Nesprin-2 declines during skin aging and its loss negatively impacts fibroblast and keratinocyte behavior in vitro R Maidhof, Y Chen, U Santhanam and J Lyga Global Research and Development, Avon Products Inc., Suffern, NY Nesprins are large multi-domain proteins that form a physical link between the cytoskeleton and nuclear membrane resulting in a stable physical connection which is essential for a wide range of cellular functions including nuclear positioning, cell polarization, motility, and

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... echanosensing. Mutations in the nesprins impair intracellular force transmission and are linked to muscular dystrophies and cardiomyopathies. In this study we investigated the expression and functional role of nesprin-2, the predominant isoform in human skin. The goal is to determine if nesprin-2 expression is affected by chronologic and photo aging and the functional consequences of nesprin-2 downregulation on human skin fibroblasts and keratinocytes. We found that nesprin-2 staining decreased in skin biopsies from sun-exposed regions versus sun-protected controls and that nesprin-2 expression was significantly lower in older-age skin cells versus young controls (80% decrease, p<0.01). To investigate functional implications of nesprin-2 downregulation we used small interfering RNA to knockdown nesprin-2 expression in fibroblasts and keratinocytes. Knockdown was confirmed by nesprin-2 protein staining and dramatically altered the actin and microtubulin cytoskeletal structure. Furthermore, the size of nuclei tended to increased (12.9%, p=0.07, n=30-36) and the circularity of nuclei decreased significantly (2.2%, p=0.005, n=30-36) in nesprin-2 knockdown cells. Knockdown resulted in misaligned collagen deposition in 2D fibroblast culture, reduced contractility in a fibroblast populated 3D collagen gel (20% less contraction, p<0.05, n=3), and slower migration/proliferation in a wound healing assay for both fibroblasts (46% less closure, p=0.003, n=10) and keratinocytes (55% less closure, p=0.002, n=10). Our results demonstrate that nesprin-2 downregulation, which occurs during both chronological and photo aging, has an important role in skin cell functionality in vitro and may be a relevant cellular target for treatment of aging skin conditions in vivo. Wound healing is a complex process that is influenced by multiple factors, including the wound's immune environment. Our previous studies showed IFN-γ's inhibitory effect on keratinocyte migration [JID 134: S133, 2013]. Herein, we investigated the mechanism by which IFN-γ impaired keratinocyte migration, by examining its effects on SH2-containing inositol 5'-phosphatase (SHIP-2) and phospho-cofilin (p-cofilin). SHIP-2 is a docking protein for the cytoskeleton and p-cofilin is an inactive form of cofilin, the enzyme which severs actin polymers into monomers; upregulation of these proteins has been associated with impaired keratinocyte migration. We also examined the effects of IFN-γ on keratinocyte proliferation. SHIP-2, cofilin and p-cofilin expression was assessed by western blot in HaCaTs, an adult, immortalized keratinocyte cell line, treated without or with IFN-γ at 20 ng/mL. Flow cytometry was performed to examine changes in cell cycle; the percentage of cells in each phase was quantified (n= at least 3 for all experiments). We found that SHIP-2 and p-cofilin expression was significantly upregulated in the IFN-γ treated group relative to control (SHIP-2 increased 2.8 ±0.6 fold (p<0.05); p-cofilin increased 2.0 ±0.3 fold (p<0.05)). Cofilin expression did not significantly change. Flow cytometric analysis showed that following IFN-γ treatment, there was a significantly increased percentage of cells in G1, and a decreased percentage of cells in S and G2, relative to control. We show here that IFN-γ upregulates both SHIP-2 and p-cofilin. These findings extend our previous data regarding IFN-γ regulation of keratinocyte migration. In addition, we showed that IFN-γ inhibits keratinocyte proliferation. Both of these effects may inhibit wound healing, and suggest that IFN-γ itself, or its downstream proteins, may be therapeutic targets for influencing wound healing dynamics. 699 Vitamin D and calcium regulation of epidermal wound healing Wound healing is essential for survival. This is a multistep process involving a number of different cell types. In the skin wounding triggers an acute inflammatory response, with the innate immune system contributing both to protection against invasive organisms and to triggering the invasion of inflammatory cells into the wounded area. These cells release a variety of cytokines and growth factors that stimulate the proliferation and migration of dermal and epidermal cells to close the wound. In particular, wounding activates stem cells in the interfollicular epidermis (IFE) and hair follicles (HF) to proliferate and send their progeny to re-epithelialize the wound. b-catenin and calcium signaling are important for this activation process. . Mice lacking the CaSR and/or the VDR when placed on a low calcium diet have delayed wound healing. This is associated with a blunted innate immune and inflammatory response to wounding in the epidermis and reduced proliferation and b-catenin transcriptional activity in the cells at the leading edge of wound closure. These effects are opposite to the roles of vitamin D and calcium signaling during keratinocyte differentiation. Surprisingly, deletion of the VDR coactivator Med1 accelerates wound healing, associated with increased proliferation and b-catenin signaling. These data suggest that vitamin D and calcium signaling are necessary components of the epidermal response to wounding, likely by regulating stem cell activation through increased b-catenin transcriptional activity. 700 Reconstitution of three-dimensional skin composed of keratinocytes, fibroblasts and melanocytes induced from Muse human pluripotent stem cell Mulitilineage-differentiating stress enduring (Muse) cells exist among human somatic tissues and are non-tumorigenic pluripotent stem cells that have self-renewal ability and can differentiate toward tridermic cells like as ES and iPS cells. We have reported in SID2014 that functional melanocytes were inducible from Muse cells derived from human adipose tissue (Muse-MC). Muse-MC could reside in the basal layer of epidermis in reconstituted 3D cultured skin (3D skin) and expressed melanocyte related proteins. To generate 3D skin sheets composed of cells derived from Muse cells, we aimed to induce fibroblasts (FB) and keratinocytes (KC) from Muse cell. We cultured Muse cells obtained from adipose tissue (hASC-Muse) in medium containing 0.3 mM ascorbic acid and 10 ng/mL TGFβ2 and observed that FB-like spindle-shaped cells after 4 weeks (Muse-FB). The Muse-FB expressed collagen 1 and 3, CD10 and CD73, representative genes of normal FB. We also generated KC from hASC-Muse by culturing with 100 μM all-trans retinoic acid and 30 ng/mL BMP4 for 5 weeks (Muse-KC). Muse-KC expressed genes related to KC such as keratin 1 and 14 and Desmoglein 3. For the next step toward future clinical usage of Muse derived skin components, we generated 3D skin using Muse-MC, -FB and -KC. A type 1 collagen layer containing Muse-FB was deposited onto insert chamber membranes and incubated for 4 days. Muse-KC and Muse-MC at the ratio of 5:1 were seeded on the collagen layer. They were incubated in keratinocyte medium for 6 days and then the Ca 2+ concentration of the media were gradually increased. The 3D skins were cultivated for another 7 days at the air-liquid interface and then fixed for evaluation. We observed the well-organized distribution of the Muse-MC, -FB and -KC in the 3D skin. These data showed that Muse cell have potentials to differentiate into major cells in human skin and suggested that Muse cells can be used to treat skin defects and to enhance wound healing. 701 High glucose environment increased thrombospondin-1 expression in keratinocytes via epigenetic regulation: Metabolic memory of impaired angiogenesis during diabetic wound healing Diabetes is an important health issue due to its increasing prevalence and association with occurrence of chronic complications. Metabolic memory described the condition in which diabetic complications still occur despite good glycemic control. Impaired healing is an important complication of diabetes associated with metabolic memory that still poses clinical challenges in many situations. In this study, we demonstrated that the expression of thrombospondin-1 (TSP1), a keratinocyte-derived anti-angiogenic molecule known to delay wound healing, is significantly increased in high glucose environment. Mechanistically, we showed that DNA hypomethylation at TSP1 promoter was responsible for increased TSP1, and this epigenetic change was induced by increased oxidative stress but not advanced glycation endproduct. Similar findings were validated in a diabetic rat model. Moreover, early antioxidant administration normalized TSP1 expression and its DNA promoter methylation status in diabetic rat skin as well as improved diabetic wound healing in vivo. Since oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes since alterations in epigenome may not be reversible by normalization of glucose levels during later stages of the disease course, contributing to occurrence of metabolic memory. More importantly, epigenetic profiling and modulation of epigenome may provide important prognostic information and offer preventive therapy, respectively, regarding complications associated with diabetes. 702 Targeted genetic alteration in hyaluronan catabolism delays wound healing in mice The breakdown and molecular size of Hyaluronan (HA) has been hypothesized to influence wound healing, but direct evidence of this has not been obtained. To test if the breakdown of HA acts as an endogenous signal of injury we generated transgenic mice that conditionally overexpressed Hyaluronidase 1 (HYAL1). When HYAL1 was constitutively expressed in early embryogenesis (EIIa/ HYAL1), these mice showed extensive degradation of HA in the dermis yet were morphologically normal. When these mice were wounded by full-thickness 6 mm punch biopsy, wound closure was delayed by approximately 2 days in EIIa/HYAL1 compared to control mice (p<0.001). Delayed repair may result from abnormal macrophage polarization in wound tissues 48 hours after wounding as we found a significantly higher M1/M2 macrophage ratio in EIIa/HYAL1 mice (p<0.05). Consistant with a role for HA fragments in this response, we showed treatment with specific sized HA fragments induced the M1 marker Nos2 but not the M2 marker Arg1 in bone marrow-derived macrophages (BMDM). Histological and flow cytometirc evaluation of the skin wounds at 96 hours showed significantly more inflammatory cell infiltration in EIIa/HYAL1 mice (p<0.05). MIP-2 mRNA and protein levels were also significantly higher in EIIa/HYAL1 (p<0.001). Furthermore, mRNA expression of CD44, a receptor of HA, was significantly higher in EIIa/HYAL1 before wounding (p<0.05) and immunohistochemistry confirmed the higher expression of CD44 in endothelial cells. Responses to HA catabolism were dependent on CD44 as the delay of wound closure was rescued by deletion of CD44. These data show that abnormal degradation of hyaluronan modulates wound healing in a CD44-dependent manner and suggests targeting this pathway may be therapeutically useful in treatment of abnormal wound repair.