To elucidate the role of selenoproteins in skin function, we targeted the removal of Trsp
gene in mouse cells that express K14 and examined the phenotypic consequences of this deletion. The expression of cre
-recombinase under the control of mammalian K5 or K14 promoter have provided a useful molecular tool in analyzing genes in the epidermis of mice 
. However, the loxP-cre
mediated recombination of genes in mice containing these promoters is female germ-line mediated, which restricts the breeding strategy 
. The knockout mice developed scaly thick skin within 6 days after birth and manifested a progressive runt phenotype and premature death. The lethality and body weights varied considerably even among knockout pups from the same litter, which could be attributed to a possible difference in the efficiency of cre
deletion or nutritional differences. Though the knockout pups appeared to be identical to control littermates at birth, skin and hair defects became apparent within 6 days after birth. Malformed hair follicles, hyperplastic epidermis, and dysplastic mucosal and tongue epithelia were clearly evident in 8 day old mice. The weight loss in the knockout mice started from day 7, while pups were still nursed by their mothers. Abnormalities in mucosal and tongue epithelia may hamper their suckling and these mice may not have an adequate intake of milk. Though this cannot be the sole cause of premature death, it is most certainly a contributing factor. We did not find any gross histopathological abnormalities in other organs. K14-cre
mediated deletion of Trsp
blocked the expression of Sec tRNA in keratinocytes, in turn preventing the synthesis of selenoproteins in this cell type. It should also be noted that selenoproteins are not only absent in K14 promoter active cells, but in all cells derived from K14 promoter active progenitors, which include differentiated keratinocytes of epidermis and hair follicles. An examination of some key selenoproteins by western blot revealed their virtual absence in keratinocytes from knockout mice.
As pointed out before, the loss of selenoproteins was not restricted to K14 promoter active cells, but to all descendents of such cells, including the suprabasal cells of epidermis and epithelial cells of hair follicles. Although hair follicle initiation and elongation appeared normal in mice lacking selenoproteins, a histological time course analysis revealed impaired hair shaft formation, changed hair follicle appearance, and early abnormal regression of follicles with a concomitant decrease in subcutaneous fat. Hair follicles are composed of multiple concentric layers of keratinocytes around the developing hair shaft arising from the proliferating matrix cells in hair follicle bulb. The most marked early morphological changes occur in the matrix cell region of follicles of knockout mice, suggesting oxidative damage leading to cessation of cell proliferation and premature onset of catagen. It is noteworthy that the proliferation markers Ki67 or BrdU were detected in the ORS of hair follicles, but strikingly absent in the area of the follicles where matrix cells are expected to be located at 9 days after birth. The ROS accumulation and lipid oxidation in knockout follicles detected in skin sections of 14 day old mice is likely to have started in younger mice. Damaged hair follicles can trigger inflammatory cells and we observed infiltrating macrophages in knockout skin from 7 day old mice and granulocytes in knockout skin from 8 day old mice. These cells were most abundant around grossly deformed hair follicles and may assist in the removal of damaged hair follicles by phagocytosis. The triggering event to initiate oxidative damage is so far unknown. Nevertheless, our observations emphasize the importance of selenoproteins in hair follicle morphogenesis in postnatal mouse skin.
Histological examination of the epidermal layer from knockout mice reveals the epidermis to be morphologically normal at birth, but gradually exhibiting a thickened cornified layer, along with progressive hyperplasia. Micro-blisters were observed in the epidermis by focal separation of cell layers within the epidermis as well as at the basement membrane, leading to detachment at the epidermal-dermal junction. This detachment in mice lacking selenoproteins may contribute to the fragility and flakiness of skin. The typically cuboidal morphology of basal keratinocytes, which express K14, is obscure in knockout mice. Ablation of selenoproteins in keratinocytes creates a wound-like condition in skin, marked by epidermal as well as stromal hyperplasia. The study of keratinocyte differentiation markers as a function of age shows that K1 expression extended to some hair follicles of knockout mice, while K6 was aberrantly expressed in the epidermis. The aberrant expression of K6 in epidermis is suggestive of chronic hyperproliferation or abnormal differentiation. K6, the ‘stress inducible’ keratin, is upregulated in wound healing 
, psoriasis 
, and is strongly expressed in squamous cell carcinomas 
. A recent study reported that prolonged expression of K6, through UVB-induction leads to wrinkle formation 
. ROS accumulation in hair follicle and epidermis is observed in knockout mice, indicating generation of stress which may induce the expression of K6, resulting in the observed epidermal phenotype. The elevated expression of K6 in knockout mouse epidermis could be a contributing factor to wrinkle formation and flakiness in skin.
Primary keratinocytes isolated from knockout mice and subsequently cultured had altered morphology, severe adhesion and growth defects and underwent apoptosis. Under standard culture conditions for mouse keratinocytes, the adhesion was severely compromised and the keratinocytes required plating on ColI/FN-coated culture surfaces for attachment and growth. Integrins are receptors for extracellular proteins, responsible for anchoring the epidermis to basement membrane. In skin, they also play important roles in wound repair, inflammation, keratinocyte differentiation and proliferation and their aberrant localization and expression is a crucial factor in numerous diseases and squamous cell carcinoma 
. Suprabasal expression of integrin beta 1 in epidermis has been attributed to hyperproliferation of keratinocyte, alteration of differentiation, inflammatory response and pathogenesis of psoriasis 
. The anomalous expression of integrin beta 1 and keratinocyte differentiation marker K6 suggests that loss of selenoproteins in skin contributes to psoriasis like conditions in the knockout progeny. In a recent study, we reported that the loss of selenoprotein T, a protein with role in redox regulation has been associated with impaired cell adhesion 
. Western blot data shows a virtual absence of SelT in knockout keratinocyte lysates and this loss may play a role in adhesion defects, yet contribution from other selenoproteins cannot be ruled out. In addition, we have previously found that the loss of selenoproteins in macrophages led to both an increase in reactive oxygen species (ROS) and an aberrant expression of ECM-related genes 
, though the role of selenoproteins in adhesion warrants an elaborate investigation. Although the attachment and growth was improved by ColI/FN treatment of the culture surfaces, the expansion of keratinocytes from knockout mice was markedly reduced in comparison to those from control mice. However, expansion was improved by continued supplementation with vitamin E, an essential antioxidant present as α-tocopherol in murine and human skin 
. Vitamin E is a well characterized antioxidant in skin along with selenoenzymes such as glutathione peroxidases and studies have shown that topical application of vitamin E and selenium improves skin surface parameters 
. We also observed that the percentage of non-keratinocytic cells (mainly melanocytes) was higher in preparations of keratinocytes from knockout mice, which could potentially generate oxidative stress, influencing the growth of keratinocytes. However, the improvement in growth by vitamin E supplementation in keratinocyte cultures from newborn mice emphasized the need for antioxidants in keratinocyte growth. Most selenoproteins that have been characterized thus far are antioxidants 
and their absence most certainly makes keratinocytes prone to oxidative damage that in turn may influence their morphology, attachment and growth. Our hypothesis is supported by enhanced accumulation of ROS in skin sections of knockout mice. This emphasizes the critical antioxidant role of selenoproteins in epidermal function and keratinocyte growth.
The premature death of mice lacking selenoproteins in K14-expressing cells and their descendents is not clearly understood, but could be a cumulative effect of numerous abnormalities in these mice. Wrinkle formation and fragility are markers of photo aging, changing the integrity of skin, which in combination with alopecia and lack of subcutaneous fat can result in a hypothermic condition. In addition, the dysplastic mucosal and tongue epithelia could inhibit their suckling, in turn hampering adequate food intake, leading to weight loss in knockout animals. Though a definite cause cannot be singled out, in all probability, the reduced food uptake and grossly disturbed skin along with some hitherto unidentified internal defects could contribute to the reduced lifespan of mice lacking selenoproteins in K14-expressing cells.
In conclusion, we have shown that loss of selenoproteins in K14-expressing cells altered normal morphogenesis in skin and its appendages, including hair follicles within several days after birth, leading to premature death. Earlier studies have linked some abnormalities of skin and hair to selenium deficiency 
and have shown the protective role of selenium as antioxidant 
and in wound healing 
. In the present study we attribute selenoproteins to the protective roles of selenium in skin and establish that deficiencies in selenoproteins beget most abnormalities associated with selenium deficiency in skin. The current study unveils the importance of selenoproteins in keratinocyte growth and viability, providing genetic evidence for the role of selenoproteins in cutaneous function and development.