Abstract
Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.
Highlights
Skin provides a protective barrier from infection, injury, and water loss
Keratinocyte-targeted expression of human laminin c2 prevents the early lethality of Lamc2 KO mice
To determine whether expression of laminin c2 under a keratinocyte promoter is sufficient to rescue the early lethality of Lamc2 KO mice, we generated a new transgenic mouse line carrying the human LAMC2 cDNA under the control of TetO-CMV regulatory element (TetO-HuLamC2, Figure 1C) to use in conjunction with mice expressing the reverse tetracycline transactivator under the control of the human keratinocyte 14 promoter (K14-rtTA, Figure 1B) [24,25,28] Each of these mouse lines was independently bred to Lamc2 heterozygous mice (Figure 1A) to obtain Lamc2 Het/ TetO-HuLamC2+ and Lamc2 Het/K14-rtTA+ mice, respectively
Summary
Skin provides a protective barrier from infection, injury, and water loss. The epidermis and dermis are separated by a thin sheet of specialized extracellular matrix called the basement membrane zone (BMZ). In addition to providing tissue boundaries and structural support, components of the basement membrane influence cell attachment, proliferation, differentiation, and migration. A defect in the structure or expression of any one of the components of the BMZ can cause tissue separation and blister formation. Junctional epidermolysis bullosa (JEB) is one of the major forms of epidermolysis bullosa, a group of genetic skin blistering diseases. Lm-332 is normally secreted by skin keratinocytes and is a critical component of the BMZ between the epidermis and the dermal layer [7,8,9]. Most of the Lm-332 mutations that cause JEB are nonsense mutations that cause premature stop codons and result in a complete loss of Lm-332 [10,11,12]
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