Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis

Abstract

Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs. stimulation) and resulted in neonatal lethality in one instance. To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages. This system, termed "gene-switch," consists of two transgenic lines. The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter. These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin. On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls. Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls. In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis. Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future.

Figure 1

Constructs of the gene-switch-TGFβ1 transgenes. (A) The regulator transgene was inserted into the ClaI site of the ML expression vector. The GLVPc regulator comprises the DNA binding domain of the GAL4 activator (residues 1–93), the ligand binding domain of the truncated human progesterone receptor, which lacks 19 amino acids at the C terminus (hPRB914), and the herpes simplex virus VP-16 transactivation domain (residue 411–487). Primers 1 and 2 were used in PCR to identify the GLVPc transgene. (B) The target transgene, the porcine TGFβ1^S223/225 cDNA, was inserted into a TK promoter with four copies of the 17-mer GAL4 consensus binding sequence situated upstream and the SV40 Poly(A) region at the 3′ terminal of the TK promoter. Primers 3 and 4 were used in PCR to identify the TGFβ1 transgene.

Figure 2

Inducible expression of the TGFβ1 transgene in bigenic epidermis. Mouse ears were treated with either 10 μg of ZK or 100% ethanol and were excised 15 h after the treatment. Shown is RPA (A) and RT-PCR (B) on epidermal RNA from treated ears. Note that TGFβ1 expression was only detected in ZK-treated bigenic epidermis. The DNA marker in B is the φX174/HaeIII fragments. (C) TGFβ1 transgene expression (brown) detected by immunohistochemistry. (Bar = 27 μm.) Tissues in B and C are from ML.GLVPc line c4486 × TK.TGFβ1 line c1849.

Figure 3

(A) Reduced BrdUrd labeling in bigenic TGFβ1 mouse epidermis. Shown is BrdUrd labeling (yellow) in bigenic adult skin 15 h after either single ZK (10 μg) or ethanol (ETOH) treatment. K14 (red) highlights the epidermis and hair follicles. (B) Aberrant K6 expression in bigenic TGFβ1 epidermis after consecutive treatments with ZK. TK.TGFβ1 or bigenic TGFβ1 mice were treated with ZK, 1 μg/day for 7 days. K6 expression (yellow) in TK.TGFβ1 skin was restricted in hair follicles. However, K6 (yellow) was aberrantly expressed in the bigenic TGFβ1 epidermis after ZK treatments. K14 (red) highlights the epidermis and hair follicles. (C) Increased CD31 staining in TGFβ1-induced bigenic skin. Newborn pups of TK.TGFβ1 or bigenic TGFβ1 were treated with 1 μg/day ZK for 7 days. CD31 (red) highlights endothelial intercellular junctions. K14 (green) highlights the epidermis and hair follicles. (Bar = 57 μm.)

Figure 4

Comparison of ZK- and ethanol-treated bigenic TGFβ1 mouse epidermis 48 h after PMA application. ZK (10 μg) or 100% ethanol (50 μl) was applied to PMA-treated skin at time points of 0, 12, 24, and 36 h after PMA application. Mice were injected with BrdUrd 48 h after PMA application and were killed 1 h after injection. Histology shows significant epidermal hyperplasia in ethanol control skin 48 h after PMA treatment (A) whereas ZK-treated skin shows a 2-fold thinner epidermis (B). BrdUrd labeling (yellow) also shows a significant reduction in ZK-treated epidermis (D) compared with that of control (C). K14 (red) highlights the epidermis and hair follicles (C and D). The endogenous TGFβ1 (brown cells) was induced by PMA in the suprabasal layers of the epidermis (E). However, ZK-treated bigenic TGFβ1 skin shows uniformly expressed TGFβ1 throughout the entire epidermis (F), which includes both the endogenous and the TGFβ1 transgene. (Bar = 57 μm.)