Report on the 14th Annual Meeting of the PASPCR

The first session of the 2007 Annual PASPCR meeting was titled Cell Biology and Pigmentation and presented exciting novel findings that will contribute to our understanding of pigmentation and pigment pattern formation at several levels. Presentations provided new information regarding the modulation of melanosome tethering in drendritic tips of melanocytes, subsequent transfer of melanosomes to keratinocytes, and signaling and cross-talk within the epidermal/melanocyte unit. Here are the highlights and take-home messages.

Chediak-Hagashi Syndrome (CHS) is characterized by mild hypomelanosis of the skin, hair and eyes. This hypomelanosis results from the retention of enlarged melanosomes in the melanocyte cell body and the absence of melanosome transfer to the keratinocytes. Here, Wendy Westbroek and colleagues (The Rab27a/Melanophilin/Myosin Va tripartite complex does not target enlarged melanosomes to actin filaments in Chediak-Higashi melanocytes) demonstrate that Rab27a/Melanophilin/Myosin Va tripartite complex, necessary for melanosome transfer to the dendrites and subsequent transfer to keratinocytes, does not form in melanocytes of patients with CHS. Absence of melanosome tethering to the actin in dendritic tips of CHS melanocytes could begin to explain the skin hypomelanosis associated with CHS.

Sheila Rocha and colleagues (RNAi knockdown semaphorin 6D decreases melanosome transfer from melanocytes to keratinocytes) describe the first instance of modulating melanosome transfer via semaphorins and present a novel means of analyzing the transfer process. Neural crest-derived origin of melanocytes is interesting in light of semaphorins having a role in neural path finding. Along with their receptors, the plexins, semaphorins may be involved in skin pigmentation. Here, Semaphorin 6D knockdown in keratinocytes resulted in significant inhibition of melanosome transfer. While melanocytes can transfer melanosomes to several keratinocytes, keratinocytes accept melanosomes from only a single melanocyte, regardless of multiple melanocyte contacts. Collectively results indicate a role for semaphorins in melanosome transfer and skin pigmentation.

Glynis Scott and colleagues (Semaphorin 7a promotes spreading and dendricity in human melanocytes through ?1-integrins and Plexin C1) describe the function of Semaphorin 7a on human melanocyte dendricity. They show that Sema7a is produced by human keratinocytes and fibroblasts in vitro, is expressed in the skin in vivo, but is not expressed in melanocytes. Melanocytes express both Sems7a receptors, ?1-integrin and Plexin C1. Both receptors have important, but opposite, effects in mediating the dendricity response to Sema7a. Binding of Sema7a to ?1-integrin stimulates dendricity, whereas silencing Plexin C1 stimulates dendricity. Interestingly, Plexin C1 is a negative regulator of cofilin, an actin severing protein. Sema 7a is identified as a novel paracrine factor for melancyte dendrite formation through opposing actions of ?1-integrin and Plexin C1 signaling. In addition, Cofilin is suggested to play an important role in mediating these effects of Plexin C1.

Janice L Brissette and colleagues (Foxn1, epithelial cells, and the patterning of pigmentation) show that epithelial cells influence melanocytes and organize the epithelial/melanocyte unit, and effectively, engineer their own pigmentation through the action of the epithelial cell-specific transcription factor, Foxn1. Foxn1 is a product of the nude locus and is an epithelial cell-specific transcription factor. Foxn1 induces Fgf2 secretion, and attracts melanocytes to the region containing Foxn1-positive cells. The melanocytes then transfer the melanin to these Foxn1-containing cells. Foxn1 is shown to regulate pigmentation via Fgf2. Foxn1 initiates terminal differentiation and serves to identify keratinocytes as the target for melanosome transfer. In this way, Foxn1 functions to activate a distinct set of traits giving rise to a pigment recipient phenotype, through which epithelial cells recruit pigment donors and stimulate melanin transfer. These pigment recipients constitute a specialized counterpart to the melanocytes and provide a blueprint that instructs melanocytes where to place the pigment.

Gregory S Barsh and colleagues (A new ligand for melanocortin receptors) have recently identified K locus involvement in the dominant inheritance of black coat color in the dog. K is distinct from, but interacts with Aguoti and Mc1r, does not correspond to the predicted location of any previously known pigmentation gene, encodes a protein secreted by keratinocyes, and whose human ortholog is a beta-defensin gene. The beta-defensins are thought to play important roles in the innate immune system, protecting the body surface from microbial attack. Beta-defensins bind with high affinity to Mc1r and modulate melanocortin signaling by preventing agouti from binding Mc1r. The result is that beta-defensins have significant effects on pigment type switching. The mechanism of K involvement (Ky<Kbr<KB) leads to interesting questions regarding defensins involvement in pattern generation. Collectively, results provide insight into comparative mammalian genetics in general, and melanocortin receptor signaling specifically.

Miri Seiberg and colleagues previously showed that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here (LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes) they show that LIGR stimulates Rh0-GTP activation and induces skin pigmentation by activating only a subset of the PAR-2 signaling pathways and with no induction of inflammatory mediators. LIGR is a more specific regulator of PAR-2 induced pigmentation compared to SLIGRL (another known PAR-2 activator). The suggestion is made that topical treatment of LIGR may result in tanning by enhancing the natural pigment content of the skin, without enhancing inflammatory processes and without the need of UV exposure.

Frank Meyskens presented work from the University of California at Irvine supporting his hypothesis that the initiation of melanoma is epigenetic and involves ultraviolet light, Fe, Cobalt/Copper. They propose that the anti-oxidant action of melanin is turned to pro-oxidant by metals cobalt and copper, producing free radical generation trough the Fenton reaction. Epidemiological studies have shown that industrial metal exposure in the printing and electrical industry and in surgical implants are associated with increased risk of melanoma.

Valerie Trapp from UC Irvine have developed an interesting three dimensional culture system in which spheroids containing melanoma cells and tumor derived endothelial cells are co-cultured and tested for the effects of anti-angiogenic molecules. Using this system, they found that tumor stromal cells producing high levels of thrombospondin 1 (TSP-1) made co-cultured melanomas highly susceptible to anti-angiogenic therapy.

Jack Longley from the University of Wisconsin proposes a paradigm shift in how we think about MAGE (Melanoma antigen). MAGE may be more than just a melanoma marker and a target for immunotherapy, but may be an important component of pathways controlling melanoma growth. MAGE expression is c-kit dependent, and siRNA knockdown of MAGE expression inhibits melanoma cell growth by inducing apoptosis. This effect may be p53 dependent , and MAGE inhibition may be an important factor in promoting apoptosis induced by drugs that induce p53.

Zalfa Abdul-Malek of the University of Cincinatti summarized a large body of work demonstratuing that the melanocortin-1 Receptor is important in protecting human melanocytes from the effects of ultraviolet radiation. Melanocytes with certain MC1R alleles are naturally susceptible to the genotoxic and cytotoxic effects of UVR, and transfection of such cells with ?wild type? MC1R restores protection. Protection can be blocked by using analogues of the agouti-signaling protein, further illustrating the importance of control of pigmentation and photoprotection at the level of the MC1R.

Thomas Hornyak from the Dermatology Branch of the National Cancer Institute addressed whether polycomb group proteins are involved in melanoma initiation or transformation. Polycomb group proteins are epigenetic gene silencers involved in normal development, stem cell maintenance, and oncogenesis. Hornyak focused on the role of two of these proteins in melanoma: BMI-1 which mediates repression of CDKN2A which encode p16, and EZH2 whose increased expression is associated with increased proliferation and decreased survival in melanoma. Interestingly, EZH2 levels were low in normal melanocytes and nevi, but elevated in melanomas. BMI-1 levels were uniformly high in melanocytes, nevi and melanomas, and siRNA silencing of BMI-1 had no effect on cell proliferation and anchorage-dependent growth. Hornyak speculates that in senescent nevi , EZH2 expression suppresses CDKN2A, decreasing the tumor suppressor p16, and leading to malignant transformation.

Xiao-qi Wang of Northwestern University questioned whether different isotypes of Gm3 gangliosides correlated with metastasis in melanoma. Using a 2 layer Matrigel invasion assay, and silencing or over expression of key components of the pathway, they have shown that GM3 increases invasion acting sequentially through urokinase plasminogen activator and receptor, p38 MAPK, and matrix metalloproteinase 2 (MMP2). The authors propose that targeting GM3 may be a therapeutic strategy for melanoma therapy.

The first talk was titled The Chemokine SDF-1 regulates the development of Neural crest progenitors of the melanocyte lineage in Mouse skin by Abdelhak Belmadani, Dongjun Ren and Richard J. Miller. Dr. Belmadani presented data on the chemokine SDF-1 (CXCL 12) and its receptor CXCR4. SCF-1 was shown to be a chemoattractant for CXCR4 bearing cell in culture. In CXCR4 mutant mice DCT positive cells were noted aberently in the epidermis presumably due to defect migrating into the hair follicle and increased apoptosis was noted in these cells. Thus this pathway may be critical for the migration of melanocytic precursors into the hair follicle.

The second talk was titled Expression of melanocyte specific proteins and growth factor receptors in tissue and cultured cells from lymphangioleiomyomatosis by Jason A. Wilson, Chul Jong Park, Ljiljana Minwalla, Amy Koshoffer, Teresa A. Smolarek, and Raymond E. Boissy. Dr. Boissy discussed the curious entity of lymphangioleiomyomatosis. This neoplastic process was found to partially express pigmentation associated genes. Expressed genes included HMB-45 and Mart-1. Three of 5 specimens also expressed KIT and 2 of 5 also expressed the alpha receptor for estrogen. Similar expression patterns were also noted in culture.

The third talk was titled A new mouse model for the detection of melanocyte stem cells by Sara K. Powell, Kacey P. Young, Maria Nelson, Rebecca S. Plummer, and Deborah Lang. Dr. Lang presented her work demonstrating that the PAX-3 promoter is ideal for the detection of melanocytic stem cells. In her system the PAX-3 promoter was linked to b-galactosidase in transgenic mice. Unlike Sox-10 that remains on in the differentiated melanocytes, PAX-3 is down regulated – limiting its expression to the melanocytic stem cell compartment. PAX-3 expression was noted to increase in response to stress induced follicle regrowth post depilation and may serve as a unique marker for the isolation of melanocytic stem cells.

The forth talk was titled Evidences for an ectopic synthesis of melanin in adipose tissue by Manpreet Randhawa, Tom Huff, Julio Valencia, Vincent Hearing, Zobair M. Younossi, Ancha Baranova. Dr. Randhawa presented data supporting the presence of melanin and pigmentary enzymes in the adipose tissue of obese individuals. Hypothetically, melanocortin stimulatory pathways in the obese state may also secondarily drive the aberrant expression of pigmentary pathways in fat cells. They were able to demonstrate the expression of Mart-1, MSH, and Tyr immunohistochemically and the presence of melanin PTCA determination in excised adipose tissue. Further they were able to demonstrate up regulation of MC1R, Tyr, TYrp-1 and MITF with alpha- MSH on mature adipocytes in vivo.

The fifth talk was titled Early, over-expression of the endothelin signaling pathway leads to the generation of ectopic melanoblasts by Avner Ittah, Shyla Mirabal, Roman J. Garcia, Lydia Kos. Dr. Kos indicated that over-expression of the endothelin-B receptor (Ednrb) alone in transgenic mice actually resulted in a decrease in the number of melanocytes. This was speculated to be secondary to sequestration of a limited amount of ET-3 by the Ednrb receptor being prematurely driven by the nestin promoter. This defect was corrected by increasing the expression of ET-3 (under control of kertin 5 promoter) but this also lead to the etopic expression of melanoblasts in the roof plate over the neural tube.

The sixth talk and final presentation was titled Use of doxycycline-regulated transgenic mice to identify and characterize melanocyte label-retaining cells by Ha-Young Hwang, Ganesh Diwakar, Thomas Hornyak. Dr. Hwang report of the use of a DCT "tet off" system coupled with a K5 driven tetracycline transactivator inducing the expression of H2B green fluorescent protein. He was able to show that is the presence of tetracycline non-proliferative cells were localized in the hair bulge region (CD34 co-localization). This system may permit the isolation of viable melanocytic stem cells by fluorescence-activated cell sorting.

The Pathology and Treatment of Pigmentary Disorders section of the PASPCR Meeting followed the Keynote Lecture by Victoria Holloway Barbosa on Sat. Sept. 15, 2007. The session began with a presentation given by Yoshinori Miyamura (Hearing Laboratory) on De novo melanogenesis increases UV protection of human skin. This abstract analyzed the protective effects of tanning fair skin using different UV sources (UVA, UVB, and SSR). Although the tanning response appeared similar clinically, UVA-induced tanning was not as photoprotective (as measured by CPD formation) as UVB- or SSR-induced tanning. These results suggest that chonic UVA tanning leads to increased pigmentation and melanin dispersion, but may also have negative effects regarding photoprotection.

The next presentation, Skin pigmentation induced by repeated UV exposures and the role of melanin in photoprotection, was given by Sergio Coelho, also from the Hearing Laboratory. Upon analyses of several biomarkers (cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53 protein, pigment content assessed by Fontana-Masson staining, melanocyte function by melanosomal protein expression, apoptosis by TUNEL assay and determination of Ser46-phosphorylated p53), these investigators found that the amounts of CPD decreased while the numbers of p53-positive cells increased with increasing cumulative doses of UV even though apoptosis, melanocyte function, and melanocyte density showed no dependence on cumulative doses. These findings led investigators to hypothesize that it is possible that the pigmentation from incremental doses of repeated UV protects against additional DNA damage as evaluated by CPD formation, but other mechanisms cannot be excluded at this time.

In a 'first ever' for the PASPCR, a high school science fair winner, James Fichtelman, presented data obtained with the help of Jim Grichnik on the use of Planaria as a model system for the study of pigment biology. In this presentation, Mr. Fichtelman showed that Planaria have different pigmentary phenotypes and exhibit anticipated responses to UVR, including tanning and tumor formation. This makes this organism worthy of consideration as a tool for the study of pigment cell biology. Another member of Dr. Grichnik?s group, Adela Cardones, presented an interesting case of alpha-MSH induced nevogenesis. This case report demonstrated in a human subject, that nevogenic cells can be stimulated by alpha-MSH and that alpha-MSH levels support the growth of a subset of nevi. Importantly, the discussion following this talk raised the question of whether such pharmacologic increases in alpha-MSH also has the potential to enhance tumorigenesis, a potential cause for concern in patients who are using alpha-MSH agonists for cosmetic purposes.

Esteban Dell'Angelica also discussed An immunoblotting assayto facilitate the molecular diagnosis of Hermansky-Pudlak syndrome. Dr. Dell'Angelica (UCLA) introduced an immunoblotting-based screening assay (developed in collaboration with the group of Bill Gahl at the NIH) to narrow down the number of candidate genes to be sequenced for each patient with a new diagnosis of Hermansky-Pudlak syndrome (HPS). Because the eight known types of HPS are due to structural defects in any of four protein complexes (AP-3 and BLOC-1 through -3), the assay attempts to determine by immunoblotting which complex is defective in each patient.

A highlight of the session was Bill Paven's presentation on Comprehensive analysis of the SRY-box 10 locus (SOX10): Implications for diseases of neural crest derivatives. Dr. Pavan presented an overview of his current research program at the NIH. Part of his laboratory is conducting a mutagenesis screen to identify genes, that when mutated, increase the severity of neural crest defects in a SOX10 mutant that models Waardenburg Syndrome type 4. Another part of his laboratory is looking to identify the transcriptional regulatory networks that both control SOX10 gene expression and are controlled by SOX10 protein levels.

The session on Genetics and Gene Expression was chaired by Drs. Richard Spritz and Vijay Setaluri. Six abstracts of the total 10 abstracts submitted in this category were chosen for oral presentation. These presentations covered 3 areas- genetics of vitiligo, genetics of disease related iris phenotypes, genetic approaches to identify novel melanocyte genes and their regulation.

Dr. Spritz reviewed the published data on the recently identified vitiligo susceptibility gene NALP1 on chromosome 17p and described the results of the additional studies that showed genetic association of NALP1 with other vitiligo-associated autoimmune diseases. He also presented data that suggested the high-risk vitiligo-susceptibility NALP1 genotype is associated with apoptosis defect in blood mononuclear cells. An additional candidate gene found to contribute to the disease risk is PTPN22. These studies illustrate the need for a multidisciplinary approach to unravel the genetics of vitiligo.

The next listed presentation in the Program on the role of microenvironment changes in vitiligo was cancelled due to conflict in Dr. Zhou's travels. The next presentation from Michael Anderson?s group demonstrated the value of the utility of studying iris phenotypes as a means of identifying new candidate genes involved in human glaucoma. In an elegant presentation, Colleen Trantow, a graduate student in Dr. Anderson?s laboratory, described her studies on previously unrecognized iris disease phenotypes in beige and nm2798 mutant mice and the potential involvement of beige and Dct genes in massive accumulation of pigment and progressive pigment dispersion in iris, respectively.

In his second presentation, Dr. Spritz presented his studies on comprehensive genetic analysis of Caucasian patients with oculocutaneous albinism and autosomal recessive ocular albinism. Based on the relative frequencies of the three different types of OCA and the pathological gene mutations associated with these OCAs, Dr. Spritz proposed that OCA1 is the most frequent cause of OCA among Caucasians.

The last 2 presentations were from Dr. William Pavan's laboratory. In this presentation, described the identification of Betelgeuse a novel locus in mouse melanocyte development using a sensitized genome-wide ENU mutagenesis screen. This screen is designed to identify ENU induced mutation(s) in additional genes that act synergistically with Sox10 to increase the melanocyte defects in Sox10LacZ/+ mice. This is a powerful screen that will provide a resource for identifying additional genes involved in a complex pathway during melanocyte development in vivo. In the last presentation of the session, Dr. Pavan presented his studies on a genomic approach to identify cis-acting transcriptional regulatory elements using a combination of gene expression pattern studies, comparative genome sequencing and informatics analysis on selected 14 neural crest-expressed genes. Dr. Pavan illustrated the strategy of whole mount in situ hybridization in wild type and neural crest transcription factor mutant mouse embryos to identify key transcriptional regulatory elements contributing to the expression patterns followed by a search for elements that are evolutionarily conserved in 7 different species. The goal is to assess if correlations exist between conserved sequence elements and sub-classes of genes defined by expression indices generated by the whole mount analysis. Overall, this session was well received. The presentations elicited spirited discussion as evidenced by the number of people clustering around the speakers at the end of the session. The pace of research in this area is bound to produce new and exciting results that will enhance our understanding of the complex biology of melanocytes.

This session was spearheaded by Dr. Rosalie Luiten, who flew in from Amsterdam to show us the latest data from her group related to T cell mediated cytotoxicity in vitiligo. These data are indeed very exciting: Dr. Luiten was able to show in an ?ex vivo? system that CD8+ cytotoxic T cells from perilesional vitiligo skin will kill melanocytes in normally pigmented skin from the same donor. To do this, isolated T cells were co-cultured with tissue explants. Impressive tissue damage was observed when T cells were added in large numbers, and melanocyte apoptosis was evidenced by confocal imagery using antibodies to CD70, gp100 and caspase-3. Data using cloned T cells from a melanoma patient showed less extensive damage to melanocytes, in line with the theory that Tcells from vitiligo patients are better able to kill their targets.

The second lecture came from Dr. Gisela Erf. This was a comprehensive overview of the impressive data demonstrating the parallels between autoimmune vitiligo in the Smyth line chicken, and progressive depigmentation in humans. The chicken remains the only true model of spontaneous autoimmune vitiligo. Importantly, from studies of this model we can predict relevant precipitating factors in vitiligo as, for example, there is a clear association between vaccination against a herpes virus and the development of depigmentation. Data related to the genetic predisposition to depigmentation in this model may be expected in the near future.

The final presentation was by Dr. Ping Yu, also a co-chair for the session. She presented data related to immune infiltrates found in halo nevi compared to melanoma tumors. The immunohistology was stunning, and very convincing data were presented to show that halo nevi have similar percentages of Treg among CD4+ T cells (50%) as control skin, whereas fewer (!) FoxP3 + CD4 T cells were found in melanoma. The apparent discrepancy to the reduced clinical efficacy of an immune response in melanoma is best explained by the significantly reduced expression of perforin by infiltrating T cells in melanoma. The common denominator among the novel data presented is the active search into mechanisms (dys) regulating the immune response in pigmentary disorders.

Andrzej Slominski began the session with a review on the role of CRH in the mammalian pigmentary system. Prof. Slominski's group has now demonstrated that the same endocrine systems operating in the 'hypothalamic-pituitary-adrenal axis' are operative in the skin. Among the large number of biological functions affected by this is hormonal regulation of the pigmentary system.

Feng Liu presented her work with Frank Meyskens that contrary to previous reports by others, they found no significant differences in MiTF expression by cultured melanocytes from black, Hispanic, and Caucasion origins, even though melanin levels varied more than 6 fold, and positively correlated with skin type. During discussion it was agreed that differences with previous reports may be attributed to differences in culture conditions.

Sulochana Devi presented work in collaboration with R Kedlaya, N Maddodi, CS Weber, H Valdivia, and V Setaluri, showing that in cultured human melanocytes melanostatin1/TRPM1-mediated Ca++ uptake, which can be regulated by UV through p53, plays a role in growth and differentiation of melanocytes and melanoma tumor progression.

Akira Hachiya along with S Kasamatsu, K Higuchi, A Ohuchi, T Kitahara, and R Boissy, reported that the production of the soluble form of C-kit, S-kit, abolishes stem cell factor-induced melanogenesis in human melanocytes. The results suggested that the production of S-kit may contribute to the regulation of human skin pigmentation.

Ana Luisa Kadekaro along with N Mosby, E Hess, R Kavanagh, V Swope and Z Abdel-Malek, presented the most interesting story of alpha MSH protection of human melanocytes against UVB-induced oxidative stress, in this case through a mechanism that involves p53 modulation.

Ashley Dills with Beverly Delidow presented a convincing study that retinoic acid induces coordinate expression of Wnt inhibitory genes in melanoma. This is the first example of coordinate RA-regulation of these factors and suggests the potential of Wnt inhibition as a strategy for melanoma therapy.

Heinze Arnheiter completed the morning, and indeed the conference, with a keynote lecture on his exciting work regarding the role of MITF isoforms during pigment cell development.