17β-Estradiol Inhibits Wound Healing in Male Mice via Estrogen Receptor-α
2010; Elsevier BV; Volume: 176; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.090432
ISSN1525-2191
AutoresStephen C. Gilliver, Elaine Emmerson, Laura Campbell, Pierre Chambon, Matthew J. Hardman, Gillian S. Ashcroft,
Tópico(s)Tendon Structure and Treatment
ResumoAlthough estrogens have long been known to accelerate healing in females, their roles in males remain to be established. To address this, we have investigated the influence of 17β-estradiol on acute wound repair in castrated male mice. We report that sustained exposure to estrogen markedly delays wound re-epithelialization. Our use of hairless mice revealed this response to be largely independent of hair follicle cycling, whereas other studies demonstrated that estrogen minimally influences wound inflammation in males. Additionally, we report reduced collagen accumulation and increased gelatinase activities in the wounds of estrogen-treated mice. Increased wound matrix metalloproteinase (MMP)-2 activity in these animals may i) contribute to their inability to heal skin wounds optimally and ii) stem, at least in part, from effects on the overall levels and spatial distribution of membrane-type 1-MMP and tissue inhibitor of MMP (TIMP)-3, which respectively facilitate and prevent MMP-2 activation. Using mice rendered null for either the α or β isoform of the estrogen receptor, we identified estrogen receptor-α as the likely effector of estrogen’s inhibitory effects on healing. Although estrogens have long been known to accelerate healing in females, their roles in males remain to be established. To address this, we have investigated the influence of 17β-estradiol on acute wound repair in castrated male mice. We report that sustained exposure to estrogen markedly delays wound re-epithelialization. Our use of hairless mice revealed this response to be largely independent of hair follicle cycling, whereas other studies demonstrated that estrogen minimally influences wound inflammation in males. Additionally, we report reduced collagen accumulation and increased gelatinase activities in the wounds of estrogen-treated mice. Increased wound matrix metalloproteinase (MMP)-2 activity in these animals may i) contribute to their inability to heal skin wounds optimally and ii) stem, at least in part, from effects on the overall levels and spatial distribution of membrane-type 1-MMP and tissue inhibitor of MMP (TIMP)-3, which respectively facilitate and prevent MMP-2 activation. Using mice rendered null for either the α or β isoform of the estrogen receptor, we identified estrogen receptor-α as the likely effector of estrogen’s inhibitory effects on healing. Although anecdotal evidence has long suggested that differences exist in the abilities of females and males (particularly the elderly) to heal acute wounds, only recently have they been substantiated by published research. Indeed, it was observed sex differences in key parameters such as restoration of the basement membrane1Ashcroft GS Mills SJ Androgen receptor-mediated inhibition of cutaneous wound healing.J Clin Invest. 2002; 110: 615-624Crossref PubMed Scopus (236) Google Scholar and elastin regeneration2Ashcroft GS Kielty CM Horan MA Ferguson MW Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAs and proteins in acute cutaneous wounds of healthy humans.J Pathol. 1997; 183: 80-89Crossref PubMed Scopus (70) Google Scholar that previously encouraged us to make detailed comparisons of healing in males and females. We discovered that, although repair is broadly similar in intact (young) male and female mice, castrated males heal acute skin wounds far better than do their ovariectomized female counterparts.3Gilliver SC Ruckshanthi JP Hardman MJ Nakayama T Ashcroft GS Sex dimorphism in wound healing: the roles of sex steroids and macrophage migration inhibitory factor.Endocrinology. 2008; 149: 5747-5757Crossref PubMed Scopus (75) Google Scholar Furthermore, males and females differed in their responsiveness to macrophage migration inhibitory factor (MIF): a potent inhibitor of repair in females, in males it has minimal influence. These studies encouraged us to conclude that sex differences in the responses to cutaneous injury do exist but that they are masked in young individuals by the combined actions of gonadal sex steroids. In males, testosterone and its more potent metabolite 5α-dihydrotestosterone inhibit repair1Ashcroft GS Mills SJ Androgen receptor-mediated inhibition of cutaneous wound healing.J Clin Invest. 2002; 110: 615-624Crossref PubMed Scopus (236) Google Scholar, 4Gilliver SC Ashworth JJ Mills SJ Hardman MJ Ashcroft GS Androgens modulate the inflammatory response during acute wound healing.J Cell Sci. 2006; 119: 722-732Crossref PubMed Scopus (107) Google Scholar; in females, estrogens such as 17β-estradiol accelerate healing.5Ashcroft GS Dodsworth J van Boxtel E Tarnuzzer RW Horan MA Schultz GS Ferguson MW Estrogen accelerates cutaneous wound healing associated with an increase in TGF-β1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (458) Google Scholar, 6Ashcroft GS Greenwell-Wild T Horan MA Wahl SM Ferguson MW Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response.Am J Pathol. 1999; 155: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar Although the effects of estrogens on female cutaneous physiology are well characterized, their roles in males are poorly understood. A handful of studies have sought to address this. In a group of aged males, locally administered 17β-estradiol was shown to reduce macroscopically determined day 7 wound areas in an excisional wounding model.6Ashcroft GS Greenwell-Wild T Horan MA Wahl SM Ferguson MW Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response.Am J Pathol. 1999; 155: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar It was recently shown that an overwhelming majority of genes displaying different wound expression between young and elderly human males are subject to estrogenic control.7Hardman MJ Ashcroft GS Estrogen, not intrinsic aging, is the major regulator of delayed human wound healing in the elderly.Genome Biol. 2008; 9: R80Crossref PubMed Scopus (85) Google Scholar In a separate study, thrice-weekly application of 17β-estradiol to sun-protected skin in aged males induced the synthesis of collagen I; increased dermal collagen bundle thickness and density; and stimulated keratinocyte proliferation.8Son ED Lee JY Lee S Kim MS Lee BG Chang IS Chung JH Topical application of 17β-estradiol increases extracellular matrix protein synthesis by stimulating TGF-α signaling in aged human skin in vivo.J Invest Dermatol. 2005; 124: 1149-1161Crossref PubMed Scopus (97) Google Scholar Although these and other studies have provided useful insights, little is yet known about the healing properties of i) systemic and ii) prolonged estrogen treatment. Having previously reported preliminary evidence that systemic 17β-estradiol treatment may impair cutaneous wound healing in castrated male mice,3Gilliver SC Ruckshanthi JP Hardman MJ Nakayama T Ashcroft GS Sex dimorphism in wound healing: the roles of sex steroids and macrophage migration inhibitory factor.Endocrinology. 2008; 149: 5747-5757Crossref PubMed Scopus (75) Google Scholar we aimed with the present study to fully characterize the effects of 17β-estradiol on the healing of acute wounds in males and to delineate the mechanisms underpinning any identified responses. Because estrogens are well-known to influence the cycling of hair follicles,9Ohnemus U Uenalan M Inzunza J Gustafsson JA Paus R The hair follicle as an estrogen target and source.Endocr Rev. 2006; 27: 677-706Crossref PubMed Scopus (137) Google Scholar which themselves were recently shown to be beneficial to repair,10Langton AK Herrick SE Headon DJ An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution.J Invest Dermatol. 2008; 128: 1311-1318Crossref PubMed Scopus (146) Google Scholar the contribution of hair to estrogen-impaired healing provided our initial focus. We report that estrogen treatment of castrated mice significantly retards wound re-epithelialization in both hairless (hr/hr) mice and strain-matched controls, confirming that systemic estrogen treatment does indeed inhibit repair and suggesting that the presence of cycling hair follicles is not critical to this response. Subsequent studies identified estrogen receptor (ER)-α as the likely effector of estrogenic inhibition and highlighted the potential involvement of increased matrix metalloproteinase (MMP)-2 activity in the reduced wound accumulation of collagen that we observed in estrogen-treated mice. All animal studies were approved by both the University of Manchester Institutional Animal Use Committee and the U.K. Government Home Office and all procedures performed in accordance with the Home Office regulations relating to animal care. Our wounding/estrogen treatment protocols are summarized in Table 1.Table 1In Vivo Estrogen Treatment ProtocolsDaySexTypeGonads−140+3+10MMF1CSX←————————————————→MMF1CSX←———————————→←————————————→MMF1Intact←————————————————→Mhr/hrCSX←————————————————→FBALB/cOVX←————————————————→FBALB/cOVX←———→MWT*Mixed-strain littermates of ER-α−/− and ER-β−/− mice.CSX←————————————————→MER-α−/−CSX←————————————————→MER-β−/−CSX←————————————————→MWT*Mixed-strain littermates of ER-α−/− and ER-β−/− mice.CSX←———→MER-α−/−CSX←———→MER-β−/−CSX←———→Arrows indicate duration of treatment with 17β-estradiol (21-day slow-release 0.05 mg pellet; s.c.) Chronic treatment, commencing on day −14/acute, on day 0.Castration/ovariectomy performed on day −14.Mice wounded on day 0.Wounds excised on days +3/+10 as indicated.* Mixed-strain littermates of ER-α−/− and ER-β−/− mice. Open table in a new tab Arrows indicate duration of treatment with 17β-estradiol (21-day slow-release 0.05 mg pellet; s.c.) Chronic treatment, commencing on day −14/acute, on day 0. Castration/ovariectomy performed on day −14. Mice wounded on day 0. Wounds excised on days +3/+10 as indicated. Six-week-old male MF1 mice were castrated. A subset additionally received 0.05 mg 17β-estradiol pellets (Innovative Research of America, Sarasota, FL), s.c. implanted using a trocar at the time of castration. Two weeks subsequently, the mice were anesthetized with isoflurane, and their dorsa shaved and cleansed with ethanol. Full-thickness 1-cm incisions were made 1 cm either side of the midline. (In a subset of mice-used as a source of day 10 wounds-estrogen pellets implanted at the time of castration were replaced with fresh 17β-estradiol pellets.) All mice received immediate analgesia in the form of buprenorphine (0.1 mg/kg) and were housed individually. Three or 10 days postwounding, the wounds were excised and bisected. Eight-week-old intact (noncastrated) male MF1 mice were wounded (as described above), and their wounds were excised and bisected 3 days postwounding. Six-week-old male hr/hr mice (on an MF1 background) were castrated as above. A subset received 0.05 mg 17β-estradiol pellets at the time of castration (see above). Two weeks later, the mice were wounded (see above). Wounds were excised and bisected 3 days postwounding. Six-week-old female BALB/c mice were ovariectomized. A subset received 0.05 mg 17β-estradiol pellets at the time of ovariectomy (see above). Two weeks subsequently, the mice were wounded (see above). A further subset of ovariectomized mice not previously treated with estrogen was implanted with 0.05 mg 17β-estradiol pellets at the time of wounding. Wounds were excised and bisected 3 days postwounding. Six-week-old male mixed-strain ER-α null (αKO)11Dupont S Krust A Gansmuller A Dierich A Chambon P Mark M Effect of single and compound knockouts of estrogen receptors α (ERα) and β (ERβ) on mouse reproductive phenotypes.Development. 2000; 127: 4277-4291Crossref PubMed Google Scholar and ER-β null (βKO)11Dupont S Krust A Gansmuller A Dierich A Chambon P Mark M Effect of single and compound knockouts of estrogen receptors α (ERα) and β (ERβ) on mouse reproductive phenotypes.Development. 2000; 127: 4277-4291Crossref PubMed Google Scholar mice and wild-type littermates were castrated as above. Subsets of αKO, βKO, and wild-type animals received 0.05 mg 17β-estradiol pellets at the time of castration (see above). Two weeks subsequently, the mice were wounded (see above). Further subsets of castrated αKO, βKO, and wild-type animals not previously treated with estrogen were implanted with 0.05 mg 17β-estradiol pellets at the time of wounding. Wounds were excised and bisected 3 days postwounding. One-half of each wound was processed in 8% formaldehyde-based fixative; the other snap-frozen in liquid nitrogen and stored at −80°C. Unwounded dorsal skin was similarly processed in fixative. Sera were isolated from blood obtained by cardiac puncture. Fixed tissue was embedded in paraffin. Five-micrometer-thick histological sections prepared from the center of each wound were stained with hematoxylin and eosin (H&E) and subjected to immunohistochemistry using polyclonal IgGs raised against MMP-2, ER-α, arginase I (Insight, Wembley, U.K.), ER-β, tissue inhibitor of MMP (TIMP)-3 (Abcam, Cambridge, U.K.), MIF and tumor necrosis factor (TNF)-α (R&D Systems, Abingdon, U.K.), and monoclonal IgGs raised against Ly6G, Mac3, and CD74 (BD Biosciences, Oxford, U.K.), and membrane-type (MT)1-MMP (Abcam). Bound IgG was subsequently detected using a colorimetric Vectastain avidin-biotin complex peroxidase kit (Vector Laboratories, Peterborough, U.K.) in conjunction with the enzyme substrate Novared (Vector Laboratories). Tissue sections treated with PBS in place of primary IgG (negative controls) yielded no signal. Wound areas, extent of re-epithelialization, cell numbers/unit area, the thicknesses of individual skin layers, and immunostained areas were lastly quantified using Image-Pro Plus software (Media Cybernetics, Marlow, U.K.).5Ashcroft GS Dodsworth J van Boxtel E Tarnuzzer RW Horan MA Schultz GS Ferguson MW Estrogen accelerates cutaneous wound healing associated with an increase in TGF-β1 levels.Nat Med. 1997; 3: 1209-1215Crossref PubMed Scopus (458) Google Scholar Paraffin-embedded tissue sections were immersed in 0.1% Sirius red (in picric acid) for 1 hour and then washed in acidified water. When examined under plane-polarized light, larger collagen fibers appear red, orange, or yellow and thinner ones green. This birefringence is highly specific to collagen.12Junqueira LCU Bignolas G Brentani RR Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.Histochem J. 1979; 11: 447-455Crossref PubMed Scopus (1921) Google Scholar Epidermal MMP-2 and MT1-MMP staining intensities were compared by four-grade semiquantitative scoring: 0, no staining; 1, weak; 2, moderate; and 3, strong. Data are presented as box-and-whisker plots. Total protein was extracted from unwounded skin and wound tissue using denaturing, nonreducing buffer. Fifty micrograms of individual and pooled protein samples (n = 5 per treatment group) was tested for gelatinase activities as described previously.13Heussen C Dowdle EB Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.Anal Biochem. 1980; 102: 196-202Crossref PubMed Scopus (1838) Google Scholar Briefly, an acrylamide gel containing 0.5 mg/ml gelatin was prepared. Test samples were separated by SDS-PAGE under nonreducing conditions alongside human MMPs 2 and 9, purified from stably transfected mouse myeloma cells.14Murphy G Willenbrock F Ward RV Cockett MI Eaton D Docherty AJ The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases.Biochem J. 1992; 283: 637-641Crossref PubMed Scopus (244) Google Scholar Following separation, gels were washed in 2.5% Triton X-100 (Sigma-Aldrich, Poole, U.K.) and, briefly, distilled H2O, before being incubated for 16 hours at 37°C in assay buffer (100 mmol/L Tris, 30 mmol/L CaCl2, 0.02% (w/v) sodium azide, 0.05% (v/v) Brij 35, pH 7.9). Finally, the gels were stained with Coomassie Brilliant Blue G for 20 minutes and then destained in 1% acetic acid and 30% methanol. Band intensities were quantified using Image-Pro Plus software. Samples separated by reducing SDS-PAGE were stained with Coomassie to serve as a loading control. Levels of 17β-estradiol in serum samples isolated from MF1 and hr/hr mice were measured using an enzyme immunoassay kit (MP Biomedicals, Cambridge, U.K.), according to the manufacturer’s guidelines. Total protein was extracted from unwounded skin and wound tissue using an SDS-based detergent buffer. One microgram of individual and pooled protein samples (n = 5 per treatment group) was tested as described previously.15Ashcroft GS Horan MA Herrick SE Tarnuzzer RW Schultz GS Ferguson MW Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans.Cell Tissue Res. 1997; 290: 581-591Crossref PubMed Scopus (153) Google Scholar Briefly, samples separated by denaturing, reducing SDS-PAGE, were blotted to 0.2-μm nitrocellulose membranes (Bio-Rad, Hemel Hempstead, UK). Membranes were blocked for 16 hours in Tris-buffered saline (containing 0.1% (v/v) Tween-20 and 5% (w/v) nonfat dry milk). Following sequential 1-hour washes in primary and peroxidase-linked secondary antibodies, antigen binding was probed using ECL Plus detection reagent (GE Healthcare, Little Chalfont, U.K.). Polyclonal IgGs raised against type I collagen (Millipore, Watford, U.K.), ER-α (Insight), ER-β, MT1-MMP, and TIMP-3 (Abcam), and MMP-2 (Merck, Nottingham, UK), and a monoclonal IgG raised against β-actin (used as a loading control) (Sigma-Aldrich) were used in conjunction with sheep anti-mouse and donkey anti-rabbit secondary IgGs (both GE Healthcare). Signal intensities were determined densitometrically. Total RNA was extracted from frozen day 3 wound tissue using TRIzol (Invitrogen, Paisley, U.K.) in conjunction with a PureLink RNA Mini Kit (Invitrogen). cDNA was synthesized from RNA (1 μg) and analyzed by quantitative real-time PCR (qPCR). Each test sample was serially diluted over 3 orders of magnitude and PCR performed using the primers listed in Table 2 and the combination of a SYBR Green I core kit (Eurogentec, Romsey, U.K.) and an Opticon qPCR thermal cycler (Genetic Research Instrumentation, Braintree, U.K.). The specificity of product amplification was assessed through melting curve analysis.Table 2Primer Sequences and Product Sizes for qPCR-Amplified GenesTarget geneForward primer sequenceReverse primer sequenceProduct size (bp)Cd745′-ATGACCCAGGACCATGTGAT-3′5′-ATCTTCCAGTTCACGCCATC-3′128Col1a1*Encodes the α1 chain of type I collagen.5′-GAGCGGAGAGTACTGGATCG-3′5′-GTTCGGGCTGATGTACCAGT-3′142Col1a2†Encodes the α2 chain of type I collagen.5′-GTGTTCAAGGTGGCAAAGGT-3′5′-GAGACCGAATTCACCAGGAA-3′131Krt15′-CGTGGTGAGAAAGCACTCAA-3′5′-TGCACTCTCCAGACATCCTG-3′199Krt145′-CCTCTGGCTCTCAGTCATCC-3′5′-TGAGCAGCATGTAGCAGCTT-3′144Krt165′-GTGGTTTTGGTGCTGGTCTT-3′5′-GTACCAGTCCCGGATCTTCA-3′179Mif5′-AGGCCACACAGCAGCTTACT-3′5′-AGCTCATGACTTTTAGCGGC-3′112Mmp1a5′-GTGCTCTCCTTCCACAGAGG-3′5′-GGTCCACGTCTCATCAAGGT-3′135Mmp1b5′-CCTTCCTTTGCTGTTGCTTC-3′5′-ATCACCTCCTTGCCATTCAC-3′162Mmp25′-CTTCGCTCGTTTCCTTCAAC-3′5′-ATGTCAGACAACCCGAGTCC-3′78Mmp135′-AGTTGACAGGCTCCGAGAAA-3′5′-GGCACTCCACATCTTGGTTT-3′105Timp15′-TCCCCAGAAATCAACGAGAC-3′5′-CATTTCCCACAGCCTTGAAT-3′88Timp25′-CACAGACTTCAGCGAATGGA-3′5′-CCAGCATGAGACCTCACAGA-3′124Timp35′-TTATCCCATTGGGGCATTTA-3′5′-TTGCTGCCTTTGACTGATTG-3′161Tnfa5′-TCTCAGCCTCTTCTCATTCCTGCT-3′5′-AGAACTGATGAGAGGGAGGCCATT-3′124Rn18s‡Encodes the 18S ribosomal RNA.5′-AGTCCCTGCCTTTGTACACA-3′5′-GATCCGAGGGCCTCACTAAC-3′69Gapdh§Encodes glyceraldehyde-3-phosphate dehydrogenase.5′-TGCACCACCAACTGCTTAGC-3′5′-GGCATGGACTGTGGTCATGAG-3′87Hprt¶Encodes hypoxanthine guanine phosphoribosyl transferase.5′-TGCTCGAGATGTCATGAAGG-3′5′-AATCCAGCAGGTCAGCAAAG-3′95Ywhaz∥Encodes tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ.5′-TTCTTGATCCCCAATGCTTC-3′5′-TTCTTGTCATCACCAGCAGC-3′107* Encodes the α1 chain of type I collagen.† Encodes the α2 chain of type I collagen.‡ Encodes the 18S ribosomal RNA.§ Encodes glyceraldehyde-3-phosphate dehydrogenase.¶ Encodes hypoxanthine guanine phosphoribosyl transferase.∥ Encodes tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ. Open table in a new tab Peritoneal macrophages were isolated from 8-week-old male mice by i.p. lavage with sterile ice-cold PBS and subsequently purified using Ficoll-Paque Premium (GE Healthcare). Cells were pooled and suspended at a concentration of 2 × 106 cells/ml in Phenol-Red-free Dulbecco’s modified Eagle’s medium (supplemented with 5% charcoal-stripped fetal calf serum), before being treated with bacterial lipopolysaccharide (LPS) (1 μg/ml) for 2 hours. Thus-activated cells were then treated with 100 nmol/L 17β-estradiol (Sigma-Aldrich), 1 μmol/L PPT (Tocris Bioscience, Bristol, U.K.), or 1 μmol/L diarylpropionitrile (Tocris Bioscience), or left untreated, for a further 3 hours. Total cellular RNA and protein samples were subsequently isolated and analyzed by qPCR and immunoblotting/zymography. Male and female mouse neonates were euthanized by decapitation and their trunk skin removed and rinsed in 70% ethanol.16Yuspa SH Harris CC Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro.Exp Cell Res. 1974; 86: 95-105Crossref PubMed Scopus (380) Google Scholar The epidermis was loosened from the dermis through overnight incubation in 5 mg/ml dispase I (Sigma-Aldrich) at 4°C. Primary keratinocytes were isolated from the epidermis according to the protocol provided by CELLnTEC. Briefly, skin was washed in CnT-57 medium (CELLnTEC, Bern, Switzerland) to remove excess dispase and the epidermis separated from the dermis with forceps. The epidermal sheet was placed on a drop of TrypLE Select (Invitrogen) in a petri dish with the basal layer facing downward and incubated at room temperature for 30 minutes. The epidermis was gently rubbed on the bottom of the dish to disturb basal cells. The resulting cell suspension was centrifuged, and the thus-pelleted cells resuspended in CnT-57 medium before being seeded to cell culture flasks coated with collagen IV (BD Biosciences). Pelage hairs were removed from dorsal skin of sacrificed adult male mice by wax-based depilation (using Veet (Reckitt Benckiser, Slough, U.K.)). The skin was excised, and the epidermis and dermis separated following overnight incubation in 0.25% trypsin/EDTA (Invitrogen) at 4°C. Isolation and culture of primary keratinocytes were performed as described previously.17Hager B Bickenbach JR Fleckman P Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976Crossref PubMed Scopus (107) Google Scholar The migration of scratch-wounded mouse epidermal keratinocytes on type I collagen was assessed as described previously.18Gilliver SC Ruckshanthi JP Hardman MJ Zeef LA Ashcroft GS 5α-Dihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization.J Pathol. 2009; 217: 73-82Crossref PubMed Scopus (59) Google Scholar The influence of 17β-estradiol at three concentrations (1, 100, and 1000 nmol/L) on cell migration was evaluated. The proliferation of mouse epidermal keratinocytes was assessed using the colorimetric MTS-based CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Southampton, U.K.), according to the manufacturer’s guidelines and as described previously.18Gilliver SC Ruckshanthi JP Hardman MJ Zeef LA Ashcroft GS 5α-Dihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization.J Pathol. 2009; 217: 73-82Crossref PubMed Scopus (59) Google Scholar The effects on cell proliferation of 17β-estradiol (1, 100, and 1000 nmol/L) were evaluated. Epidermal keratinocytes were seeded to type I collagen-coated 6-well plates and cultured to ∼ 90% confluence, before being treated with 17β-estradiol (1 or 1000 nmol/L). Total cellular RNA and protein samples were isolated 24 hours subsequently and were respectively analyzed by qPCR and immunoblotting. Simfit (version 6.7.15) (William Bardsley, University of Manchester, Manchester, U.K.) was used to test for statistical significance by one-way and two-way analysis of variance, unpaired Student’s t-tests and, for nonparametric data, systematic, pairwise Mann-Whitney U-tests with Bonferroni correction. Posthoc testing was performed using Bonferroni-corrected unpaired Student’s t-tests. To investigate the roles of estrogen in the repair of acute skin wounds in males, we initially treated castrated male mice systemically with 17β-estradiol. Corroborating our previous findings,3Gilliver SC Ruckshanthi JP Hardman MJ Nakayama T Ashcroft GS Sex dimorphism in wound healing: the roles of sex steroids and macrophage migration inhibitory factor.Endocrinology. 2008; 149: 5747-5757Crossref PubMed Scopus (75) Google Scholar 17β-estradiol markedly impaired healing (assessed in terms of day 3 and 10 wound areas) (Figure 1, A and B). Moreover, it greatly retarded wound re-epithelialization (Figure 1C). To determine whether exogenous estrogen could influence healing against a background of normal circulating steroid levels, we similarly treated intact male mice with 17β-estradiol. We found that 17β-estradiol increased day 3 wound areas in intact animals (Figure 1, D and E) without significantly affecting the extent of re-epithelialization at that time point (Figure 1F). To probe the potential involvement of hair follicles in this apparent inhibition of repair by estrogens, we likewise treated hr/hr mice (strain-matched) with 17β-estradiol. Intriguingly, estrogen delayed re-epithelialization in these animals without obviously influencing day 3 wound areas (Figure 1, G–I). This suggests that hair follicles do not contribute significantly to estrogenic inhibition of re-epithelialization but may play roles in additional repair processes that estrogen controls. As others have previously shown,10Langton AK Herrick SE Headon DJ An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution.J Invest Dermatol. 2008; 128: 1311-1318Crossref PubMed Scopus (146) Google Scholar healing was significantly delayed in hr/hr mice relative to controls (Figure 1, B and H). We subsequently found that three concentrations of 17β-estradiol did not influence the migration of scratch-wounded epidermal keratinocytes cultured from male mice (Figure 1J), or the proliferation of such cells (Figure 1K), suggesting that the inhibitory effect of estrogen in vivo is realized indirectly or via an alternative mechanism. The effectiveness of our in vivo estrogen treatment protocol was determined by enzyme immunoassay of isolated sera (Table 3).Table 3Serum 17β-Estradiol Levels following Systemic ReplacementMF1hr/hrCSXCSX + E2CSXCSX + E217β-Estradiol (ng/ml)18.15 (±6.10)63.21 (±9.48)*P < 0.01;19.27 (±4.89)118.8 (±50.6)†P < 0.05 (versus CSX). n = 5 per treatment group.Data are presented as mean (±SD), measured 3 days post-wounding. Statistical significance was determined by unpaired Student’s t-tests:* P < 0.01;† P < 0.05 (versus CSX). n = 5 per treatment group. Open table in a new tab Data are presented as mean (±SD), measured 3 days post-wounding. Statistical significance was determined by unpaired Student’s t-tests: In an attempt to identify the mechanisms by which systemically administered estrogen impairs healing in castrated male mice, we examined its effects on wound inflammation. We found day 3 wound numbers of Ly6G-positive neutrophils to be comparable in estrogen-treated and untreated mice (Figure 2A). Similarly, day 3 wound Mac3-positive macrophage population sizes (Figure 2B) were unaffected by estrogen, although greater numbers of arginase 1-positive (alternatively activated (AA)) macrophages infiltrated the wounds of estrogen-treated animals than those of untreated controls (Figure 2C). Local levels of the proinflammatory cytokine TNF-α did not differ significantly between estrogen-treated mice and controls (Figure 2D). Because estrogen exerts much of its influence on healing in female mice through its regulation of MIF expression,19Hardman MJ Waite A Zeef L Burow M Nakayama T Ashcroft GS Macrophage migration inhibitory factor: a central regulator of wound healing.Am J Pathol. 2005; 167: 1561-1574Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar we proceeded to study its effects on MIF and CD74, a putative MIF receptor.20Leng L Metz CN Fang Y Xu J Donnelly S Baugh J Delohery T Chen Y Mitchell RA Bucala R MIF signal transduction initiated by binding to CD74.J Exp Med. 2003; 197: 1467-1476Crossref PubMed Scopus (787) Google Scholar Although treatment with 17β-estradiol did not influence day 3 wound MIF levels (Figure 2E), it did elicit a reduction in wound numbers of CD74-positive cells (Figure 2F), suggesting that estrogens may dampen the response to MIF in males. Having previously shown that healing in castrated male MIF null mice is unresponsive to exogenous MIF,3Gilliver SC Ruckshanthi JP Hardman MJ Nakayama T Ashcroft GS Sex dimorphism in wound healing: the roles of sex steroids and macrophage migration inhibitory factor.Endocrinology. 2008; 149: 5747-5757Crossref PubMed Scopus (75) Google Scholar our present findings reinforce our conclusion that MIF, so detrimental to repair in females, is of minimal influence in males. In further studies, exogenous 17β-estradiol did not influence wound expression of the Tnfa, Mif, or Cd74 genes (Figure 2G). And, although it tended t
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