Insulin-Like Growth Factor-1 Promotes Wound Healing in Estrogen-Deprived Mice: New Insights into Cutaneous IGF-1R/ERα Cross Talk  Elaine Emmerson, Laura Campbell, Faith C.J. Davies, Nina L. Ross, Gillian S. Ashcroft, Andrée Krust, Pierre Chambon, Matthew J. Hardman  Journal of Investigative Dermatology  Volume 132, Issue 12, Pages 2838-2848 (December 2012) DOI: 10.1038/jid.2012.228 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Local IGF-1 promotes cutaneous healing in estrogen-deprived mice. Local IGF-1 accelerates healing to an extent equal to systemic 17β-estradiol (+E2) replacement, quantified by (a) a significant reduction in wound area and (b) increase in re-epithelialization versus untreated ovariectomized (Ovx) mice. (c) Representative hematoxylin and eosin–stained sections of incisional wounds (day 3). Arrows indicate wound margins. IGF-1 reduces (d) wound neutrophil and (e) macrophage numbers to a similar extent as 17β-estradiol–treated wounds. Mean+SEM, n=6 per group. *P<0.05, **P<0.01. Bar=500μm. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The IGF-1 receptor (IGF-1R) antagonist JB3 differentially influences local inflammation and healing outcome depending on the route of administration. (a) IGF-1R–positive cells were significantly increased in IGF-1–treated ovariectomized (Ovx) wounds, but were unchanged with 17β-estradiol treatment (+E2). (b) Representative immunohistochemistry for wound IGF-1 receptor (IGF-1R; arrows, positive cells). Local JB3 blocks IGF-1's healing-promoting effects, whereas systemic JB3 does not, quantified by (c) wound area and (d) re-epithelialization. (e) Representative hematoxylin and eosin–stained sections of incisional wounds (day 3; arrows, wound margins). (f) Neither systemic nor local JB3 treatment altered IGF-1's anti-inflammatory potential with respect to neutrophils, whereas (g) systemic, but not local, JB3 prevented IGF-1-mediated reduction in wound macrophages. Systemic JB3 alone (g) further increased wound macrophage influx, but (f) had no effect on neutrophils. Mean+SEM, n=5–6 per group. *P<0.05. (b) Bar=25μm; (e) bar=500μm. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IGF-1 modulates local estrogen receptor (ER) expression, with IGF-1 effects on healing being partially ER dependent. (a–d) Local IGF-1 has no effect on cutaneous ERα expression but significantly increases the number of ERβ-expressing wound cells. (e–h) Representative immunohistochemistry for ERα (e, dermis; g, epidermis) and ERβ (f, dermis; h, epidermis). Arrows indicate positive cells. (i–l) ICI partially blocks the healing-promoting effects of IGF-1, quantified by (i) a significant increase in wound area and (l) macrophage recruitment versus IGF-1-treated ovariectomized (Ovx) mice. ICI had no effect on (j) re-epithelialization or (k) neutrophil influx. Mean+SEM, n=5–6 per group. *P<0.05. (e, f) Bar=25μm; (g, h) bar=50μm. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IGF-1-mediated promotion of healing in vivo requires estrogen receptor alpha (ERα). (a–c) Local IGF-1 promotes healing in ovariectomized (Ovx) wild-type (WT) littermate controls and Ovx ERβ knockout (ERβ-/-) mice, quantified by (a) a significant reduction in wound area and (c) wound macrophage recruitment compared with Ovx. Conversely, IGF-1 fails to promote healing in ERα knockout (ERα-/-) mice, quantified by (a) no change in wound area and (c) a significant increase in macrophages (c) compared with Ovx. Although IGF-1 promoted re-epithelialization in Ovx WT mice, there was no difference between Ovx and IGF-1–treated ERα-/- and ERβ-/- mice. (d) Representative hematoxylin and eosin–stained sections of incisional wounds (day 3). Arrows indicate wound margins. Mean+SEM, n=4–7 per group. *P<0.05. Bar=500μm. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cell-specific estrogen receptor alpha (ERα)-null mice reveal complex roles for IGF-1 in vivo. (a) Local IGF-1 fails to promote healing in epidermal-specific ovariectomized (Ovx) ERα knockout mice (K14;ERα-/-) while conversely enhancing healing in Ovx inflammatory cell–specific ERα knockout mice (LysM;ERα-/-), quantified by wound area. (b) IGF-1 failed to promote re-epithelialization in both Ovx K14;ERα-/- and Ovx LysM;ERα-/- mice. (c) Local IGF-1 fails to dampen wound macrophage recruitment in Ovx LysM;ERα-/- mice and actually significantly increases cell numbers in Ovx K14;ERα-/- mice. Representative day 3 (d) hematoxylin and eosin staining and (e) macrophage (Mac3) immunohistochemistry. Arrows indicate wound margins. Mean+SEM, n=5–7 per group. *P<0.05. (d) Bar=500μm; (e) bar=50μm. WT, wild type. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cell-specific roles for IGF-1 receptor (IGF-1R) and estrogen receptors (ERs) are confirmed in vitro. (a) IGF-1–promoted human keratinocyte “scratch wound” migration in vitro was inhibited by coincubation with IGF-1R antagonist but not ERα- or ERβ-selective antagonists. (b) By contrast, IGF-1–promoted human fibroblast migration was inhibited by ERα-selective antagonism, whereas IGF-1R or ERβ antagonism had no effect. Representative crystal violet–stained (c) keratinocyte and (d) fibroblast scratches. (e, f) IGF-1 downregulated lipopolysaccharide-induced proinflammatory cytokine expression in peritoneal macrophages in vitro. The ERα-selective antagonist blocked IGF-1s anti-inflammatory activity, preventing reduction in (e) macrophage migration inhibitory factor and (f) tumor necrosis factor-α expression, whereas IGF-1R- and ERβ antagonists had no effect. Mean+SEM, n=3–5 replicates per group and two individual experiments. *P<0.05, **P<0.01. Bar=400μm. Journal of Investigative Dermatology 2012 132, 2838-2848DOI: (10.1038/jid.2012.228) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions