Transrepression function of the glucocorticoid receptor regulates eyelid development and keratinocyte proliferation but is not sufficient to prevent skin chronic inflammation

Abstract

Glucocorticoids (GCs) play a key role in skin homeostasis and stress responses acting through the GC receptor (GR), which modulates gene expression by DNA binding-dependent (transactivation) and -independent (transrepression) mechanisms. To delineate which mechanisms underlie the beneficial and adverse effects mediated by GR in epidermis and other epithelia, we have generated transgenic mice that express a mutant GR (P493R, A494S), which is defective for transactivation but retains transrepression activity, under control of the keratin 5 promoter (K5-GR-TR mice). K5-GR-TR embryos exhibited eyelid opening at birth and corneal defects that resulted in corneal opacity in the adulthood. Transgenic embryos developed normal skin, although epidermal atrophy and focal alopecia was detected in adult mice. GR-mediated transrepression was sufficient to inhibit keratinocyte proliferation induced by acute and chronic phorbol 12-myristate 13-acetate exposure, as demonstrated by morphometric analyses, bromodeoxyuridine incorporation, and repression of keratin 6, a marker of hyperproliferative epidermis. These antiproliferative effects were mediated through negative interference of GR with MAPK/activator protein-1 and nuclear factor-kappaB activities, although these interactions occurred with different kinetics. However, phorbol 12-myristate 13-acetate-induced inflammation was only partially inhibited by GR-TR, which efficiently repressed IL-1beta and MMP-3 genes while weakly repressing IL-6 and TNF-alpha. Our data highlight the relevance of deciphering the mechanisms underlying GR actions on epithelial morphogenesis as well as for its therapeutic use to identify more restricted targets of GC administration.

Fig. 1.

Generation of K5-GR-TR Transgenic Mice Expressing a Mutant GR in Keratinocytes that Is Defective in Transactivation but Effective in Transrepression A, Scheme of the construct K5-GR-TR. The cDNA of GR containing a double mutation (P493R, A494S) was placed under control of bovine K5 (bk5) regulatory sequences. B, PB keratinocytes were transiently transfected with either MMTV-CAT or 8x-κB-CAT reporter plasmids along with expression vectors encoding for empty vector (pcDNA3), K5-GR, or K5-GR-TR. After depletion of endogenous GCs, cells were treated with either vehicle or 1 μm Dex for 6 h to determine MMTV-CAT activity. In transfections using 8x-κB-CAT reporter, vehicle or TNF-α (10 ng/ml) were added for 4 h before measuring CAT activity (a.u, arbitrary units). Data presented are average of three independent experiments, each performed in triplicate. Error bars indicate sd. Asterisks denote statistically significant differences relative to pCDNA3-transfected cells, as analyzed by Student’s t test (*, P < 0.0005). Expression levels for the WT and mutant GR upon transfection were checked by immunoblotting (lower panel). C, RT-PCR analysis showing the endogenous [mouse GR (mGR)] and transgene [rat GR (rGR)] transcript levels in the skin of WT and K5-GR-TR adult littermates. D, Immunoblotting using whole-cell extracts from tail and dorsal skin of three transgenic founders (298, 306, and 599) to check the relative expression levels of GR. E, Immunolocalization of GR in dorsal skin paraffin sections from newborn WT and transgenic mice. Arrows indicate the nuclear localization of GR in the interfollicular basal epithelium and the outer root sheath of HFs in transgenic skin. Scale bar, 25 μm.

Fig. 2.

Lack of Eyelid Closure in K5-GR-TR Embryos A, Eyelid fusion failed in 18.5-dpc K5-GR-TR HM embryos as compared with WT mice. B, Immunostaining using K5 in WT and K5-GR-TR embryos demonstrating the epithelia to which transgene expression is targeted: cornea (a and d), conjunctiva (b and e), and eyelid epithelium (c and f). Fused eyelids in WT embryos are pointed to by an arrowhead. a–f are also shown at higher magnification in the lower panel. Note that K5 staining was lacking in the cornea epithelium, whereas it appeared as more restricted in K5-GR-TR eye embryos. Additional ocular defects, including microphthalmia, dysplastic retinal tissue, and attachment of eyelid epithelium to the posterior side of the cornea were observed in K5-GR-TR transgenic embryos (arrow). C, Immunostaining using P-c-Jun and P-JNK antibodies revealed no differences in the expression of these phosphorylated proteins in the eyelid epithelium of K5-GR-TR vs. WT embryos. Arrows indicate nuclear localization of P-c-Jun and P-JNK. D, Adult transgenic mice exhibited corneal opacity. E, The secretory Meibomian glands of the eyelids were unaffected in the transgenics (arrowheads). Scale bar, 50 μm.

Fig. 3.

Normal Embryo Skin Development and Adult Skin Alterations in K5-GR-TR Mice A, Normal development of the epidermis and HFs in 18.5-dpc K5-GR-TR HM mice. Immunostaining with K5 and K10 showed no alterations in transgenic embryo skin as compared with controls. B, Adult HM transgenic mice developed patchy alopecia along the whole coat (left) and throughout the face, with scarce and underdeveloped vibrissae (right). C, Immunostaining using an anti-K5 antibody showed a discontinuous pattern in the epidermis of adult HM mice (arrows). Scale bar, 50 μm.

Fig. 4.

GR-TR Inhibits PMA-Induced Proliferation upon Acute and Chronic Treatment. A and B, Responses of adult K5-GR-TR and K5-GR to acute PMA treatment. Age-matched adult WT, K5-GR-TR HT mice of lines 298 and 306, and K5-GR mice (line 285) were used for these experiments. The proliferation rate of interfollicular epidermal keratinocytes was evaluated in dorsal skin paraffin sections from the indicated genotypes, as indicated in Materials and Methods. Arrows indicate BrdU-positive interfollicular keratinocytes. Results of the quantification were graphed as the percentage of positive BrdU keratinocytes vs. total nuclei. Mean values ± sd are shown (n = 5). Asterisks denote statistically significant differences relative to PMA-treated WT mice, as determined by Student’s t test (*, P < 0.05; **, P < 0.005). C, Responses of adult K5-GR-TR and K5-GR mice to chronic PMA (1 wk). Age-matched adult WT, K5-GR-TR HT and HM mice of line 298, and K5-GR mice (line 285) were used for these experiments. Hematoxylin and eosin staining shows dramatic epidermal hyperplasia in WT mice that was greatly reduced in HT and HM skin as well as in K5-GR mice. K6 staining was evaluated as a marker of hyperproliferative skin. Bar, 50 μm except for K6 immunostaining, where the bar represents 25 μm. D, Determination of the mRNA levels of k6β by RT-PCR in WT and K5-GR-TR HM skin that was treated with either vehicle (−) or PMA for 1 wk. Experiments were performed in at least three individuals of each genotype. Quantitation of RT-PCR is shown at right. Mean values ± sd are shown (n = 3), and statistically significant differences (P < 0.05) are indicated by asterisks. E–H, Responses of adult K5-GR-TR and K5-GR to PMA (1 wk) and PMA plus Dex (1 wk) (P+D). Age-matched adult WT, K5-GR-TR HT and HM mice of line 298, K5-GR-TR mice of line 306, and K5-GR mice (line 285) were used for these experiments. Morphometric analyses were performed to determine epidermal width (E), dermal cellularity (G), and number of HFs per millimeter (H) in mouse skin of the indicated genotypes. Percentage of BrdU incorporation was also examined in the same skin samples (F). Note that the inhibition of epidermal keratinocyte growth correlated with transgene dosage (compare HT vs. HM) and was consistently observed in the two independent transgenic lines (298 and 306). Morphometric and BrdU measurements were performed in at least five individuals of each genotype. Mean values ± sd are shown. Asterisks indicate statistical significance of the response of each genotype as compared with PMA-treated WT mice, as determined by Student’s t test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

Fig. 5.

GR-TR Differentially Regulates Proinflammatory Cytokines in Vivo Determination of the mRNA levels of IL-6, TNF-α, cycD1, IL-1β, and MMP-3 in mouse skin of the indicated genotypes. Mice were treated with either vehicle or PMA for 4 or 48 h or pretreated with Dex for 24 h before PMA application. For quantitation of RT-PCR, experiments were performed in at least three individuals of each genotype. Mean values ± sd are shown. Values representing gene expression levels of untreated mice from each genotype were set to 1; fold induction is thus represented relative to this basal level. Asterisks denote statistically significant differences relative to WT mice with the same treatment, as determined by Student’s t test (*, P < 0.05; **, P < 0.005).

Fig. 6.

GR-TR Abrogates the PMA-Induced Activation of JNK at Early Time Points A, Immunoblotting using whole-cell extracts from adult K5-GR-TR and WT dorsal skin that were either untreated or treated with PMA at the indicated times to check the expression of ERK, P-ERK, JNK, P-JNK, c-Jun, and c-Fos. B, Quantitation of the relative levels of P-ERK/ERK and P-JNK/JNK was determined in at least three individuals of each genotype.

Fig. 7.

GR-TR Reduces NF-κB Activation at Later Stages A, EMSA showing NF-κB-binding activity of total skin extracts from WT and transgenic mice that were either untreated or PMA-treated for the indicated times. The composition of the NF-κB complexes (p50/p65 and p50/p50 dimers) was determined by supershift assays (right panel). PI, Preimmune serum. Free probe is indicated at the bottom of the gel. B, Cytoplasmic and nuclear extracts were prepared from adult K5-GR-TR and WT skin to examine the expression of p65 and ΙκBα by using specific antibodies. C, RT-PCR analysis showing ikba transcript levels in skin from adult WT, K5-GR, and K5-GR-TR mice.