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Montagna Symposium 2014—Skin Aging: Molecular Mechanisms and Tissue Consequences Barbara A. Gilchrest1, Judith Campisi2, Howard Y. Chang3,GaryJ.Fisher4 and Molly F. Kulesz-Martin5 Journal of Investigative Dermatology (2015) 135, 950–953; doi:10.1038/jid.2014.546

The 63rd annual Montagna Symposium theory proved extremely difficult to test that are regulated by NF-kB, focusing on on the Biology of Skin, ‘‘Skin Aging: because mutations and epimutations Lethe, which is induced by NF-kBand Molecular Mechanisms and Tissue Con- occur at low frequency, turning each in turn dampens the NF-kBresponse, sequences,’’ was held from 9–13 Octo- tissue into genome mosaics. Dr Vijg helping cells forget that they were ber 2014, in Gleneden Beach, Oregon. presented data from his group on their stressed in the past. Chang concluded The meeting brought together basic single-cell approach to the study of by describing a new technology that can gerontologists, dermatologists, and skin somatic DNA mutations and epimuta- map chromatin changes in just a few biologists working on mechanisms and tions in aging tissues. Making use of the thousand cells, finding that many age- problems of skin aging, industry scien- most recent next-generation sequencing associated changes are only evident in tists attempting to create products to methods, their data indicated that the the long-lived compartment of address unmet needs in the field, and frequency of somatic mutations is much tissue. trainees wishing to acquire a better higher than previously thought, with Ruby Ghadially discussed functional understanding of the aging process and many mutations inactivating gene func- studies of human epithelial stem cells. its consequences in the skin. The many tion. Dr Vijg discussed these results in Although human skin stem cells can be recent advances in both basic and the context of some broader philosophi- enriched by one or more cell surface applied aspects of cutaneous aging led cal and ethical questions posed by the markers, their functional characteriza- to productive exchanges among these study of aging. For example, in view of a tion had previously depended on participants, broadened everyone’s hor- gradual erosion of genome and epigen- in vitro colony-forming assays. Dr Gha- izons, and stimulated several new col- ome integrity, is it feasible to develop dially showed that intradermal injection laborations. The Symposium was interventions to delay, halt, or even of skin stem cells can recapitulate some chaired by Barbara A. Gilchrest with revert aging, and are there ethical costs? aspects of wound healing and lead to Session Chairs Judith Campisi, Howard This discussion provided participants the formation of epidermal inclusion Chang, and Gary Fisher. with an engaging, big-picture introduc- cysts where cells undergo keratiniza- The meeting opened with a Keynote tion to the meeting’s theme. tion. This assay system also allowed presentation by Jan Vijg. Dr Vijg began Session Chair Howard Chang drew comparison of the proliferative capacity by pointing out the great progress that the connection between aging and stu- of epithelial stem cells in vivo, albeit in has been made over the last two cen- dies of epigenetics—the mechanisms of a xenograft setting, and can be used to turies in increasing both life expectancy gene memory over time—and then compare stem cells in different settings, and health span––i.e., the average time introduced recent findings that the including aging. span an individual can expect to remain human genome encodes thousands of If seeing is believing, Valentina Greco functional. However, further progress long noncoding RNAs, many of which continued the session with an exciting depends entirely on gaining a deeper have functions in chromatin regulation. presentation on in vivo imaging of stem understanding of the intrinsic mechan- Chang reported that the stress-respon- cell niche. Dr Greco developed cutting- isms that underlie the aging process and sive NF-kBis edge two-photon microscopy methods predisposition to disease. Among the increasingly active with age in the skin and transgenic animals with fluorescent many theories of how we age, one of and other tissues and that interruption of nuclei in skin cells in order to make the oldest is based on the accumulation NF-kB activity in aged skin can restore movies of how individual cells in of errors in genome and epigenome. many features of young skin. He hair follicles move and develop. Although conceptually fairly simple, this then described long noncoding RNAs Direct visualization of cell dynamics

1Dermatology Service, Massachusetts General Hospital, Boston, Massachusetts, USA; 2Buck Institute for Research on Aging, and Lawrence Berkeley National Laboratory, Novato, California, USA; 3Department of Dermatology, Stanford University, Stanford, California, USA; 4Department of Dermatology, University of Michigan, Ann Arbor, Michigan, USA and 5Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA Correspondence: Molly F. Kulesz-Martin, Department of Dermatology, Oregon Health & Science University, L468R, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA. E-mail: [email protected]

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overturned several dogmas in the skin pharmacologic alleviation of endoplas- sequences de novo. Long-term exposure stem cell field, including the idea that mic reticulum stress. to T-oligos mimics the effects of short, stem cells are somehow uniquely speci- Session Chair Judith Campisi intro- dysfunctional telomeres, but short-term fied. In fact, if existing hair follicle stem duced the multifaceted stress response exposure to T-oligos elevates DNA cells are destroyed, other cells can take termed cellular senescence. Senescent repair capacity, increases telomerase their place, influenced by the unique cells are characterized by an essentially activity, and elongates telomeres. These environment of the niche. During hair irreversible arrest of cell proliferation studies indicate another doubled-edged follicle regression in catagen, Dr Greco and a complex senescence-associated sword. At low levels, DNA damage showed that only the outer root sheath secretory phenotype composed of signaling, such as that caused by short- cells die, whereas the inner root sheath numerous growth factors, inflammatory term T-oligo exposure, is protective, cells move up the follicle. The signal for cytokines, and proteases. The senes- increasing DNA repair systems, telomer- outer root sheath cells to die emanates cence growth arrest is known to be an ase expression, and telomere length. At from mesenchymal cells in the dermal important tumor suppressive mechan- higher levels, such as that caused by papilla. These results raised the concept ism. The senescence-associated secre- long-term T-oligo and excessive sun that the mesenchymal cells in the der- tory phenotype, however, is a double- exposures, the protective effects are over- mal papilla may be the intrinsic clock edged sword. Using a mouse model that whelmed and DNA damage signaling for hair follicle cycling over time. allows the visualization, sorting, and then drives apoptosis and senescence. Ray Monnat followed with studies of selective elimination of senescent cells, Steven Artandi closed the session by the Werner Syndrome, a disease with Campisi presented evidence that skin elucidating how telomerase expression many features of premature aging aris- wounding is accompanied by a rapid and assembly are controlled. In both ing from loss of function mutations in but transient appearance of senescent mice and humans, the identification the WRN helicase gene. A consequence cells, primarily fibroblasts and endothe- of telomerase-positive cells has been of this progeroid syndrome is the lial cells that secreted platelet-derived elusive owing to the very low expression increased incidence of cancers, includ- growth factor AA, a newly identified of the holoenzyme components. The ing melanoma, with an over 50-fold senescence-associated secretory pheno- Artandi laboratory recently overcame increase in Werner Syndrome patients. type factor that was essential for optimal this limitation by generating a knock-in Dr Monnat identified several aspects of wound closure. By contrast, the chronic reporter mouse model in which the DNA metabolism that are altered in presence of senescent cells, which can open reading frame of TERT, the reverse Werner Syndrome cells. He found that occur in response to whole-body radia- transcriptase subunit of the enzyme, is the rate of the DNA replication fork tion, systemic genotoxic chemotherapy, replaced by fluorescent proteins, allow- progression is altered, and many genes or oxidative stress, is deleterious and ing tracking of telomerase-positive stem exhibit altered expression levels. The can promote tumor metastasis and and transit amplifying cells and deter- changes in gene expression apparently retard skin wound healing. mining their role in telomere length had large effects on genes encoding Anne Chang followed with a presen- maintenance. New evidence suggests tRNA synthetase pathways; thus, unex- tation on the marked individual varia- that telomerase is assembled in sub- pectedly, a defect in DNA metabolism tion in the ability to maintain skin nuclear domains such as Cajal bodies, or replication may indirectly impact youthfulness during aging, a phenotype an important step in the optimal expres- protein translation. that thus far has been poorly character- sion and localization of the enzyme. Danica Chen concluded the session ized. At least some of the ability to These findings promise deeper insights with a presentation on aging and sir- maintain healthy, youthful-appearing into telomerase regulation during carci- tuins. Although sirtuins were first recog- skin into advanced age appears to have nogenesis and natural aging. nized for their roles in chromatin a genetic basis. Chang suggested that a Session Chair Gary Fisher presented regulation in the nucleus, Chen reported shift in focus from skin aging to skin three-dimensional videos of second har- the novel roles of the mitochondrial youthfulness can uncover new key monic generation signals and atomic resident Sirt3 protein in aging. She pathways that regulate skin health force microscopy nanoscale images that showed that Sirt3 is important to deace- and potential interventions to restore illustrated deterioration of tylate superoxide dismutase 2, a key skin youthfulness pathways in aged fibrils with aging. Multiphoton fluores- enzyme to detoxify reactive oxygen individuals. cence microscopy images further species, and that Sirt3 is necessary to Barbara A. Gilchrest discussed the revealed that attachment of fibroblasts prevent the build-up of reactive oxygen promise of T-oligos, oligonucleotides to collagen fibrils is disrupted by frag- species in aged hematopoietic stem that are homologous to the vertebrate mentation, leading to reduced fibroblast cells. Another sirtuin, Sirt7, responds to telomere DNA repeat sequence spreading. Fibroblasts respond to loss of stress signals in the endoplasmic reticu- (TTAGGG). Human telomeres shorten attachment by upregulating production lum and signals the translational progressively with repeated cell division of collagen-degrading matrix metallo- machinery to slow down accordingly. owing to the biochemistry of DNA proteinases and downregulating produc- Sirt7-deficient animals exhibit fatty liver replication and because most human tion of collagen, leading to further and altered hematopoietic stem cell cells only minimally express telomerase, collagen deficit, with attendant decline function, which can be reversed by the enzyme that adds telomeric of skin health. Human intervention

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studies demonstrated that fibroblasts determined. We eagerly await the glycation formation. Thus, using such in aged skin could be ‘‘rejuvenated’’ results of ongoing studies to determine agents to extend skin health during by enhancing structural support and whether transgenic expression of naked aging remains a possibility. mechanical force within the dermis, by mole rat HAS2 will extend the life span To open Barbara A. Gilchrest’s ses- injection of cross-linked hyaluronic acid of mice. sion, Vladimir Botchkarev discussed (HA) dermal filler. Thus, functional Yong Li presented the role of prosta- epigenetic regulation of skin stem cell decline of dermal fibroblasts may be glandins in loss of skin collagen during activity in skin development, aging, and largely a reflection of their tissue envir- aging, a novel counterpart to the activity cancer. Specifically, his group looked at onment and not irreversible. of these well-known lipid mediators of how p63 and epigenetic regulators The theme of the reversibility of some immunity and inflammation in the skin. interact to promote, establish, and main- features of skin aging was further cDNA microarray analysis of the skin tain epidermal differentiation program advanced by Sewon Kang, who pre- from persons of varying ages revealed in and how these sented an overview of retinoids and that the gene responsible for prostaglan- epigenetic mechanisms provide novel their ability to improve the appearance din E2 synthesis, PTGES1, strongly posi- approaches for the treatment of age- of aged human skin. Although the tively correlated with age. Compelling associated skin disorders. mechanisms by which retinoids improve data showed that PTGES1 expression is Calvin B. Harley outlined methods for appearance are not precisely known, highly sensitive to fibroblast spreading. measuring telomerase activity and telo- the induction of collagen appears to be The potential for clinical translation of mere length and the potential of these a critical factor. Retinoic acid stimulated these findings is high. Nonsteroidal methods and measurements to improve production of type VII collagen anchor- anti-inflammatory drugs, which are clinical practice in dermatology and ing fibrils and type I collagen. Retinoid well-known to inhibit prostaglandin E2 beyond. Beginning with an overview of dermatitis, a side effect due to the production, provided proof of concept, telomere biology, Dr Harley went on to actions of retinoids on the epidermis, with one such drug raising collagen present telomere measurement techni- could be partially mitigated by combina- levels in aged human skin. ques such as quantitative reverse trans- tion with inhibitors of EGFR, potentially Richard Clark described his recent criptase in real time and quantitative improving tolerability and therapeutic studies on fibronectin-derived peptide fluorescent in situ hybridization and benefit. P12. Fibronectin provides a substrate their applications in cancer care and Vera Gorbunova presented fascinat- for dermal cell attachment, migration, the wellness market, as well as a poten- ing connections between longevity, and growth during wound healing. P12 tial role for these methodologies in such essentially complete resistance to all peptide displays strong binding to plate- areas as improved skin biopsies and types of cancers, and the unique proper- let-derived growth factor BB, and this telomere vaccines. ties of hyaluronic acid, a long chain binding enhances its growth promoting J. Silvio Gutkind presented his group’s polysaccharide composed of repeating activity. Aged pre-senescent fibroblasts multi-institutional clinical trial exploring units of the D-glucuronic acid and lack sufficient traction force to expose the anti-tumor effects of rapamycin on N-acetylglucosamine, in the naked mole P12 and therefore respond poorly to newly diagnosed head and neck squa- rat. This mammal has a life expectancy platelet-derived growth factor BB. mous cell carcinoma, which also exceeding 30 years. High viscosity of Responsiveness to platelet-derived yielded data about the role of mTOR culture media from naked mole rat growth factor BB could be markedly in skin aging, specifically, that the inhi- fibroblasts was traced to secretion of improved by addition of exogenous bition of mTOR may prevent premature large amounts of very long chains P12. This raises the intriguing possibility aging of the skin without increasing (molecular weights 6–12 times greater that P12 may be therapeutic to improve cancer incidence. than in mice or humans) of HA. wound healing in the elderly. Maria Eriksson presented her group’s Increased length of HA chains was Vincent Monnier wrapped up the work on the Hutchinson-Gilford pro- found to be due to two amino acid session by discussing glycation of extra- geria syndrome (HGPS) as a model for substitutions within the active site of cellular matrix molecules, such as normal aging, including recent studies the major HA-synthesizing enzyme collagen and elastin, resulting in accu- on the expression of HGPS mutation in HAS2. Both increased expression of mulation of advanced glycation end basal cells of the interfollicular epider- HAS2 and reduced expression of HA- products during aging. This accumula- mis resulting in the upregulation of degrading activities were found to be tion alters the structure and mechanical lamin B receptor in the suprabasal cell responsible for high HA content in the properties of dermal collagen and elas- layer, and on the development of trans- naked mole rat. Interestingly, inhibition tin, deleterious to both the physical genic mouse models with expression of of high molecular weight HA produc- properties and functions of dermal cells. this mutation only in certain cell types, tion conferred oncogenic transformation Unfortunately, to date, strategies to shedding light on systemic responses on normally resistant naked mole rat remove advanced glycation end pro- underlying aging phenotypes. fibroblasts. These data reveal that long ducts in vivo have been unsuccessful; Dr Eriksson’s talk was followed by chain HA has unique anti-cancer however, understanding of the chemis- three short talks. Rosemarie Osborne properties; however, the precise mech- try of glycation has led to identifica- described her group’s efforts to define anism of this activity remains to be tion of several molecules that retard molecular pathways involved in

952 Journal of Investigative Dermatology (2015), Volume 135 BA Gilchrest et al. Montagna Symposium 2014: Skin Aging

intrinsic skin aging and photoaging, Betty Yu presented data showing a plat- for Investigative Dermatology Travel using transcriptional profiling tools that form that employs topically applied Award for Young Investigators: Hideo elucidate the expression of genes asso- invisible cross-linked polymer films to Kudo, MD, Ph.D., Kumamoto Univer- ciated with changes in cellular energy enhance hydration, provide support, sity/Pharmaceuticals Medical Devices production. Abbas Raza presented his and otherwise improve compromised Agency, Japan. lab’s current work on acyclothymidine skin. dinucleosides as structural mimetics of Barbara A. Gilchrest and Sewon Kang CONFLICT OF INTEREST DNA, with data showing that topical moderated the culminating Future H.Y.C. receives grant support from the Life Exten- application of acyclothymidine dinu- Directions panel. Panelists Chris de sion Foundation and has financial interests in Epinomics, Inc., and RaNA Therapeutics. The cleosides resulted in DNA photoprotec- Bryune, John Doux, and Serge Lichtstei- other authors state no conflict of interest. tion in both mouse and human skin ner led a discussion on current exposed to UVB. Abdoelwaheb el approaches and opportunities/barriers Ghalbzouri described his group’s work to new dermatologic products, drugs, ACKNOWLEDGMENTS The Montagna Symposium on the Biology of Skin on reticular and papillary fibroblasts in and cosmeceuticals and implications for is supported by the National Institute of Arthritis human skin equivalents. translation from bench to bedside. This and Musculoskeletal and Skin Diseases and the The meeting’s final session began lively and candid exchange aired National Institute of Aging (R13 AR009431). Other 2014 supporters included the following: The Este´e with five short talks on current trends experiences, shared perspectives of Lauder Companies; Galderma International; in industry research. Raaj Khusial pre- obstacles, and set forth new mechan- Advancing Innovation in Dermatology; DUSA sented work on a novel role for autop- isms for synergy between industry, aca- Pharmaceuticals; Johnson and Johnson Consumer hagy in epidermal differentiation. demia, and government researchers, and Personal Products Worldwide; KYTHERA Bio- pharmaceuticals; Living Proof; The Procter and Michael Milane outlined a phase 2 bringing the meeting to a noteworthy Gamble Company; Curtis T. Thompson; Valeant clinical study evaluating the safety and conclusion. Pharmaceuticals North America LLC; Maruho efficacy of aminolevulinic acid photo- Society for Investigative Dermatology Company; The Orentreich Family Foundation; dynamic therapy for reducing the inci- Eugene M. Farber Travel Awards for and Joel S. Gordon. Loa Nowina-Sapinski and Clara Stemwedel assisted in the preparation of this dence of facial actinic keratoses in high- Young Investigators: Nicole K. Brogden, manuscript. The Montagna Symposium on the risk patients. Florence Nadal-Wollbold PharmD, Ph.D., University of Iowa; Biology of Skin, directed by Molly F. Kulesz- presented data from an industry- Marco Demaria, Ph.D., Buck Institute Martin, is an annual nonprofit scientific meeting, inaugurated in 1950 by William Montagna, that academic collaboration on the role of for Research on Aging; Vyacheslav gathers leading cutaneous biologists and dermatol- carbonyl stress in actinic elastosis and Labunskyy, Ph.D., Boston University ogists to discuss new findings, techniques, and photoaging. Tracy Shafizadeh gave an School of Medicine; George Man, Uni- goals in skin biology. Podcast interviews of Sym- overview of the metabolome and how it versity of California, Berkeley; Michael posium invited speakers are available at http:// www.nature.com/jid/skinpod/index.html, and blog may be useful in the development of C. Velarde, Ph.D., Buck Institute for posts from the meeting may be found at http:// therapeutics and skin care products. Research on Aging. Japanese Society www.scilogs.com/jid/category/meeting-notes/.

Montagna Symposium 2015 15–19 October 2015, Salishan Spa & Golf Resort, Gleneden Beach, Oregon ‘‘Harnessing Stem Cells to Reveal Novel Skin Biology and Disease Treatment’’

Program Chairs Xiao-Jing Wang, MD, Ph.D., University of Colorado, Denver Valerie Horsley, Ph.D.,

Program Committee Mayumi Ito, Ph.D., New York University John McGrath, MD, King’s College London Rui Yi, Ph.D., University of Colorado, Boulder

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