old mouse back skin section. DCFH-DA is stained green and nucleus is stained blue with DAPI. (B) Measuring lipid peroxidation through ELISA assay for 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation in protein lysates from freshly isolated keratinocytes from control (CT) and knockout (KO) mice. The data represents the mean values ± S.D. for three independent experiments carried out in duplicate. Scale bar: 50 µm.
Figure 9 Selenoprotein ablation generates ROS and induces lipid peroxidation in knockout mice skin. (A) Immunofluorescent detection of DCFH-DA to measure ROS in 14 day old mouse back skin section. DCFH-DA is stained green and nucleus is stained blue with DAPI. (B) Measuring lipid peroxidation through ELISA assay for 4-hydroxynonenal (4-HNE), a byproduct of lipid peroxidation in protein lysates from freshly isolated keratinocytes from control (CT) and knockout (KO) mice. The data represents the mean values ± S.D. for three independent experiments carried out in duplicate. Scale bar: 50 µm.CC0 · PubMed Central

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  1. HPM Gollnick, 1996, Systemic b carotene plus topical UV-sunscreen are an optimal protection against harmful effects of natural UV-sunlight: results of the Berlin-Eilath study., Eur J Dermatol, 6, 200

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Metadata

Authors
Sengupta, A., Lichti, U. F., Carlson, B. A., Ryscavage, A. O., Gladyshev, V. N., Yuspa, S. H., & Hatfield, D. L.
Affiliations
  • National Institutes of Health (US)
  • Brigham and Women's Hospital (US)
Corresponding author
  • Yuspa, Stuart H.
  • Hatfield, Dolph L.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2010-08-18
Volume
5
Issue
8
Pages
e12249
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0012249
PMC
PMC2923614
OpenAlex
W2140893882
Cited by
109
References
41
Citations by year
2026: 7 · 2025: 7 · 2024: 9 · 2023: 9 · 2022: 5 · 2021: 7 · 2020: 4 · 2019: 8 · 2018: 10 · 2017: 6 · 2016: 6 · 2015: 8 · 2014: 3 · 2013: 6 · 2012: 8
FWCI
6.07
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, public-domain)
  • PubMed Central (publishedVersion, public-domain)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Cancer Institute
  • National Institute of General Medical Sciences (R01GM061603)
Topics
  • Health Sciences › Nursing › Nutrition and Dietetics › Selenium in Biological Systems
  • Physical Sciences › Chemistry › Organic Chemistry › Organoselenium and organotellurium chemistry
  • Health Sciences › Medicine › Dermatology › Hair Growth and Disorders
Keywords
selenoproteins, selenocysteine, keratinocytes, skin development, hair follicle morphogenesis, hair loss, selenium deficiency, keratin 14, epidermal hyperplasia, antioxidant enzymes, keratinocyte viability, knockout mice
MeSH terms
Epidermal Cells, Animals, Animals, Newborn, Antioxidants, Cell Adhesion, Cell Differentiation, Epidermis, Female, Gene Expression Regulation, Macrophages, Male, Organ Specificity, Recombination, Genetic, RNA, Messenger, RNA, Transfer, Amino Acid-Specific, Selenium, Skin, Lipid Peroxidation, Biomarkers, Keratinocytes, Gene Deletion, Reactive Oxygen Species, Hair Follicle, Cell Proliferation, Selenoproteins, Mice, Keratin-14, Gene Knockout Techniques

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