A resposta imune dirigida a autoantígenos pode contribuir para a patogênese das doenças autoimunes. Porém, também é discutido o papel imunorregulador da autoimunidade em processos inflamatórios e na rejeição do aloenxerto. Nós pesquisamos os autoanticorpos IgG e IgM reativos a peptídeos da miosina cardíaca (MC) e da proteína de choque térmico 60 (Hsp60) no soro de indivíduos sadios (IS, n=30; 3 momentos com intervalos de 6 meses) e indivíduos transplantados cardíacos (Tx, n=65, > 2 amostras/indivíduo, de diferentes períodos Tx: pré-Tx, T1: < 1 ano, T2: 1 a 5 anos e T3: >5 anos), por ensaio imunoenzimático (ELISA). Todos os sujeitos do estudo tiveram anticorpos IgG ou IgM que reconheceram pelo menos um dos peptídeos avaliados. Os anticorpos IgG de indivíduos Tx reconheceram mais peptídeos do que dos IS, para a MC (12,2 ± 8,5, intervalo: 132 peptídeos versus 5,2 ± 3,0, intervalo: 0-14; p<0,0001), e para a Hsp60 (6,0 ± 4,4, intervalo: 0-18 versus 3,9 ± 3,0, intervalo: 0-12; p=0,0208). A frequência de indivíduos positivos para os anticorpos IgG foi maior no grupo Tx do que nos sadio (p<0,05), com reatividade para a maioria dos peptídeos da MC e da Hsp60. Em contraste, a frequência de indivíduos positivos para os anticorpos IgM foi maior no grupo de IS do que no Tx (p<0,05), principalmente para a reatividade dirigida aos peptídeos da MC. Os indivíduos do grupo Tx reconheceram todos os peptídeos da MC, inclusive alguns não reconhecidos pelos sadios (S2: 19, 21, 22, 25, 27, e 29). A variabilidade temporal da autoimunidade humoral aos peptídeos desses antígenos foi maior no grupo Tx (p<0,001), indicando maior estabilidade do perfil no estado fisiológico. No grupo Tx, a frequência de indivíduos positivos para anticorpos IgG e o número de peptídeos reconhecidos foram maiores nos períodos de pré- Tx e T1 e na rejeição (p<0,05). Em contraste, para os anticorpos IgM, a frequência de indivíduos positivos e número de peptídeos reconhecidos foram maiores nos períodos de T1, T2 e no momento sem rejeição (p<0,05). Em resumo, no estado fisiológico, observamos um predomínio de autoanticorpos dirigidos à MC e à Hsp60 do tipo IgM, enquanto que no período pré-Tx e durante a rejeição o predomínio foi de IgG. Com base nesses resultados, interpretamos que o ambiente inflamatório da doença cardíaca e da rejeição possa induzir uma maior expressão de Hsp60 e exposição da MC - decorrente da necrose de cardiomiócitos - a células do sistema imune. A resposta imune desencadeada, neste contexto, culminaria na mudança do isotipo IgM, predominante no estado fisiológico, para o isotipo IgG, predominante no quadro de inflamação. Em conclusão, identificamos um perfil distinto da autoimunidade humoral dirigida à miosina cardíaca e à Hsp60, no estado fisiológico e no transplante cardíaco. Novos estudos permitirão avaliar a atividade funcional desses autoanticorpos no enxerto e nas células do sistema imune, talvez desempenhando um papel na rejeição ou na manutenção da homeostase, no contexto fisiológico / The immune response directed to self antigens can contribute to the pathogenesis of autoimmune diseases. However, autoimmunity may also have an immunoregulatory role in allograft rejection and in other inflammatory processes. We analyzed IgG and IgM autoantibodies reactive to peptides from the human cardiac myosin (CM) and the heat shock protein 60 (Hsp60) in the sera of healthy individuals (HI, n=30, 3 time points with 6 month intervals) and heart transplant individuals (Tx, n=65, >2 samples/individual, from different Tx periods: pre-Tx, T1: <1 year post-Tx, T2: 1 to 5 years and T3: >5 years), by Enzyme-Linked Immunosorbent Assay (ELISA). All subjects from both groups had IgG or IgM antibodies that recognized at least one of peptides studied. The numbers of peptides recognized by IgG antibodies was higher in the Tx group than in the HI, for CM (12.2 ± 8.5, range: 132 peptides versus 5.2 ± 3.0, range: 014 peptides; p <0.0001) and for Hsp60 (6.0 ± 4.4, range: 0-18 peptides versus 3.9 ± 3.0, range: 012 peptides; p=0.0208). The frequency of individuals displaying IgG antibodies was higher in the Tx group than in HI (p<0.05), for both CM and Hsp60. In contrast, the frequency of individuals with IgM antibodies was higher in HI than in the Tx group (p<0.05), mainly for CM. The Tx individuals recognized all CM peptides, including those not recognized by healthy individuals (S2: 19, 21, 22, 25, 27, e 29). Time variability of humoral autoimmunity directed to peptides of both antigens was higher in the Tx group (p<0,001), indicating a more stable profile in the physiologic state. In the Tx group, the frequency of individuals with IgG autoantibodies and the number of peptides recognized were higher in the pre-Tx and T1 periods and during rejection (p<0.05). In contrast, for IgM antibodies, the frequency of individuals and the number of peptides recognized were higher in the T1, T2 and in the period with no rejection (p<0.05). In summary, IgM autoantibodies directed to CM and Hsp60 were predominant in the physiologic state, in contrast with the predominance of IgG autoantibodies in the pre-Tx period and during rejection. We suggest that the inflammatory environment found in both cardiac diseases and rejection favors the increase of Hsp60 expression and the exposure of cardiac myosin antigens due to cardiomyocyte necrosis. The immune response triggered in this context induces cell activation and isotype switch, from IgM, predominant in the physiologic state, to IgG, more detected in the inflammatory process. In conclusion, we identified a distinct profile of humoral autoimmunity to cardiac myosin and to Hsp60 in the physiologic state and in cardiac transplantation. Further studies will allow us to evaluate the functional activity of these antibodies in the graft and in cells of the immune system; they may have a role in rejection or in the maintenance of homeostasis, in the physiologic context.

Les moteurs moléculaires sont des protéines capables de produire une force : elles transforment l'énergie chimique de l'hydrolyse de l'ATP en énergie mécanique. Cette thèse se focalise sur l'étude d'une famille de moteurs moléculaires, les myosines, qui se déplacent le long des filaments d'actine et assurent d'importantes fonctions cellulaires.La myosine VI est une myosine très particulière car elle est la seule à se déplacer vers l'extrémité négative des filaments d'actine. Elle est produite dans la cellule sous forme auto-inhibée, inactive. Dans la cellule, son activité est également régulée par plusieurs protéines interagissant avec la queue C-terminale de la myosine VI. Ces protéines, présentes à des endroits précis de la cellule, recrutent la myosine VI et dictent l'action qu'elle doit effectuer. Des analyses de SAXS, de dispersion de la lumière, de microscopie, d'interaction et de mutagénèse ont permis de mieux comprendre le mécanisme régulant l'adoption de l'état auto-inhibé, ainsi que son activation par le calcium. L'interaction avec différents partenaires a été caractérisée. GIPC1, le partenaire le plus étudié, promeut de façon indirecte la dimérisation et l'activation de la myosine VI.Pendant ma thèse, j'ai également été impliqué dans deux autres projets qui s'inscrivent dans la logique du projet de thèse et qui ont mené à la publication de quatre articles. Deux chapitres, plus brefs, sont donc dédiés à ces projets. Le deuxième chapitre porte sur la régulation de l'activité de la myosine VII par ses partenaires cellulaires. Finalement, le troisième chapitre est dédié à l'étude de la modification allostérique de l'activité des myosines par des petites molécules. / Molecular motors are essential agents of force production in the cells: they convert the chemical energy released by the hydrolysis of ATP into mechanical work. This thesis focuses on myosins, a family of molecular motors responsible for actin-based motility. Myosin VI is unique among all of the myosin superfamily members in that it moves in the opposite direction of all other known myosins. Previous work revealed myosin VI tail ability to fold back, constituting a potential auto-inhibited state that prevents motor activity. Several myosin VI partners, binding to the C-terminal tail of the myosin, have been identified and shown to recruit the motor for different functions. In the first chapter of this thesis, the mechanism allowing the regulation of myosin VI activity has been studied using biochemical and biophysical analysis (SAXS, light scattering, microscopy, binding assays and mutagenesis). GIPC1, the most studied myosin VI partners, promotes myosin dimerization and activation. During my PhD, I have been also involved in two other projects, in line with my thesis project, that have led to the publication of four articles. Two shorter chapters are therefore devoted to these projects. The second chapter of my thesis explores myosin VII activity regulation by its cellular partners. Finally, the third chapter is devoted to the allosteric regulation of myosins activity by small molecules.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xi, 82 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

A resposta imune dirigida a autoantígenos pode contribuir para a patogênese das doenças autoimunes. Porém, também é discutido o papel imunorregulador da autoimunidade em processos inflamatórios e na rejeição do aloenxerto. Nós pesquisamos os autoanticorpos IgG e IgM reativos a peptídeos da miosina cardíaca (MC) e da proteína de choque térmico 60 (Hsp60) no soro de indivíduos sadios (IS, n=30; 3 momentos com intervalos de 6 meses) e indivíduos transplantados cardíacos (Tx, n=65, > 2 amostras/indivíduo, de diferentes períodos Tx: pré-Tx, T1: < 1 ano, T2: 1 a 5 anos e T3: >5 anos), por ensaio imunoenzimático (ELISA). Todos os sujeitos do estudo tiveram anticorpos IgG ou IgM que reconheceram pelo menos um dos peptídeos avaliados. Os anticorpos IgG de indivíduos Tx reconheceram mais peptídeos do que dos IS, para a MC (12,2 ± 8,5, intervalo: 132 peptídeos versus 5,2 ± 3,0, intervalo: 0-14; p<0,0001), e para a Hsp60 (6,0 ± 4,4, intervalo: 0-18 versus 3,9 ± 3,0, intervalo: 0-12; p=0,0208). A frequência de indivíduos positivos para os anticorpos IgG foi maior no grupo Tx do que nos sadio (p<0,05), com reatividade para a maioria dos peptídeos da MC e da Hsp60. Em contraste, a frequência de indivíduos positivos para os anticorpos IgM foi maior no grupo de IS do que no Tx (p<0,05), principalmente para a reatividade dirigida aos peptídeos da MC. Os indivíduos do grupo Tx reconheceram todos os peptídeos da MC, inclusive alguns não reconhecidos pelos sadios (S2: 19, 21, 22, 25, 27, e 29). A variabilidade temporal da autoimunidade humoral aos peptídeos desses antígenos foi maior no grupo Tx (p<0,001), indicando maior estabilidade do perfil no estado fisiológico. No grupo Tx, a frequência de indivíduos positivos para anticorpos IgG e o número de peptídeos reconhecidos foram maiores nos períodos de pré- Tx e T1 e na rejeição (p<0,05). Em contraste, para os anticorpos IgM, a frequência de indivíduos positivos e número de peptídeos reconhecidos foram maiores nos períodos de T1, T2 e no momento sem rejeição (p<0,05). Em resumo, no estado fisiológico, observamos um predomínio de autoanticorpos dirigidos à MC e à Hsp60 do tipo IgM, enquanto que no período pré-Tx e durante a rejeição o predomínio foi de IgG. Com base nesses resultados, interpretamos que o ambiente inflamatório da doença cardíaca e da rejeição possa induzir uma maior expressão de Hsp60 e exposição da MC - decorrente da necrose de cardiomiócitos - a células do sistema imune. A resposta imune desencadeada, neste contexto, culminaria na mudança do isotipo IgM, predominante no estado fisiológico, para o isotipo IgG, predominante no quadro de inflamação. Em conclusão, identificamos um perfil distinto da autoimunidade humoral dirigida à miosina cardíaca e à Hsp60, no estado fisiológico e no transplante cardíaco. Novos estudos permitirão avaliar a atividade funcional desses autoanticorpos no enxerto e nas células do sistema imune, talvez desempenhando um papel na rejeição ou na manutenção da homeostase, no contexto fisiológico / The immune response directed to self antigens can contribute to the pathogenesis of autoimmune diseases. However, autoimmunity may also have an immunoregulatory role in allograft rejection and in other inflammatory processes. We analyzed IgG and IgM autoantibodies reactive to peptides from the human cardiac myosin (CM) and the heat shock protein 60 (Hsp60) in the sera of healthy individuals (HI, n=30, 3 time points with 6 month intervals) and heart transplant individuals (Tx, n=65, >2 samples/individual, from different Tx periods: pre-Tx, T1: <1 year post-Tx, T2: 1 to 5 years and T3: >5 years), by Enzyme-Linked Immunosorbent Assay (ELISA). All subjects from both groups had IgG or IgM antibodies that recognized at least one of peptides studied. The numbers of peptides recognized by IgG antibodies was higher in the Tx group than in the HI, for CM (12.2 ± 8.5, range: 132 peptides versus 5.2 ± 3.0, range: 014 peptides; p <0.0001) and for Hsp60 (6.0 ± 4.4, range: 0-18 peptides versus 3.9 ± 3.0, range: 012 peptides; p=0.0208). The frequency of individuals displaying IgG antibodies was higher in the Tx group than in HI (p<0.05), for both CM and Hsp60. In contrast, the frequency of individuals with IgM antibodies was higher in HI than in the Tx group (p<0.05), mainly for CM. The Tx individuals recognized all CM peptides, including those not recognized by healthy individuals (S2: 19, 21, 22, 25, 27, e 29). Time variability of humoral autoimmunity directed to peptides of both antigens was higher in the Tx group (p<0,001), indicating a more stable profile in the physiologic state. In the Tx group, the frequency of individuals with IgG autoantibodies and the number of peptides recognized were higher in the pre-Tx and T1 periods and during rejection (p<0.05). In contrast, for IgM antibodies, the frequency of individuals and the number of peptides recognized were higher in the T1, T2 and in the period with no rejection (p<0.05). In summary, IgM autoantibodies directed to CM and Hsp60 were predominant in the physiologic state, in contrast with the predominance of IgG autoantibodies in the pre-Tx period and during rejection. We suggest that the inflammatory environment found in both cardiac diseases and rejection favors the increase of Hsp60 expression and the exposure of cardiac myosin antigens due to cardiomyocyte necrosis. The immune response triggered in this context induces cell activation and isotype switch, from IgM, predominant in the physiologic state, to IgG, more detected in the inflammatory process. In conclusion, we identified a distinct profile of humoral autoimmunity to cardiac myosin and to Hsp60 in the physiologic state and in cardiac transplantation. Further studies will allow us to evaluate the functional activity of these antibodies in the graft and in cells of the immune system; they may have a role in rejection or in the maintenance of homeostasis, in the physiologic context.

Orientador: Mário Tyago Murakami / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T11:13:33Z (GMT). No. of bitstreams: 1 Nascimento_AndreyFabricioZiem_D.pdf: 59337766 bytes, checksum: b26927ac8e7083ceb39d09631e1b378f (MD5) Previous issue date: 2014 / Resumo: As miosina de classe V, amplamente distribuídas em sistemas eucarióticos desde leveduras até vertebrados, são um dos mais caracterizados motores moleculares da superfamília das miosinas e desempenham um papel chave no transporte intracelular de vesículas, organelas e RNA mensageiro. As miosinas V consistem de duas cadeias pesadas idênticas que se dimerizam através da formação de uma estrutura coiled-coil e sua arquitetura tridimensional pode ser dividida em três distintos domínios: a porção N-terminal ou domínio motor que apresenta os sítios de interação com ATP e actina; a porção central ou pescoço formada por 6 domínios IQ, responsável pela regulação e interação com calmodulina; e a porção C-terminal que inclui a região coiled-coil e o domínio de ligação de cargas celulares também conhecido como domínio cauda globular (GTD). Uma das mais importantes questões pertinentes até hoje é como ocorre a sinalização e a interação entre as vesículas/organelas e o domínio globular C-terminal das miosinas V. Neste estudo, foram resolvidas as estruturas dos domínios cauda globular das miosinas Va, Vb e Vc humanas, revelando pequenas mudanças estruturais que levam a diferenciação funcional e, ainda, um novo mecanismo redox que controla a dimerização do GTD de forma independente do coiled-coil, que é exclusivo para a miosina Vc. As alterações estruturais induzidas pela fosforilação do GTD também foram exploradas, mostrando que o estado fosforilado possuí uma flexibilidade menor, podendo estar envolvido na regulação do estado inibido e/ou reconhecimento de cargas nucleares. Além disso, os sítios de ligação a carga e ao domínio motor foram estruturalmente anotados, indicando uma conservação de resíduos envolvidos na interação com adaptadores para o transporte de peroxissomos e proporcionando detalhes da inibição da atividade motora pelo GTD. Estes resultados contribuem para a compreensão dos determinantes estruturais para o transporte de carga, autoinibição e mecanismos de regulação dos motores de miosina V. Além dos resultados obtidos com a cauda globular, alguns problemas cristalográficos como o problema da fase, pseudosimetria e ordem-desordem cristalina foram abordados (descrito nos Apêndices 9.3 e 9.4). Patologias cristalinas como ordem-desordem parcial ou total podem estar relacionadas à valores elevados de Rfactor e Rfree, mesmo após a conclusão do refinamento, ou mesmo à dificuldade de resolução da estrutura. Problemas de ordem-desordem rotacional parcial e pseudosimetria foram observados em cristais de uma hidrolase glicosídica (TpAbn). Nesse caso, valores satisfatórios de Rfactor e Rfree foram obtidos somente após um minucioso processamento dos dados e redução da simetria. Além disso, os dados dos GTDs das miosinas de classe V foram utilizados como caso teste para o desenvolvimento de novas metodologias de faseamento ab initio em média e baixa resolução (2 ¿ 3 Å) em colaboração com o grupo de Prof. Dr. Isabel Usón (IBMB, Barcelona, Espanha). Utilizando o programa ARCIMBOLDO foi possível resolver a estrutura do GTD-MioVb a 2,1 Å utilizando apenas duas hélices de poli-Ala de 22 resíduos (7,5% do conteúdo da unidade assimétrica), mostrando o grande potencial desta metodologia para dados de média a baixa resolução / Abstract: The class V myosins (MyoVs) are widely distributed in eukaryotic organisms from yeast to vertebrates, being one of the most characterized molecular motors of the myosin superfamily. MyoVs play a central role in the intracellular transport of vesicles, organelles, messenger RNA and proteins. MyoVs consist of a coiled-coil-stabilized dimer of two identical heavy chains and their general structure can be divided into three distinct domains: the N-terminal portion or motor domain which binds both actin and ATP; the central portion or neck formed by 6 IQ domains, responsible for the regulation and interaction with calmodulin; and the C-terminal portion that includes the coiled-coil region and the cargo-binding domain also known as globular tail domain (GTD). One of the most important issues still obscure so far is how occurs the interaction between the cargoes and the globular C-terminal domain of myosin V. Here, we have solved the globular tail domain structures of the three human MyoV paralogs (Va, Vb and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the GTD dimerization process, which is unique for the MyoVc subclass. The structural changes induced by the phosphorylation of GTD have also been explored, showing that the phosphorylated state is less flexible and may be involved in the regulation of the auto-inhibition mechanism and/or in the recognition of nuclear cargoes. Moreover, the cargo- and motor-binding sites were structurally assigned indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high-resolution insights into motor domain inhibition by GTD. These results contribute to the understanding of the structural requirements for cargo transport, auto-inhibition and regulatory mechanisms in myosin V motors. In addition to the results obtained with the GTD structures, some crystallographic problems, such as the phase problem, pseudosymmetry and lattice order-disorder were discussed (described in Appendices 9.3 and 9.4). Crystal pathologies such as partial or total order-disorder may be related to high values of Rfactor and Rfree, even at late stages of crystallographic refinement, or even hindering the structure determination. Problems of partial rotational order-disorder and pseudosymmetry were found in TpAbn crystals where only after a careful data processing and symmetry reduction was possible to obtain satisfactory values of residuals (Rfactor and Rfree). Moreover, data from MyoV GTDs were used as a test case for the development of new methodologies for ab initio phasing at medium and low resolution (2 ¿ 3 Å) in collaboration with the group of Prof. Dr. Isabel Usón (IBMB, Barcelona, Spain). Using the program ARCIMBOLDO we have solved the GTD-MioVb structure at 2.1 Å using only two 22-residue-long poly-Ala helix fragments (7.5% of asymmetric unit content), showing the great potential of this methodology for data at medium to low resolution / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

The importance of insulin delivery and action is best characterized in Type 2 Diabetes, a disease that is becoming a pandemic both nationally and globally. Obesity is a principal risk factor for Type 2 Diabetes, and adipocyte function abnormalities due to adipose hypertrophy and hyperplasia, have been linked to obesity. Numerous reports suggest that the intracellular and systemic consequences of adipocyte function abnormalities include adipocyte insulin resistance, enhanced production of free fatty acids, and production of inflammatory mediators. A hallmark of adipocyte insulin sensitivity is the stimulation of glucose transporter isoform 4 (GLUT4) trafficking events to promote glucose uptake. In the Type 2 diabetic and insulin resistant states the mechanism behind insulin-stimulated GLUT4 trafficking is compromised. Therefore, understanding the role of factors involved in glucose-uptake in adipose tissue is of great importance. Studies from our laboratory suggest an important role for the unconventional myosin, Myo1c, in promoting insulin-mediated glucose uptake in cultured adipocytes. Our observations suggest that depletion of Myo1c in cultured adipocytes results in a significant reduction in the ability of adipocytes to take up glucose following insulin treatment, suggesting Myo1c is required for insulin-mediated glucose uptake. A plausible mechanism by which Myo1c promotes glucose uptake in adipocytes has been suggested by further work from our laboratory in which expression of fluorescently-tagged Myo1c in cultured adipocytes induces significant membrane ruffling at the cell periphery, insulin-independent GLUT4 translocation to the cell periphery, and accumulation of GLUT4 in membrane ruffling regions. Taken together Myo1c seems to facilitate glucose uptake through remodeling of cortical actin. In the first part of this thesis I, in collaboration with others, uncovered a possible mechanism through which Myo1c regulates adipocyte membrane ruffling. Here we identified a novel protein complex in cultured adipocytes, comprising Myo1c and the mTOR binding partner, Rictor. Interestingly our studies in cultured adipocytes suggest that the Rictor-Myo1c complex is biochemically distinct from the Rictor-mTOR complex of mTORC2. Functionally, only depletion of Rictor but not Myo1c results in decreased Akt phosphorylation at serine 473, but depletion of either Rictor or Myo1c results in compromised cortical actin dynamic events. Furthermore we observed that whereas the overexpression of Myo1c in cultured adipocytes causes remarkable membrane ruffling, Rictor depletion in cells overexpressing Myo1c significantly reduces these ruffling events. Taken together our findings suggest that Myo1c, in conjunction with Rictor, modulates cortical actin remodeling events in cultured adipocytes. These findings have implications for GLUT4 trafficking as GLUT4 has been previously observed to accumulate in Myo1c-induced membrane ruffles prior to fusion with the plasma membrane. During our studies of adipocyte function we noticed that current siRNA electroporation methods present numerous limitations. To silence genes more effectively we employed a lentivirus-mediated shRNA delivery system, and to standardize this technology in cultured adipocytes we targeted Myo1c and MAP4K4. Using this technology we were able to achieve clear advantages over siRNA oligonucleotide electroporation techniques in stability and permanence of gene silencing. Furthermore we showed that the use of lentiviral vectors in cultured adipocytes did not affect insulin signaling or insulin-mediated glucose uptake events. Despite our inability to use lentiviral vectors to achieve gene silencing in mice we were able to achieve adipose tissue-specific gene silencing effects in mice following manipulation of the lentiviral conditional silencing vector, and then crossing resulting founders with aP2-Cre mice. Interestingly however, only founders from the MAP4K4 conditional shRNA vector, but not founders from the Myo1c conditional shRNA vector, showed gene knockdown, possibly due to position-effect variegation. Taken together, findings from these studies are important because they present an alternative means of achieving gene silencing in cultured adipocytes, with numerous advantages not offered by siRNA oligonucleotide electroporation methods. Furthermore, the in vivo, adipose tissue-specific RNAi studies offer a quick, inexpensive, and less technically challenging means of achieving adipose tissue-specific gene ablations relative to traditional gene knockout approaches.

Bone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 were shown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4 and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes and keratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinase expression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associated with increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as well as by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulate melanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These data reveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, in melanocytes behaviour.