Topical 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibition Corrects Cutaneous Features of Systemic Glucocorticoid Excess in Female Mice

Topical 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibition Corrects Cutaneous Features of... Abstract Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11β-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11β-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11β-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11β-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11β-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11β-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds. Systemic glucocorticoid (GC) therapy remains mainstream treatment of many inflammatory diseases (e.g., lupus, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, polymyalgia rheumatica, and giant cell arteritis). Recent estimates indicate that 1.2% of the US population (>2.5 million people) were prescribed oral GCs between 1999 and 2008, 28% of whom reported use of >5 years (1). Despite anti-inflammatory benefits, chronic GC excess drives adverse side effects, including weight gain, hypertension, hyperglycemia, osteoporosis, muscle weakness, glaucoma, and depression (2). GC therapy also substantially increases health care costs. In patients with systemic lupus erythematosus, GC use was associated with a threefold increase in annual expenditure (3). In a study of patients with severe asthma, high-dose GC use was associated with annual increments of $5479 relative to low GC exposure (4). Moreover, the incidence and impact of GC-related side effects are underreported (5) and further compounded by GC overprescription (6). In skin, GC excess causes acne, thinning, dryness, atrophic striae, telangiectasia, bruising, impaired wound healing (WH), and increased infection risk (2, 7). Skin bruising and thinning have also been reported with low-dose (<7.5 mg/d) GC therapy (8). In a survey of asthma physicians, cutaneous manifestations were the second most frequently reported complication of inhaled GCs (9). Despite recent advances in GC mimetics and GC-independent immune suppressants, the “holy grail” of dissociating GC anti-inflammatory benefits from side effects remains elusive (10). It is now established that human skin has corticosteroidogenic capability that is regulated in a complex way (11, 12), although this function is lacking in mouse skin (13). Peripheral GC availability is also regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts biologically inert cortisone and 11-dehydrocorticosterone to cortisol and corticosterone in humans and mice, respectively. In intact tissues and cells, 11β-HSD1 functions predominantly as a reductase in a nicotinamide adenine dinucleotide phosphate–dependent manner (14). We previously reported increased 11β-HSD1 activity during mouse skin WH (13) and in aged mouse and human skin (15, 16), as well as accelerated WH in aged 11β-HSD1–null mice (16). However, the role of 11β-HSD1 in impaired WH is unknown. We propose that 11β-HSD1 is a causative agent of cutaneous side effects during systemic GC excess such as skin atrophy and impaired WH. Because human 11β-HSD1 inhibitors are readily available, such a demonstration could lead to rapid progress in ameliorating this major clinical problem. Here, we provide what, to our knowledge, is the first empirical evaluation of this causal link. We investigate effects of topical 11β-HSD1 inhibition by carbenoxolone (CBX) on skin function in mice treated with oral corticosterone and examine inflammatory, growth factor, and extracellular matrix gene expression responses during WH. We demonstrate that 11β-HSD1 inhibition accelerates WH and reduces skin damage in steroid-treated mice without compromising the inflammatory response. Materials and Methods Declarations Studies presented in this article were approved by the Institutional Animal Care and Use Committee and San Francisco Veterans Affairs Medical Center Veterinary Medical Unit. Materials were obtained from Sigma-Aldrich unless otherwise stated. Animal studies Female SKH1 hairless mice (8 to 10 weeks old) were obtained from Charles River Laboratories (Wilmington, MA) and acclimatized for 2 weeks. Mice were group housed, supplied with a basal chow diet ad libitum, and exposed to a standard 12-hour light/dark cycle. Drinking water was supplemented with corticosterone (100 μg/mL) or vehicle (VEH) (0.66% ethanol) for 6 weeks (replaced twice weekly). Seven days prior to wounding, 100 μg/cm2 topical CBX (20 μL) or VEH (70% propylene glycol, 30% ethanol) was massaged into 2 cm2 skin until fully absorbed. Treatments were repeated bidaily for the remainder of the experiment and replaced with 10 μL per wound postwounding. Twenty-four hours prior to wounding, epidermal barrier function was measured using a Tewameter 300 (Courage + Khazaka, Cologne, Germany) to evaluate transepidermal water loss (TEWL). Epidermal barrier recovery was determined at 2 and 4 hours following disruption by sequential D-Squame tape stripping to a TEWL level of >30 g/h/m2. Epidermal integrity was defined as the number of tape strips required to achieve barrier disruption. WH was conducted as previously described (13). Briefly, mice anesthetized under 2% isoflurane were wounded on both dorsal flanks by a 5-mm punch biopsy (Acuderm, Fort Lauderdale, FL). Wounds received 20 μL bupivacaine 0.25% (analgesic) and were monitored daily. At 2, 4, and 9 days’ postwounding, mice were culled by cervical dislocation, and wounds were digitally imaged and excised for 11β-HSD1 activity assay, RNA extraction, or histology. Wound areas were determined by ImageJ (NIH, Bethesda, MD). Experiments were also replicated in control mice (untreated drinking water) treated with VEH or CBX. 11β-HSD1 activity assay Radioactive conversion of tritiated 11β-HSD1 substrate is the gold-standard measure of 11β-HSD1 activity as tissue steroid extraction efficiencies are variable and less sensitive. Freshly isolated skin (20 to 40 mg) was incubated immediately in 1 mL high glucose and pyruvate Dulbecco’s modified Eagle medium with 100 nM 11-dehydrocorticosterone (Steraloids, Newport, RI) and ∼1500 cpm [3H] 11-dehydrocorticosterone, generated in house as previously described (13). Samples were incubated at 37°C for 13 hours. Subsequently, tissues were weighed and steroids extracted and separated by thin-layer chromatography in 186 mL chloroform and 14 mL ethanol for 90 minutes (comigrated with 10 mM 11-dehydrocorticosterone/corticosterone standards). Plate regions identified under ultraviolet were excised and percentage conversion of 11-dehydrocorticosterone to corticosterone was determined after liquid scintillation. Quantitative polymerase chain reaction Fresh skin tissue (20 to 40 mg) was snap-frozen and stored at −80°C. Samples were homogenized in 1 mL Trizol and RNA extracted using a PureLink RNA Mini Kit (Life Technologies, Grand Island, NY). Complementary DNA (cDNA) was generated from 1.2 μg RNA using a Tetro cDNA Synthesis Kit (Bioline, Taunton, MA). Quantitative polymerase chain reaction was conducted in 10-μL reactions using a SensiFAST Probe Kit (Bioline) with 900 nM TaqMan primers (with 250 nM FAM probe) or 50 nM 18S ribosomal RNA primers (with 200 nM VIC probe) mix (Life Technologies) and 10 ng cDNA. Duplicate polymerase chain reactions were performed and analyzed, as previously described (13). Serum corticosterone For serum corticosterone, 400 μL of blood was obtained by terminal cardiac puncture at 11 am and incubated for 1 hour at 5°C before centrifuging at 1000 × relative centrifugal force for 5 minutes. Corticosterone levels were determined using a corticosterone EIA Kit (Cayman Chemical, Ann Arbor, MI). Histology Freshly isolated samples were stored in Formalde-Fresh (Fisher Scientific, Pittsburgh, PA) and processed into paraffin blocks. Then, 5-μm sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (Leica, Buffalo Grove, IL and Thermo Scientific, Kalamazoo, MI). Sections were examined with a Zeiss microscope (Jena, Germany) and digital images captured with AxioVision software (Carl Zeiss Vision, Munich, Germany). For epidermal thickness, an average of four measurements were taken per image, and dermal area was quantified using ImageJ. For quantification of epidermal cellularity (>90% of which is composed of keratinocytes), the total number of epidermal nuclei was counted per field of view and normalized to epidermal length (to account for rete ridge undulation). Proliferation was evaluated by incubating rehydrated sections with biotinylated primary antibody against proliferating cell nuclear antigen (CalTag Laboratories, Burlingame, CA) overnight at 4°C. After 3× Tris-buffered saline washes, staining was detected with the ABC-Peroxidase Kit (Vector Laboratories, Burlingame, CA) and quantified using ImageJ. Statistical analysis Following confirmation of data displaying a normal distribution, significance levels were determined by Student t test or one-way or two-way analysis of variance using GraphPad Prism (GraphPad Software, La Jolla, CA) on untransformed data. For two-way analysis of variance, post hoc testing included analysis of differences between time points in each treatment group and differences between treatment groups at each time point with P values adjusted for multiple testing as detailed in the figure legends. Variation is displayed as standard error based on at least three biological replicates. Results Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess In agreement with known suppressive effects of GC treatment on hypothalamic-pituitary-adrenal axis signaling, systemic GC treatment [corticosterone (CORT)] reduced circulating corticosterone levels by 70% compared with VEH mice (Fig. 1A) with body weight unaffected between groups (data not shown). Figure 1. View largeDownload slide Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess. (A) Serum corticosterone levels were downregulated in mice treated with oral CORT compared with VEH (n = 3 to 6, Student t test). (B) 11β-HSD1 and (C) H6PDH mRNA expression were upregulated by CORT in unwounded skin compared with VEH mice. Increased expression postwounding was exacerbated by CORT independently of 11β-HSD1 inhibitor (CBX) treatment (n = 3 to 6, two-way analysis of variance): (D) 11β-HSD1 enzyme activity was elevated by CORT in unwounded skin and increased during healing independently of CORT. Activity was inhibited by CBX in unwounded skin and during healing (n = 3 to 5, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 1. View largeDownload slide Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess. (A) Serum corticosterone levels were downregulated in mice treated with oral CORT compared with VEH (n = 3 to 6, Student t test). (B) 11β-HSD1 and (C) H6PDH mRNA expression were upregulated by CORT in unwounded skin compared with VEH mice. Increased expression postwounding was exacerbated by CORT independently of 11β-HSD1 inhibitor (CBX) treatment (n = 3 to 6, two-way analysis of variance): (D) 11β-HSD1 enzyme activity was elevated by CORT in unwounded skin and increased during healing independently of CORT. Activity was inhibited by CBX in unwounded skin and during healing (n = 3 to 5, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. The effect of GC excess on 11β-HSD1 expression and activity during mouse skin WH was previously unexplored. At baseline, 11β-HSD1 messenger RNA (mRNA) was 10-fold higher in CORT skin compared with VEH (Fig. 1B). Hexose-6-phosphate dehydrogenase (H6PDH), which supplies 11β-HSD1 cofactor, showed a similar increase in expression at baseline (Fig. 1C). Correspondingly, 11β-HSD1 activity was also 42% greater in unwounded CORT skin compared with VEH (Fig. 1D). In VEH-treated mice, 11β-HSD1 mRNA and activity increased by 43-fold and 2.6-fold at day 2 and by 17-fold and 1.7-fold at day 4 postwounding, respectively (Fig. 1B and 1D). H6PDH mRNA was also increased by sevenfold at days 2 and 4 postwounding (Fig. 1C). CORT further increased 11β-HSD1 mRNA by 11-fold and 20-fold (independently of CBX) at days 4 and 9 postwounding, respectively (Fig. 1B). H6PDH mRNA was also further upregulated by fivefold at day 4 and ninefold (independently of CBX) at day 9 postwounding in CORT mice (Fig. 1C). However, 11β-HSD1 activity was not affected by CORT treatment during WH (Fig. 1D). In CORT mice, CBX treatment inhibited 11β-HSD1 activity by 61% in unwounded skin and by 91% and 55% at days 2 and 4 postwounding, respectively (Fig. 1D). In control mice (untreated drinking water), CBX inhibited 11β-HSD1 activity by 30% in unwounded skin and by 93%, 94%, and 73% at days 2, 4, and 9 postwounding (Supplemental Fig. 1). These findings indicate that GC excess induces cutaneous 11β-HSD1 expression and activity in unwounded skin that is inhibited effectively by CBX treatment. 11β-HSD1 inhibition limits GC-mediated skin thinning GCs cause skin thinning and dermal atrophy. However, the effect of 11β-HSD1 inhibition on epidermal thinning and proliferation following GC exposure was previously unreported. Epidermal thickness was reduced by 36% in CORT mice compared with VEH and was restored by 59% with CBX (Fig. 2A and 2B). A 27% CORT-mediated reduction in epidermal cellularity was also prevented by CBX (Fig. 2A and 2C) with a similar trend in proliferating cell nuclear antigen staining (Supplemental Fig. 2). Dermal area decreased by 21% with CORT treatment compared with VEH, and this was also prevented by CBX (Fig. 2D). No difference in epidermal thickness or epidermal cellularity was observed in control mice treated with CBX (Supplemental Fig. 3). Figure 2. View largeDownload slide 11β-HSD1 inhibition limits GC-mediated skin thinning. (A) Representative hematoxylin and eosin staining showing decreased epidermal thickness and epidermal cellularity in CORT mice and improvement following 7 days of CBX treatment [n = 4, one-way analysis of variance (ANOVA)], (B) quantification of epidermal thickness (n = 4), (C) quantification of epidermal cellularity (n = 4, one-way ANOVA), and (D) quantification of dermal area (n = 4, one-way ANOVA). Scale bar: 50 μm. Multiple comparisons for B to D: * = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 2. View largeDownload slide 11β-HSD1 inhibition limits GC-mediated skin thinning. (A) Representative hematoxylin and eosin staining showing decreased epidermal thickness and epidermal cellularity in CORT mice and improvement following 7 days of CBX treatment [n = 4, one-way analysis of variance (ANOVA)], (B) quantification of epidermal thickness (n = 4), (C) quantification of epidermal cellularity (n = 4, one-way ANOVA), and (D) quantification of dermal area (n = 4, one-way ANOVA). Scale bar: 50 μm. Multiple comparisons for B to D: * = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. These results suggest that 11β-HSD1 inhibition can limit GC-mediated skin thinning despite sustained exposure to systemic GC excess. Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function A functional epidermal barrier is indispensable for protection against dehydration, exposure to irritants, and invasion by microorganisms. The regulation of epidermal barrier function, integrity, and recovery by corticosterone excess or 11β-HSD1 inhibition has not been investigated. TEWL is the gold-standard measure of epidermal barrier function. Baseline TEWL was largely unaffected by CORT or CBX (Fig. 3A). Interestingly, the number of tape strips required to disrupt the barrier (indicative of barrier integrity) was 50% greater in CORT mice, and this was prevented by CBX treatment (Fig. 3B). We observed a modest increase in barrier recovery in the CORT/CBX group 2 hours after barrier disruption, but no difference was observed between CORT and CORT/CBX or between any of the groups 4 hours after disruption (Fig. 3C). No statistically significant effect of CBX on barrier function (baseline TEWL), barrier integrity, or barrier recovery was observed in control mice (Supplemental Fig. 4). Figure 3. View largeDownload slide Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function. (A) Baseline TEWL in CORT mice and following 7 days of CBX treatment [n = 8 to 10, one-way analysis of variance (ANOVA)], (B) increased barrier integrity (number of tape strips required to disrupt barrier) in CORT mice and reversal by CBX cotreatment (n = 8 to 10, one-way ANOVA), and (C) barrier recovery relative to baseline TEWL (n = 7 to 8, two-way ANOVA). Multiple comparisons for A and B: # = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test) and for C: * = vs 2-hour time point in each treatment group (Sidak test) and # = vs VEH/VEH at each time point. Significance: *P < 0.05, ***P < 0.001. Figure 3. View largeDownload slide Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function. (A) Baseline TEWL in CORT mice and following 7 days of CBX treatment [n = 8 to 10, one-way analysis of variance (ANOVA)], (B) increased barrier integrity (number of tape strips required to disrupt barrier) in CORT mice and reversal by CBX cotreatment (n = 8 to 10, one-way ANOVA), and (C) barrier recovery relative to baseline TEWL (n = 7 to 8, two-way ANOVA). Multiple comparisons for A and B: # = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test) and for C: * = vs 2-hour time point in each treatment group (Sidak test) and # = vs VEH/VEH at each time point. Significance: *P < 0.05, ***P < 0.001. These findings suggest that 11β-HSD1 mediates increased epidermal integrity during systemic GC excess. 11β-HSD1 inhibition improves skin wound healing during GC excess GC excess drives impaired WH, and chronic wound management remains an unmet clinical need in diabetic and elderly patients. However, the regulation of impaired WH during GC excess by 11β-HSD1 is unknown. Systemic GC excess induced a striking WH delay, which was improved by CBX treatment (Fig. 4A). Figure 4. View largeDownload slide 11β-HSD1 inhibition improves skin wound healing during GC excess. (A) Representative wound appearance at day 9 (n = 8), (B) quantification of wound areas showing cessation of healing from day 4 in CORT mice and improvement of healing by CBX treatment (n = 8 to 24, two-way analysis of variance), and (C) representative day 9 hematoxylin and eosin staining showing advanced granulation tissue (arrows) formation in mice cotreated with CBX compared with CORT alone (n = 3). Scale bar: (A) 13 mm, (C) 50 μm. * = vs day 4. Multiple comparisons for B: * = vs day 4 in each treatment group (Dunnett test), # = vs VEH/VEH at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 4. View largeDownload slide 11β-HSD1 inhibition improves skin wound healing during GC excess. (A) Representative wound appearance at day 9 (n = 8), (B) quantification of wound areas showing cessation of healing from day 4 in CORT mice and improvement of healing by CBX treatment (n = 8 to 24, two-way analysis of variance), and (C) representative day 9 hematoxylin and eosin staining showing advanced granulation tissue (arrows) formation in mice cotreated with CBX compared with CORT alone (n = 3). Scale bar: (A) 13 mm, (C) 50 μm. * = vs day 4. Multiple comparisons for B: * = vs day 4 in each treatment group (Dunnett test), # = vs VEH/VEH at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Initially, WH progressed at similar rates between all groups with a 36%, 39%, and 33% reduction in wound area at day 4 compared with day 2 in VEH, CORT, and CORT/CBX mice, respectively (Fig. 4B). No change in wound area was observed between days 4 and 6 for either group. Although healing continued from day 6 for VEH and CORT/CBX mice, no further healing was observed for CORT/VEH mice. By day 9, wound areas were 94% and 48% healed compared with day 4 in VEH and CORT/CBX mice, respectively (Fig. 4B). Hematoxylin and eosin staining of day 9 wounds revealed more advanced granulation tissue formation, improved structural organization, and extracellular matrix deposition in CORT/CBX compared with CORT mice (Fig. 4C). CBX treatment did not affect WH in control mice (Supplemental Fig. 5). In summary, 11β-HSD1 inhibition improved mouse skin WH despite sustained systemic GC exposure. Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing To further investigate the mechanisms underlying improved WH with 11β-HSD1 inhibition, we analyzed changes in cytokine, extracellular matrix, and growth factor gene expression. Proinflammatory cytokine interleukin (IL)-1β mRNA increased at days 2, 4, and 9 postwounding by 750-fold, 587-fold, and 90-fold, respectively, independently of CORT or CBX treatment (Fig. 5A). A similar expression profile was seen for tumor necrosis factor α mRNA (Fig. 5B). Figure 5. View largeDownload slide Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing. (A) IL-1β and (B) tumor necrosis factor α (TNFα) mRNA increased during WH independently of CORT or CBX. (C) IL-6 mRNA showed a trend toward induction by CORT at day 2, which was suppressed by CBX. (D) KGF mRNA induction at day 4 was suppressed by CORT but not CORT/CBX and induced by CORT but not CORT/CBX at day 9. (E) Collagen type I α1 (COL-1α1) and (F) collagen type III α1 (COL-3α1) mRNA was induced by CORT but not CORT/CBX at day 9. (G) MMP-9 mRNA induction by CORT at day 9 was reversed by CORT/CBX. (H) TIMP-1 mRNA induction at day 4 was suppressed by CORT and reversed by CORT/CBX (n = 3 to 6, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 5. View largeDownload slide Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing. (A) IL-1β and (B) tumor necrosis factor α (TNFα) mRNA increased during WH independently of CORT or CBX. (C) IL-6 mRNA showed a trend toward induction by CORT at day 2, which was suppressed by CBX. (D) KGF mRNA induction at day 4 was suppressed by CORT but not CORT/CBX and induced by CORT but not CORT/CBX at day 9. (E) Collagen type I α1 (COL-1α1) and (F) collagen type III α1 (COL-3α1) mRNA was induced by CORT but not CORT/CBX at day 9. (G) MMP-9 mRNA induction by CORT at day 9 was reversed by CORT/CBX. (H) TIMP-1 mRNA induction at day 4 was suppressed by CORT and reversed by CORT/CBX (n = 3 to 6, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. The immunomodulatory cytokine IL-6 regulates a broad range of biological functions, including antibody-secreting B-cell differentiation and generation of Th17 cells. IL-6 mRNA expression was undetectable in the majority of unwounded skin samples but increased at days 2 and 4 before declining at day 9 postwounding in VEH mice (Fig. 5C). IL-6 displayed a trend toward higher expression at day 2 postwounding in CORT compared with VEH mice, which was prevented by CBX (Fig. 5C). IL-6 mRNA was also 14-fold greater in CORT mice at day 9 postwounding compared with VEH mice (Fig. 5C). Keratinocyte growth factor (KGF) is produced during WH and stimulates wound reepithelialization. KGF mRNA expression peaked at day 4 with a 9.9-fold and a 3.5-fold induction in VEH and CORT/CBX mice compared with unwounded skin, respectively (Fig. 5D). No induction in KGF was seen at day 4 in CORT mice. At day 9 postwounding, KGF was 17-fold higher in CORT compared with VEH mice, and this was reversed by CBX (Fig. 5D). Type I and III collagen constitute >70% of the dermis by dry weight. Collagen type I α1 and collagen type III α1 mRNA expression increased 15-fold at day 4 postwounding in VEH mice compared with unwounded skin (Fig. 5E and 5F). This was delayed in CORT mice, which displayed a 3.5-fold and 14-fold induction of collagen type I α1 and collagen type III α1 at day 9 postwounding compared with VEH controls both reversed by CBX (Fig. 5E and 5F). Extracellular matrix turnover is regulated by collagen-degrading matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of MMPs (TIMPs). MMP-9 mRNA in VEH mice increased 7.7-fold and 9.5-fold at days 2 and 4 postwounding, respectively, before returning to baseline levels by day 9 (Fig. 5G). However, in CORT (but not CORT/CBX) mice, MMP-9 expression at day 9 remained elevated at 7.2-fold higher than VEH mice (Fig. 5G). TIMP-1 displayed a similar mRNA profile, with 20-fold and 33-fold higher expression at days 2 and 4 postwounding, respectively, in VEH mice compared with unwounded skin (Fig. 5H). At day 9 postwounding, MMP-9 mRNA was also 10-fold greater in CORT (but not CORT/CBX) compared with VEH mice (Fig. 5H). These findings oppose reports of increased inflammatory cytokine expression following 11β-HSD1 blockade in other models. Improved WH appears to be mediated, in part, by restoration of KGF signaling and appropriate collagen, MMP, and TIMP expression. Discussion Although GCs have been used for >65 years for their anti-inflammatory properties (17), adverse side-effects limit their use to low-dose, short-term intervention (2). GC-mediated skin atrophy, bruising, impaired WH, and increased infection risk are of concern to both patients and physicians alike (18). In a survey of patients taking GCs for >60 days, skin bruising or thinning was the second most prevalent self-reported side effect (8). We have demonstrated for the first time that topical 11β-HSD1 inhibition can limit these cutaneous side effects. This key insight opens a wide range of avenues for 11β-HSD1 inhibitors as adjunct therapies to safely increase steroid doses or treatment duration. Morgan et al. (19) recently demonstrated that global 11β-HSD1–null mice were protected from the systemic side effects of oral corticosterone. However, this study did not evaluate inflammation, which others report to be exacerbated by 11β-HSD1 inhibition (20). Here, we evaluated the ability of 11β-HSD1 inhibition to reverse cutaneous effects of GC excess. Reduced morning serum corticosterone levels in CORT mice are consistent with the reported adrenal suppression at this sampling time (21), and total body weight was unaffected by GC treatment as previously described (19). The forward-feedback induction of 11β-HSD1 mRNA and activity by GCs in unwounded skin is also in agreement with our previous observations in human skin (15) and may exacerbate the 11β-HSD1–mediated effects observed in unwounded CORT mouse skin. Our results demonstrate that 11β-HSD1 inhibition prevents GC-mediated reductions in epidermal cellularity and dermal area, despite sustained exposure to systemic GC excess. Interestingly, we observed that CBX also reversed a GC-mediated induction in epidermal integrity. This is supported by studies demonstrating that exogenous GC treatment promotes epidermal keratinocyte differentiation and increases corneocyte numbers (22), reflecting a greater degree of disruption required for epidermal barrier perturbation. Conversely, endogenous GC excess or topical GC treatment causes GC-mediated decreases in epidermal integrity, indicating that model-specific differences should also be taken into consideration (23–26). Baseline TEWL (epidermal barrier function) was unaffected by CORT treatment, in agreement with previous reports (23–26). In contrast to our findings, other studies reported impaired barrier recovery following endogenous GC excess and GC treatment, although this was associated with impaired barrier integrity (23, 25, 26). In our model, integrity and barrier recovery were both stimulated by systemic GC excess. This may be due to differences in GC potency, formulation, treatment duration, administration, strain, sex, age, diet, or environment. Although integrity appeared to be 11β-HSD1 mediated, the modest induction in barrier recovery was not reversed by CBX. This may be due to regulation by other hypothalamic-pituitary-adrenal axis pathways such as β2-adrenergic receptor signaling (27). Between days 0 and 4, wound closure was similar between all groups, suggesting that the initial contractile phase of WH (28) was unaffected. We observed termination of WH kinetics from day 4 postwounding in CORT mice, supported by previous reports of GC-mediated impairment of keratinocyte proliferation, migration, and increased epidermal differentiation (29, 30). Strikingly, our results demonstrate that CBX treatment improved WH in CORT mice. The lack of complete WH rescue by CBX may be due to exposure to serum corticosterone before cellular compartmentalization is fully restored. Others have also demonstrated benefits of 11β-HSD1 blockade on cutaneous WH in animal models of metabolic disease and endogenous GC excess (31, 32), but 11β-HSD1 inhibition, inflammatory effects, and mechanistic insights were lacking in these studies. Our findings indicate that the cutaneous side effects of GC excess are mediated through increased 11β-HSD1 substrate availability as 11β-HSD1 activity was not further increased by CORT treatment and CBX treatment did not affect skin thickness, epidermal integrity, or WH in control mice not exposed to systemic GC excess. This suggests that 11-dehydrocorticosterone availability is insufficient in young, healthy mice to regulate skin function. Recent reports have raised concerns over increased inflammation as a consequence of 11β-HSD1 blockade (33–36). We found no evidence of this our model; there was no exacerbation of IL-1β, tumor necrosis factor α, or IL-6 mRNA expression during early WH and no delay in resolution of these proinflammatory cytokines in CBX mice. On the contrary, IL-6 expression increased during CORT mouse WH. This is supported by reports of proinflammatory GC actions (37, 38), indicating greater regulatory complexity than previously thought (39–41). KGF is a key factor that promotes wound reepithelialization and is indispensable to the WH process (30, 42, 43). We found a lack of induction of KGF at day 4 postwounding in CORT mice (coinciding with the cessation of wound closure), which was restored by CBX. The induction of collagen type I and III mRNA during the granulation phase of WH were attenuated in CORT mice, consistent with previous reports (30, 44), and were also normalized by CBX, further supported by our histological findings. MMP-9 and TIMP-1 mRNA expression were elevated at day 9 postwounding in CORT mice and were normalized by CBX treatment. Although the underlying mechanisms remain poorly understood, elevated MMP-9 and TIMP-1 mRNA are associated with slower healing in mouse wounds (45), and MMP-9 is elevated in diabetic foot ulcers (46). GCs are able to activate the mineralocorticoid receptor (MR) with strong affinity, and recent work by Boix et al. (47) elegantly demonstrated that MR epidermal knockout mice were resistant to GC-induced epidermal thinning. Differentiating between GC receptor and MR-mediated effects was beyond the scope of the current study but is an area of ongoing interest. CBX has been used extensively in human and mammalian models. Administered systemically, it also inhibits 11β-HSD2 (which conducts the opposing reaction to 11β-HSD1), resulting in renal mineralocorticoid excess due to GC-mediated MR activation (48). However, 11β-HSD2 is not expressed in unwounded mouse skin or during WH (13), and topical administration limits the risk of systemic exposure. CBX is also able to block gap junctions but at several orders of magnitude less potently than 11β-HSD1 (48). Furthermore, we observed no difference between CBX and VEH mice not exposed to CORT, suggesting our findings are most likely due to cutaneous 11β-HSD1 inhibition. In summary, we report the ability of topical 11β-HSD1 inhibition to limit cutaneous side effects of systemic GC excess, including skin thinning and delayed WH. Our findings may improve the safety profile of systemic steroids and the prognosis of chronic wounds, particularly those associated with elevated circulating GC levels (49). Abbreviations: 11β-HSD1 11β-hydroxysteroid dehydrogenase type 1 CBX carbenoxolone cDNA complementary DNA CORT corticosterone GC glucocorticoid H6PDH hexose-6-phosphate dehydrogenase IL interleukin KGF keratinocyte growth factor MMP matrix metalloproteinase MR mineralocorticoid receptor mRNA messenger RNA TEWL transepidermal water loss TIMP tissue inhibitor of matrix metalloproteinase VEH vehicle WH wound healing. Acknowledgments Financial Support: This work was supported by Department of Defence Grant W81XWH-11-2-0189 (to P.M.E). Author Contributions: A.T., Y.U., and W.M.H. designed the research study. A.T. performed the experiments, analyzed the data, and prepared the manuscript. M.H. prepared tissue sections for histology. T.M. and P.M.E. contributed essential equipment. All authors evaluated and approved the final manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Overman RA, Yeh JY, Deal CL. 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Topical 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibition Corrects Cutaneous Features of Systemic Glucocorticoid Excess in Female Mice

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Oxford University Press
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00607
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Abstract

Abstract Glucocorticoid (GC) excess drives multiple cutaneous adverse effects, including skin thinning and poor wound healing. The ubiquitously expressed enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates mouse corticosterone from 11-dehydrocorticosterone (and human cortisol from cortisone). We previously demonstrated elevated 11β-HSD1 activity during mouse wound healing, but the interplay between cutaneous 11β-HSD1 and systemic GC excess is unexplored. Here, we examined effects of 11β-HSD1 inhibition by carbenoxolone (CBX) in mice treated with corticosterone (CORT) or vehicle for 6 weeks. Mice were treated bidaily with topical CBX or vehicle (VEH) 7 days before wounding and during wound healing. CORT mice displayed skin thinning and impaired wound healing but also increased epidermal integrity. 11β-HSD1 activity was elevated in unwounded CORT skin and was inhibited by CBX. CORT mice treated with CBX displayed 51%, 59%, and 100% normalization of wound healing, epidermal thickness, and epidermal integrity, respectively. Gene expression studies revealed normalization of interleukin 6, keratinocyte growth factor, collagen 1, collagen 3, matrix metalloproteinase 9, and tissue inhibitor of matrix metalloproteinase 4 by CBX during wound healing. Importantly, proinflammatory cytokine expression and resolution of inflammation were unaffected by 11β-HSD1 inhibition. CBX did not regulate skin function or wound healing in the absence of CORT. Our findings demonstrate that 11β-HSD1 inhibition can limit the cutaneous effects of GC excess, which may improve the safety profile of systemic steroids and the prognosis of chronic wounds. Systemic glucocorticoid (GC) therapy remains mainstream treatment of many inflammatory diseases (e.g., lupus, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, polymyalgia rheumatica, and giant cell arteritis). Recent estimates indicate that 1.2% of the US population (>2.5 million people) were prescribed oral GCs between 1999 and 2008, 28% of whom reported use of >5 years (1). Despite anti-inflammatory benefits, chronic GC excess drives adverse side effects, including weight gain, hypertension, hyperglycemia, osteoporosis, muscle weakness, glaucoma, and depression (2). GC therapy also substantially increases health care costs. In patients with systemic lupus erythematosus, GC use was associated with a threefold increase in annual expenditure (3). In a study of patients with severe asthma, high-dose GC use was associated with annual increments of $5479 relative to low GC exposure (4). Moreover, the incidence and impact of GC-related side effects are underreported (5) and further compounded by GC overprescription (6). In skin, GC excess causes acne, thinning, dryness, atrophic striae, telangiectasia, bruising, impaired wound healing (WH), and increased infection risk (2, 7). Skin bruising and thinning have also been reported with low-dose (<7.5 mg/d) GC therapy (8). In a survey of asthma physicians, cutaneous manifestations were the second most frequently reported complication of inhaled GCs (9). Despite recent advances in GC mimetics and GC-independent immune suppressants, the “holy grail” of dissociating GC anti-inflammatory benefits from side effects remains elusive (10). It is now established that human skin has corticosteroidogenic capability that is regulated in a complex way (11, 12), although this function is lacking in mouse skin (13). Peripheral GC availability is also regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts biologically inert cortisone and 11-dehydrocorticosterone to cortisol and corticosterone in humans and mice, respectively. In intact tissues and cells, 11β-HSD1 functions predominantly as a reductase in a nicotinamide adenine dinucleotide phosphate–dependent manner (14). We previously reported increased 11β-HSD1 activity during mouse skin WH (13) and in aged mouse and human skin (15, 16), as well as accelerated WH in aged 11β-HSD1–null mice (16). However, the role of 11β-HSD1 in impaired WH is unknown. We propose that 11β-HSD1 is a causative agent of cutaneous side effects during systemic GC excess such as skin atrophy and impaired WH. Because human 11β-HSD1 inhibitors are readily available, such a demonstration could lead to rapid progress in ameliorating this major clinical problem. Here, we provide what, to our knowledge, is the first empirical evaluation of this causal link. We investigate effects of topical 11β-HSD1 inhibition by carbenoxolone (CBX) on skin function in mice treated with oral corticosterone and examine inflammatory, growth factor, and extracellular matrix gene expression responses during WH. We demonstrate that 11β-HSD1 inhibition accelerates WH and reduces skin damage in steroid-treated mice without compromising the inflammatory response. Materials and Methods Declarations Studies presented in this article were approved by the Institutional Animal Care and Use Committee and San Francisco Veterans Affairs Medical Center Veterinary Medical Unit. Materials were obtained from Sigma-Aldrich unless otherwise stated. Animal studies Female SKH1 hairless mice (8 to 10 weeks old) were obtained from Charles River Laboratories (Wilmington, MA) and acclimatized for 2 weeks. Mice were group housed, supplied with a basal chow diet ad libitum, and exposed to a standard 12-hour light/dark cycle. Drinking water was supplemented with corticosterone (100 μg/mL) or vehicle (VEH) (0.66% ethanol) for 6 weeks (replaced twice weekly). Seven days prior to wounding, 100 μg/cm2 topical CBX (20 μL) or VEH (70% propylene glycol, 30% ethanol) was massaged into 2 cm2 skin until fully absorbed. Treatments were repeated bidaily for the remainder of the experiment and replaced with 10 μL per wound postwounding. Twenty-four hours prior to wounding, epidermal barrier function was measured using a Tewameter 300 (Courage + Khazaka, Cologne, Germany) to evaluate transepidermal water loss (TEWL). Epidermal barrier recovery was determined at 2 and 4 hours following disruption by sequential D-Squame tape stripping to a TEWL level of >30 g/h/m2. Epidermal integrity was defined as the number of tape strips required to achieve barrier disruption. WH was conducted as previously described (13). Briefly, mice anesthetized under 2% isoflurane were wounded on both dorsal flanks by a 5-mm punch biopsy (Acuderm, Fort Lauderdale, FL). Wounds received 20 μL bupivacaine 0.25% (analgesic) and were monitored daily. At 2, 4, and 9 days’ postwounding, mice were culled by cervical dislocation, and wounds were digitally imaged and excised for 11β-HSD1 activity assay, RNA extraction, or histology. Wound areas were determined by ImageJ (NIH, Bethesda, MD). Experiments were also replicated in control mice (untreated drinking water) treated with VEH or CBX. 11β-HSD1 activity assay Radioactive conversion of tritiated 11β-HSD1 substrate is the gold-standard measure of 11β-HSD1 activity as tissue steroid extraction efficiencies are variable and less sensitive. Freshly isolated skin (20 to 40 mg) was incubated immediately in 1 mL high glucose and pyruvate Dulbecco’s modified Eagle medium with 100 nM 11-dehydrocorticosterone (Steraloids, Newport, RI) and ∼1500 cpm [3H] 11-dehydrocorticosterone, generated in house as previously described (13). Samples were incubated at 37°C for 13 hours. Subsequently, tissues were weighed and steroids extracted and separated by thin-layer chromatography in 186 mL chloroform and 14 mL ethanol for 90 minutes (comigrated with 10 mM 11-dehydrocorticosterone/corticosterone standards). Plate regions identified under ultraviolet were excised and percentage conversion of 11-dehydrocorticosterone to corticosterone was determined after liquid scintillation. Quantitative polymerase chain reaction Fresh skin tissue (20 to 40 mg) was snap-frozen and stored at −80°C. Samples were homogenized in 1 mL Trizol and RNA extracted using a PureLink RNA Mini Kit (Life Technologies, Grand Island, NY). Complementary DNA (cDNA) was generated from 1.2 μg RNA using a Tetro cDNA Synthesis Kit (Bioline, Taunton, MA). Quantitative polymerase chain reaction was conducted in 10-μL reactions using a SensiFAST Probe Kit (Bioline) with 900 nM TaqMan primers (with 250 nM FAM probe) or 50 nM 18S ribosomal RNA primers (with 200 nM VIC probe) mix (Life Technologies) and 10 ng cDNA. Duplicate polymerase chain reactions were performed and analyzed, as previously described (13). Serum corticosterone For serum corticosterone, 400 μL of blood was obtained by terminal cardiac puncture at 11 am and incubated for 1 hour at 5°C before centrifuging at 1000 × relative centrifugal force for 5 minutes. Corticosterone levels were determined using a corticosterone EIA Kit (Cayman Chemical, Ann Arbor, MI). Histology Freshly isolated samples were stored in Formalde-Fresh (Fisher Scientific, Pittsburgh, PA) and processed into paraffin blocks. Then, 5-μm sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (Leica, Buffalo Grove, IL and Thermo Scientific, Kalamazoo, MI). Sections were examined with a Zeiss microscope (Jena, Germany) and digital images captured with AxioVision software (Carl Zeiss Vision, Munich, Germany). For epidermal thickness, an average of four measurements were taken per image, and dermal area was quantified using ImageJ. For quantification of epidermal cellularity (>90% of which is composed of keratinocytes), the total number of epidermal nuclei was counted per field of view and normalized to epidermal length (to account for rete ridge undulation). Proliferation was evaluated by incubating rehydrated sections with biotinylated primary antibody against proliferating cell nuclear antigen (CalTag Laboratories, Burlingame, CA) overnight at 4°C. After 3× Tris-buffered saline washes, staining was detected with the ABC-Peroxidase Kit (Vector Laboratories, Burlingame, CA) and quantified using ImageJ. Statistical analysis Following confirmation of data displaying a normal distribution, significance levels were determined by Student t test or one-way or two-way analysis of variance using GraphPad Prism (GraphPad Software, La Jolla, CA) on untransformed data. For two-way analysis of variance, post hoc testing included analysis of differences between time points in each treatment group and differences between treatment groups at each time point with P values adjusted for multiple testing as detailed in the figure legends. Variation is displayed as standard error based on at least three biological replicates. Results Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess In agreement with known suppressive effects of GC treatment on hypothalamic-pituitary-adrenal axis signaling, systemic GC treatment [corticosterone (CORT)] reduced circulating corticosterone levels by 70% compared with VEH mice (Fig. 1A) with body weight unaffected between groups (data not shown). Figure 1. View largeDownload slide Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess. (A) Serum corticosterone levels were downregulated in mice treated with oral CORT compared with VEH (n = 3 to 6, Student t test). (B) 11β-HSD1 and (C) H6PDH mRNA expression were upregulated by CORT in unwounded skin compared with VEH mice. Increased expression postwounding was exacerbated by CORT independently of 11β-HSD1 inhibitor (CBX) treatment (n = 3 to 6, two-way analysis of variance): (D) 11β-HSD1 enzyme activity was elevated by CORT in unwounded skin and increased during healing independently of CORT. Activity was inhibited by CBX in unwounded skin and during healing (n = 3 to 5, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 1. View largeDownload slide Cutaneous 11β-HSD1 expression and activity increase during wound healing and systemic GC excess. (A) Serum corticosterone levels were downregulated in mice treated with oral CORT compared with VEH (n = 3 to 6, Student t test). (B) 11β-HSD1 and (C) H6PDH mRNA expression were upregulated by CORT in unwounded skin compared with VEH mice. Increased expression postwounding was exacerbated by CORT independently of 11β-HSD1 inhibitor (CBX) treatment (n = 3 to 6, two-way analysis of variance): (D) 11β-HSD1 enzyme activity was elevated by CORT in unwounded skin and increased during healing independently of CORT. Activity was inhibited by CBX in unwounded skin and during healing (n = 3 to 5, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. The effect of GC excess on 11β-HSD1 expression and activity during mouse skin WH was previously unexplored. At baseline, 11β-HSD1 messenger RNA (mRNA) was 10-fold higher in CORT skin compared with VEH (Fig. 1B). Hexose-6-phosphate dehydrogenase (H6PDH), which supplies 11β-HSD1 cofactor, showed a similar increase in expression at baseline (Fig. 1C). Correspondingly, 11β-HSD1 activity was also 42% greater in unwounded CORT skin compared with VEH (Fig. 1D). In VEH-treated mice, 11β-HSD1 mRNA and activity increased by 43-fold and 2.6-fold at day 2 and by 17-fold and 1.7-fold at day 4 postwounding, respectively (Fig. 1B and 1D). H6PDH mRNA was also increased by sevenfold at days 2 and 4 postwounding (Fig. 1C). CORT further increased 11β-HSD1 mRNA by 11-fold and 20-fold (independently of CBX) at days 4 and 9 postwounding, respectively (Fig. 1B). H6PDH mRNA was also further upregulated by fivefold at day 4 and ninefold (independently of CBX) at day 9 postwounding in CORT mice (Fig. 1C). However, 11β-HSD1 activity was not affected by CORT treatment during WH (Fig. 1D). In CORT mice, CBX treatment inhibited 11β-HSD1 activity by 61% in unwounded skin and by 91% and 55% at days 2 and 4 postwounding, respectively (Fig. 1D). In control mice (untreated drinking water), CBX inhibited 11β-HSD1 activity by 30% in unwounded skin and by 93%, 94%, and 73% at days 2, 4, and 9 postwounding (Supplemental Fig. 1). These findings indicate that GC excess induces cutaneous 11β-HSD1 expression and activity in unwounded skin that is inhibited effectively by CBX treatment. 11β-HSD1 inhibition limits GC-mediated skin thinning GCs cause skin thinning and dermal atrophy. However, the effect of 11β-HSD1 inhibition on epidermal thinning and proliferation following GC exposure was previously unreported. Epidermal thickness was reduced by 36% in CORT mice compared with VEH and was restored by 59% with CBX (Fig. 2A and 2B). A 27% CORT-mediated reduction in epidermal cellularity was also prevented by CBX (Fig. 2A and 2C) with a similar trend in proliferating cell nuclear antigen staining (Supplemental Fig. 2). Dermal area decreased by 21% with CORT treatment compared with VEH, and this was also prevented by CBX (Fig. 2D). No difference in epidermal thickness or epidermal cellularity was observed in control mice treated with CBX (Supplemental Fig. 3). Figure 2. View largeDownload slide 11β-HSD1 inhibition limits GC-mediated skin thinning. (A) Representative hematoxylin and eosin staining showing decreased epidermal thickness and epidermal cellularity in CORT mice and improvement following 7 days of CBX treatment [n = 4, one-way analysis of variance (ANOVA)], (B) quantification of epidermal thickness (n = 4), (C) quantification of epidermal cellularity (n = 4, one-way ANOVA), and (D) quantification of dermal area (n = 4, one-way ANOVA). Scale bar: 50 μm. Multiple comparisons for B to D: * = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 2. View largeDownload slide 11β-HSD1 inhibition limits GC-mediated skin thinning. (A) Representative hematoxylin and eosin staining showing decreased epidermal thickness and epidermal cellularity in CORT mice and improvement following 7 days of CBX treatment [n = 4, one-way analysis of variance (ANOVA)], (B) quantification of epidermal thickness (n = 4), (C) quantification of epidermal cellularity (n = 4, one-way ANOVA), and (D) quantification of dermal area (n = 4, one-way ANOVA). Scale bar: 50 μm. Multiple comparisons for B to D: * = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. These results suggest that 11β-HSD1 inhibition can limit GC-mediated skin thinning despite sustained exposure to systemic GC excess. Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function A functional epidermal barrier is indispensable for protection against dehydration, exposure to irritants, and invasion by microorganisms. The regulation of epidermal barrier function, integrity, and recovery by corticosterone excess or 11β-HSD1 inhibition has not been investigated. TEWL is the gold-standard measure of epidermal barrier function. Baseline TEWL was largely unaffected by CORT or CBX (Fig. 3A). Interestingly, the number of tape strips required to disrupt the barrier (indicative of barrier integrity) was 50% greater in CORT mice, and this was prevented by CBX treatment (Fig. 3B). We observed a modest increase in barrier recovery in the CORT/CBX group 2 hours after barrier disruption, but no difference was observed between CORT and CORT/CBX or between any of the groups 4 hours after disruption (Fig. 3C). No statistically significant effect of CBX on barrier function (baseline TEWL), barrier integrity, or barrier recovery was observed in control mice (Supplemental Fig. 4). Figure 3. View largeDownload slide Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function. (A) Baseline TEWL in CORT mice and following 7 days of CBX treatment [n = 8 to 10, one-way analysis of variance (ANOVA)], (B) increased barrier integrity (number of tape strips required to disrupt barrier) in CORT mice and reversal by CBX cotreatment (n = 8 to 10, one-way ANOVA), and (C) barrier recovery relative to baseline TEWL (n = 7 to 8, two-way ANOVA). Multiple comparisons for A and B: # = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test) and for C: * = vs 2-hour time point in each treatment group (Sidak test) and # = vs VEH/VEH at each time point. Significance: *P < 0.05, ***P < 0.001. Figure 3. View largeDownload slide Impact of GC excess and 11β-HSD1 inhibition on epidermal barrier function. (A) Baseline TEWL in CORT mice and following 7 days of CBX treatment [n = 8 to 10, one-way analysis of variance (ANOVA)], (B) increased barrier integrity (number of tape strips required to disrupt barrier) in CORT mice and reversal by CBX cotreatment (n = 8 to 10, one-way ANOVA), and (C) barrier recovery relative to baseline TEWL (n = 7 to 8, two-way ANOVA). Multiple comparisons for A and B: # = vs VEH/VEH and † = vs VEH/CORT (Tukey post hoc test) and for C: * = vs 2-hour time point in each treatment group (Sidak test) and # = vs VEH/VEH at each time point. Significance: *P < 0.05, ***P < 0.001. These findings suggest that 11β-HSD1 mediates increased epidermal integrity during systemic GC excess. 11β-HSD1 inhibition improves skin wound healing during GC excess GC excess drives impaired WH, and chronic wound management remains an unmet clinical need in diabetic and elderly patients. However, the regulation of impaired WH during GC excess by 11β-HSD1 is unknown. Systemic GC excess induced a striking WH delay, which was improved by CBX treatment (Fig. 4A). Figure 4. View largeDownload slide 11β-HSD1 inhibition improves skin wound healing during GC excess. (A) Representative wound appearance at day 9 (n = 8), (B) quantification of wound areas showing cessation of healing from day 4 in CORT mice and improvement of healing by CBX treatment (n = 8 to 24, two-way analysis of variance), and (C) representative day 9 hematoxylin and eosin staining showing advanced granulation tissue (arrows) formation in mice cotreated with CBX compared with CORT alone (n = 3). Scale bar: (A) 13 mm, (C) 50 μm. * = vs day 4. Multiple comparisons for B: * = vs day 4 in each treatment group (Dunnett test), # = vs VEH/VEH at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 4. View largeDownload slide 11β-HSD1 inhibition improves skin wound healing during GC excess. (A) Representative wound appearance at day 9 (n = 8), (B) quantification of wound areas showing cessation of healing from day 4 in CORT mice and improvement of healing by CBX treatment (n = 8 to 24, two-way analysis of variance), and (C) representative day 9 hematoxylin and eosin staining showing advanced granulation tissue (arrows) formation in mice cotreated with CBX compared with CORT alone (n = 3). Scale bar: (A) 13 mm, (C) 50 μm. * = vs day 4. Multiple comparisons for B: * = vs day 4 in each treatment group (Dunnett test), # = vs VEH/VEH at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Initially, WH progressed at similar rates between all groups with a 36%, 39%, and 33% reduction in wound area at day 4 compared with day 2 in VEH, CORT, and CORT/CBX mice, respectively (Fig. 4B). No change in wound area was observed between days 4 and 6 for either group. Although healing continued from day 6 for VEH and CORT/CBX mice, no further healing was observed for CORT/VEH mice. By day 9, wound areas were 94% and 48% healed compared with day 4 in VEH and CORT/CBX mice, respectively (Fig. 4B). Hematoxylin and eosin staining of day 9 wounds revealed more advanced granulation tissue formation, improved structural organization, and extracellular matrix deposition in CORT/CBX compared with CORT mice (Fig. 4C). CBX treatment did not affect WH in control mice (Supplemental Fig. 5). In summary, 11β-HSD1 inhibition improved mouse skin WH despite sustained systemic GC exposure. Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing To further investigate the mechanisms underlying improved WH with 11β-HSD1 inhibition, we analyzed changes in cytokine, extracellular matrix, and growth factor gene expression. Proinflammatory cytokine interleukin (IL)-1β mRNA increased at days 2, 4, and 9 postwounding by 750-fold, 587-fold, and 90-fold, respectively, independently of CORT or CBX treatment (Fig. 5A). A similar expression profile was seen for tumor necrosis factor α mRNA (Fig. 5B). Figure 5. View largeDownload slide Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing. (A) IL-1β and (B) tumor necrosis factor α (TNFα) mRNA increased during WH independently of CORT or CBX. (C) IL-6 mRNA showed a trend toward induction by CORT at day 2, which was suppressed by CBX. (D) KGF mRNA induction at day 4 was suppressed by CORT but not CORT/CBX and induced by CORT but not CORT/CBX at day 9. (E) Collagen type I α1 (COL-1α1) and (F) collagen type III α1 (COL-3α1) mRNA was induced by CORT but not CORT/CBX at day 9. (G) MMP-9 mRNA induction by CORT at day 9 was reversed by CORT/CBX. (H) TIMP-1 mRNA induction at day 4 was suppressed by CORT and reversed by CORT/CBX (n = 3 to 6, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. Figure 5. View largeDownload slide Effect of systemic GC excess and 11β-HSD1 inhibition on cytokine, extracellular matrix, and growth factor gene expression during wound healing. (A) IL-1β and (B) tumor necrosis factor α (TNFα) mRNA increased during WH independently of CORT or CBX. (C) IL-6 mRNA showed a trend toward induction by CORT at day 2, which was suppressed by CBX. (D) KGF mRNA induction at day 4 was suppressed by CORT but not CORT/CBX and induced by CORT but not CORT/CBX at day 9. (E) Collagen type I α1 (COL-1α1) and (F) collagen type III α1 (COL-3α1) mRNA was induced by CORT but not CORT/CBX at day 9. (G) MMP-9 mRNA induction by CORT at day 9 was reversed by CORT/CBX. (H) TIMP-1 mRNA induction at day 4 was suppressed by CORT and reversed by CORT/CBX (n = 3 to 6, two-way analysis of variance). Multiple comparisons for B to D: * = vs baseline (day 0) in each treatment group (Dunnett test), # = vs VEH/VEH at each time point, and † = vs VEH/CORT at each time point (Tukey post hoc test). Significance: *P < 0.05, **P < 0.01, ***P < 0.001. A.U., arbitrary units. The immunomodulatory cytokine IL-6 regulates a broad range of biological functions, including antibody-secreting B-cell differentiation and generation of Th17 cells. IL-6 mRNA expression was undetectable in the majority of unwounded skin samples but increased at days 2 and 4 before declining at day 9 postwounding in VEH mice (Fig. 5C). IL-6 displayed a trend toward higher expression at day 2 postwounding in CORT compared with VEH mice, which was prevented by CBX (Fig. 5C). IL-6 mRNA was also 14-fold greater in CORT mice at day 9 postwounding compared with VEH mice (Fig. 5C). Keratinocyte growth factor (KGF) is produced during WH and stimulates wound reepithelialization. KGF mRNA expression peaked at day 4 with a 9.9-fold and a 3.5-fold induction in VEH and CORT/CBX mice compared with unwounded skin, respectively (Fig. 5D). No induction in KGF was seen at day 4 in CORT mice. At day 9 postwounding, KGF was 17-fold higher in CORT compared with VEH mice, and this was reversed by CBX (Fig. 5D). Type I and III collagen constitute >70% of the dermis by dry weight. Collagen type I α1 and collagen type III α1 mRNA expression increased 15-fold at day 4 postwounding in VEH mice compared with unwounded skin (Fig. 5E and 5F). This was delayed in CORT mice, which displayed a 3.5-fold and 14-fold induction of collagen type I α1 and collagen type III α1 at day 9 postwounding compared with VEH controls both reversed by CBX (Fig. 5E and 5F). Extracellular matrix turnover is regulated by collagen-degrading matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of MMPs (TIMPs). MMP-9 mRNA in VEH mice increased 7.7-fold and 9.5-fold at days 2 and 4 postwounding, respectively, before returning to baseline levels by day 9 (Fig. 5G). However, in CORT (but not CORT/CBX) mice, MMP-9 expression at day 9 remained elevated at 7.2-fold higher than VEH mice (Fig. 5G). TIMP-1 displayed a similar mRNA profile, with 20-fold and 33-fold higher expression at days 2 and 4 postwounding, respectively, in VEH mice compared with unwounded skin (Fig. 5H). At day 9 postwounding, MMP-9 mRNA was also 10-fold greater in CORT (but not CORT/CBX) compared with VEH mice (Fig. 5H). These findings oppose reports of increased inflammatory cytokine expression following 11β-HSD1 blockade in other models. Improved WH appears to be mediated, in part, by restoration of KGF signaling and appropriate collagen, MMP, and TIMP expression. Discussion Although GCs have been used for >65 years for their anti-inflammatory properties (17), adverse side-effects limit their use to low-dose, short-term intervention (2). GC-mediated skin atrophy, bruising, impaired WH, and increased infection risk are of concern to both patients and physicians alike (18). In a survey of patients taking GCs for >60 days, skin bruising or thinning was the second most prevalent self-reported side effect (8). We have demonstrated for the first time that topical 11β-HSD1 inhibition can limit these cutaneous side effects. This key insight opens a wide range of avenues for 11β-HSD1 inhibitors as adjunct therapies to safely increase steroid doses or treatment duration. Morgan et al. (19) recently demonstrated that global 11β-HSD1–null mice were protected from the systemic side effects of oral corticosterone. However, this study did not evaluate inflammation, which others report to be exacerbated by 11β-HSD1 inhibition (20). Here, we evaluated the ability of 11β-HSD1 inhibition to reverse cutaneous effects of GC excess. Reduced morning serum corticosterone levels in CORT mice are consistent with the reported adrenal suppression at this sampling time (21), and total body weight was unaffected by GC treatment as previously described (19). The forward-feedback induction of 11β-HSD1 mRNA and activity by GCs in unwounded skin is also in agreement with our previous observations in human skin (15) and may exacerbate the 11β-HSD1–mediated effects observed in unwounded CORT mouse skin. Our results demonstrate that 11β-HSD1 inhibition prevents GC-mediated reductions in epidermal cellularity and dermal area, despite sustained exposure to systemic GC excess. Interestingly, we observed that CBX also reversed a GC-mediated induction in epidermal integrity. This is supported by studies demonstrating that exogenous GC treatment promotes epidermal keratinocyte differentiation and increases corneocyte numbers (22), reflecting a greater degree of disruption required for epidermal barrier perturbation. Conversely, endogenous GC excess or topical GC treatment causes GC-mediated decreases in epidermal integrity, indicating that model-specific differences should also be taken into consideration (23–26). Baseline TEWL (epidermal barrier function) was unaffected by CORT treatment, in agreement with previous reports (23–26). In contrast to our findings, other studies reported impaired barrier recovery following endogenous GC excess and GC treatment, although this was associated with impaired barrier integrity (23, 25, 26). In our model, integrity and barrier recovery were both stimulated by systemic GC excess. This may be due to differences in GC potency, formulation, treatment duration, administration, strain, sex, age, diet, or environment. Although integrity appeared to be 11β-HSD1 mediated, the modest induction in barrier recovery was not reversed by CBX. This may be due to regulation by other hypothalamic-pituitary-adrenal axis pathways such as β2-adrenergic receptor signaling (27). Between days 0 and 4, wound closure was similar between all groups, suggesting that the initial contractile phase of WH (28) was unaffected. We observed termination of WH kinetics from day 4 postwounding in CORT mice, supported by previous reports of GC-mediated impairment of keratinocyte proliferation, migration, and increased epidermal differentiation (29, 30). Strikingly, our results demonstrate that CBX treatment improved WH in CORT mice. The lack of complete WH rescue by CBX may be due to exposure to serum corticosterone before cellular compartmentalization is fully restored. Others have also demonstrated benefits of 11β-HSD1 blockade on cutaneous WH in animal models of metabolic disease and endogenous GC excess (31, 32), but 11β-HSD1 inhibition, inflammatory effects, and mechanistic insights were lacking in these studies. Our findings indicate that the cutaneous side effects of GC excess are mediated through increased 11β-HSD1 substrate availability as 11β-HSD1 activity was not further increased by CORT treatment and CBX treatment did not affect skin thickness, epidermal integrity, or WH in control mice not exposed to systemic GC excess. This suggests that 11-dehydrocorticosterone availability is insufficient in young, healthy mice to regulate skin function. Recent reports have raised concerns over increased inflammation as a consequence of 11β-HSD1 blockade (33–36). We found no evidence of this our model; there was no exacerbation of IL-1β, tumor necrosis factor α, or IL-6 mRNA expression during early WH and no delay in resolution of these proinflammatory cytokines in CBX mice. On the contrary, IL-6 expression increased during CORT mouse WH. This is supported by reports of proinflammatory GC actions (37, 38), indicating greater regulatory complexity than previously thought (39–41). KGF is a key factor that promotes wound reepithelialization and is indispensable to the WH process (30, 42, 43). We found a lack of induction of KGF at day 4 postwounding in CORT mice (coinciding with the cessation of wound closure), which was restored by CBX. The induction of collagen type I and III mRNA during the granulation phase of WH were attenuated in CORT mice, consistent with previous reports (30, 44), and were also normalized by CBX, further supported by our histological findings. MMP-9 and TIMP-1 mRNA expression were elevated at day 9 postwounding in CORT mice and were normalized by CBX treatment. Although the underlying mechanisms remain poorly understood, elevated MMP-9 and TIMP-1 mRNA are associated with slower healing in mouse wounds (45), and MMP-9 is elevated in diabetic foot ulcers (46). GCs are able to activate the mineralocorticoid receptor (MR) with strong affinity, and recent work by Boix et al. (47) elegantly demonstrated that MR epidermal knockout mice were resistant to GC-induced epidermal thinning. Differentiating between GC receptor and MR-mediated effects was beyond the scope of the current study but is an area of ongoing interest. CBX has been used extensively in human and mammalian models. Administered systemically, it also inhibits 11β-HSD2 (which conducts the opposing reaction to 11β-HSD1), resulting in renal mineralocorticoid excess due to GC-mediated MR activation (48). However, 11β-HSD2 is not expressed in unwounded mouse skin or during WH (13), and topical administration limits the risk of systemic exposure. CBX is also able to block gap junctions but at several orders of magnitude less potently than 11β-HSD1 (48). Furthermore, we observed no difference between CBX and VEH mice not exposed to CORT, suggesting our findings are most likely due to cutaneous 11β-HSD1 inhibition. In summary, we report the ability of topical 11β-HSD1 inhibition to limit cutaneous side effects of systemic GC excess, including skin thinning and delayed WH. Our findings may improve the safety profile of systemic steroids and the prognosis of chronic wounds, particularly those associated with elevated circulating GC levels (49). Abbreviations: 11β-HSD1 11β-hydroxysteroid dehydrogenase type 1 CBX carbenoxolone cDNA complementary DNA CORT corticosterone GC glucocorticoid H6PDH hexose-6-phosphate dehydrogenase IL interleukin KGF keratinocyte growth factor MMP matrix metalloproteinase MR mineralocorticoid receptor mRNA messenger RNA TEWL transepidermal water loss TIMP tissue inhibitor of matrix metalloproteinase VEH vehicle WH wound healing. Acknowledgments Financial Support: This work was supported by Department of Defence Grant W81XWH-11-2-0189 (to P.M.E). Author Contributions: A.T., Y.U., and W.M.H. designed the research study. A.T. performed the experiments, analyzed the data, and prepared the manuscript. M.H. prepared tissue sections for histology. T.M. and P.M.E. contributed essential equipment. All authors evaluated and approved the final manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Overman RA, Yeh JY, Deal CL. 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EndocrinologyOxford University Press

Published: Jan 1, 2018

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