The thyroid hormone receptors modulate the skin response to retinoids

Abstract

Background: Retinoids play an important role in skin homeostasis and when administered topically cause skin hyperplasia, abnormal epidermal differentiation and inflammation. Thyroidal status in humans also influences skin morphology and function and we have recently shown that the thyroid hormone receptors (TRs) are required for a normal proliferative response to 12-O-tetradecanolyphorbol-13-acetate (TPA) in mice.

Methodology/principal findings: We have compared the epidermal response of mice lacking the thyroid hormone receptor binding isoforms TRα1 and TRβ to retinoids and TPA. Reduced hyperplasia and a decreased number of proliferating cells in the basal layer in response to 9-cis-RA and TPA were found in the epidermis of TR-deficient mice. Nuclear levels of proteins important for cell proliferation were altered, and expression of keratins 5 and 6 was also reduced, concomitantly with the decreased number of epidermal cell layers. In control mice the retinoid (but not TPA) induced parakeratosis and diminished expression of keratin 10 and loricrin, markers of early and terminal epidermal differentiation, respectively. This reduction was more accentuated in the TR deficient animals, whereas they did not present parakeratosis. Therefore, TRs modulate both the proliferative response to retinoids and their inhibitory effects on skin differentiation. Reduced proliferation, which was reversed upon thyroxine treatment, was also found in hypothyroid mice, demonstrating that thyroid hormone binding to TRs is required for the normal response to retinoids. In addition, the mRNA levels of the pro-inflammatory cytokines TNFα and IL-6 and the chemotactic proteins S1008A and S1008B were significantly elevated in the skin of TR knock-out mice after TPA or 9-cis-RA treatment and immune cell infiltration was also enhanced.

Conclusions/significance: Since retinoids are commonly used for the treatment of skin disorders, these results demonstrating that TRs regulate skin proliferation, differentiation and inflammation in response to these compounds could have not only physiological but also therapeutic implications.

Figure 1. Reduced skin proliferation after 9-cis-RA and TPA treatment in TR-deficient mice.

A) Dorsal skins of wild-type mice (Wt) and knock-out mice lacking TRα1 and TRβ (KO), were treated topically with vehicle (acetone), 9-cis-RA or TPA. Morphometric analysis were performed to determine epidermal thickness (left panel) and proliferation rate in the basal layer was quantitated and is represented as the percentage of positive BrdU cells vs. total cells (right panel). Data are shown as mean values ± SE, and asterisks denote statistically significant differences relative to Wt mice (*, P<0.05; ***, P<0.001). B) The upper panels show representative H&E staining of 9-cis-RA and TPA treated mice. The magnification in wild-type mice shows parakeratosis induced by 9-cis-RA, which was absent in KO mice. The lower panels show representative BrdU immunostaining in the same groups.

Figure 2. Reduced hyperplasia in response to retinoids in mice lacking TRs.

Epidermal thickness was measured in wild-type and TR-deficient mice after topical treatment with all-trans-RA (ATRA) and 9-cis-RA. Data are shown as mean values ± SE, and ***(P<0.001) represents statistically significant differences relative to Wt mice.

Figure 3. Altered expression of proliferation-related proteins in the skin of TR-deficient mice after 9-cis-RA treatment.

Total and nuclear skin cell lysates from Wt and KO mice treated topically with 9-cis-RA were used for Western blotting with the indicated antibodies. Actin and lamin B were used as a loading control for total and nuclear extracts, respectively.

Figure 4. Hypothyroid mice and TR-deficient mice present similarly reduced proliferation in response to 9-cis-RA.

Epidermal thickness (left panel) and BrdU incorporation (right panel) were determined in TR KO mice, mice treated with MMI and perchlorate for 4 months (hT) and in a group of animals receiving the anti-thyroidal drugs plus thyroxine (hT+T4).

Figure 5. Expression of the proliferation marker keratin 5 is reduced in TR KO mice in response to proliferative stimuli.

Examples of double immunofluorescence images of keratin 5 (K5, red) and β-catenin (green) expression in dorsal skin of wild-type (Wt) and TR-deficient mice (KO) after topical treatment with vehicle, 9-cis-RA or TPA. The slides were counterstained with dapi (blue) and the merged images are shown.

Figure 6. Keratin 6 and 10 expression in TR-deficient mice.

A) The proliferation marker keratin 6 (K6, red) and the early differentiation marker keratin 10 (K10, green) were detected by immunofluorescence in wild-type and TR KO mice before and after application of 9-cis-RA or TPA. Nuclei were stained with dapi (blue). Panel B) represents a larger magnification of the merged images shown in A) for a better observation of K6 and K10 colocalization and the decrease in K10 expression observed in TR-deficient mice after 9-cis-RA treatment.

Figure 7. TR deletion alters expression of loricrin.

A) Localization by immunofluorescence of the terminal differentiation marker loricrin in the most external epidermal layer of skins from wild-type and TR KO mice treated with 9-cis-RA and TPA. Loricrin expression (red) demonstrates that 9-cis-RA (but not TPA) promotes altered differentiation and that this is more marked in mice lacking TRs. Panel B) shows that loricrin is totally absent in large areas of the skin of some TR KO animals after 9-cis-RA treatment.

Figure 8. Increased expression of proinflammatory and chemotactic molecules in skins of TR KO mice.

Relative mRNA levels of TNFα, IL-6, S100A8 and S100A9 in wild-type and TR KO mice treated with vehicle, 9-cis-RA or TPA. Values representing expression levels of vehicle-treated wil-type mice were set to 1, and fold induction is represented relative to this basal level.

Figure 9. Immune cell infiltration is enhanced in skins of TR deficient mice treated with 9-cis-RA.

Double immunofluorescence images of keratin 5 (K5) with mac-1 (left panel), cd45 (middle panel) and cd3ε (right panel) in skins from wild-type and TR KO mice treated with 9-cis-RA or vehicle alone. Nuclei were stained with dapi.