Akinori Kawakami

Regulation of MITF stability by the USP13 deubiquitinase

The microphthalmia-associated transcription factor (MITF) is essential for melanocyte development. Mutation-induced MAPK pathway activation is common in melanoma and induces MITF phosphorylation, ubiquitination, and proteolysis. Little is known about the enzymes involved in MITF ubiquitination/deubiquitination. Here we report the identification of a deubiquitinating enzyme, named ubiquitin-specific protease 13 (USP13) that appears to be responsible for MITF deubiquitination, utilizing a short hairpin RNA library against known deubiquitinating enzymes. Through deubiquitination, USP13 stabilizes and upregulates MITF protein levels. Conversely, suppression of USP13 (through knockdown) leads to dramatic loss of MITF protein, but not messenger RNA. Through its effects on MITF deubiquitination, USP13 was observed to modulate expression of MITF downstream target genes and, thereby, to be essential for melanoma growth in soft agar and in nude mice. These observations suggest that as a potentially drugable protease, USP13 might be a viable therapeutic target for melanoma.

Key Discoveries in Melanocyte Development

Melanocytes are melanin pigment-producing cells. Mammalian melanocytes are categorized as ‘‘cutaneous’’ (follicular and epidermal) and ‘‘extracutaneous’’ (e.g., choroidal, cochlear). Epidermal melanocytes contribute to photoprotection and thermoregulation by packaging melanin pigment into melanosomes and delivering them to neighboring keratinocytes. Melanocytes are derived from the neural crest that is a migratory multipotent population that gives rise to multiple cell lineages, including neurons, glial cells, medullary secretory cells, smooth muscle cells, and bone and cartilage cells. Coat color mutants in different species have been useful for identifying genes involved in melanocyte development. Embryonic transplantation experiments played a significant role in early efforts to investigate melanocyte development. Rawles (1947) conducted a series of elegant transplantation experiments, which revealed that melanocytes originate from the neural crest. The investigator transplanted various axial levels of the embryonic central nervous system, adjacent tissues of the somite and lateral plate, and limb-bud regions, separately and in combination, from various developmental stages of black mouse embryos, to the coelom of White Leghorn chick embryos. Only tissues containing the neural crest or cells migrating from the neural crest were found to produce melanophores. In addition, Rawles found that the portions of embryo that produce pigment cells vary at different developmental stages of donor embryos. The quail nucleus exhibits condensed heterochromatin, which could serve as a marker to distinguish quail cells from chick cells. Teillet and Le Douarin (1970) studied melanoblast migration from the neural crest using a quail-chick xenograft transplantation model. The investigators transplanted different axial levels of quail neural tube and neural crest to White Leghorn chick embryos. They found that (1) at embryonic days E4 and E5, the transplanted quail cells localize mainly in the mesenchyme; (2) at E6 when the dermis and the epidermis are formed, quail cells (melanoblasts) begin to migrate into the epidermis; (3) at E9 quail cells (melanoblasts) increase their cytoplasmic volume and start producing melanin pigments; (4) at E10 and E11 quail cells (melanoblasts) localize in the basal layer of epidermis and become dendritic. Cell type-specific markers are useful tools to study the development of certain cell types. However, it is challenging to identify these markers. Steel et al. (1992) found Tyrp-2/Dct to be a specific marker of melanoblasts, the precursors of melanocytes. Using in situ hybridization, the investigators found that Tyrp-2 expression was detectable in melanoblasts as early as 10 days post coitum. The finding of a marker for early melanoblasts enabled scientists to look for the mechanisms of coat color mutations. Steel and W mutants exhibited a white spotting color pattern. W and Steel encode, respectively, a receptor tyrosine kinase, Kit, and its ligand, which is known by several different names: steel factor, stem cell factor, mast cell growth factor, and Kit ligand. The mutation Steel-dickie (Sl) is a deletion of its transmembrane and cytoplasmic domains so that only a secreted form of stem cell factor is produced. Steel et al. (1992) found that the number of melanoblasts in (Sl/ Sl) mutants began to decrease at around 11 days post coitum. They also found that the melanoblasts caudal of the optic vesicle failed to migrate toward the vesicle. These results suggested that the cell surface form of stem cell factor is important for both the survival and migration of melanoblasts. Wehrle-Haller and Weston (1995) performed in situ hybridization with Kit, Tyrp-2, and stem cell factor probes in Sl (null) and Sl mutants to examine the early dispersal and fate of melanoblasts in order to elucidate the function of stem cell factor in more detail. They concluded that soluble stem cell factor is sufficient for responsive melanoblast precursors to initiate their dispersal onto the lateral migration pathway, and that cell-bound stem cell factor was necessary for the survival of melanoblasts in the newly formed dermal mesenchyme. Dorsky et al. (1998) found that cranial neural crest cells destined to encode pigment cells were located adjacent to the Wnt-1 and Wnt-3a expression domain, whereas neurons were far from the Wnt-expressing domain in zebrafish. Most lateral cells, which become neurons when forcibly overexpressing an activated form of b-catenin, adopted a pigment-cell fate. Conversely, when the investigators overexpressed a mutant form of Tcf-3 or a dominant-negative Wnt-1 to inhibit Wnt signaling in medial neural crest cells, the number of pigment cells decreased dramatically. Thus, Dorsky et al. provided key evidence that Wnt signaling plays an essential early role in pigment cell formation.

The master role of microphthalmia-associated transcription factor in melanocyte and melanoma biology

Certain transcription factors have vital roles in lineage development, including specification of cell types and control of differentiation. Microphthalmia-associated transcription factor (MITF) is a key transcription factor for melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes to promote melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis, including genes encoding proteins involved in apoptosis (eg, BCL2) and the cell cycle (eg, CDK2). Loss-of-function mutations of MITF cause Waardenburg syndrome type IIA, whose phenotypes include depigmentation due to melanocyte loss, whereas amplification or specific mutation of MITF can be an oncogenic event that is seen in a subset of familial or sporadic melanomas. In this article, we review basic features of MITF biological function and highlight key unresolved questions regarding this remarkable transcription factor.

Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase‐related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild‐type (Rab7WT), constitutively active (Rab7Q67L) or dominant‐negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio‐distribution of melanosomal proteins between Rab7WT‐expressing cells and mutant Rab7‐expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

Spontaneous regression of bowenoid papulosis in a patient with acquired immunodeficiency syndrome after an increase in peripheral CD4+ T lymphocytes

XXX Correspondence Melanoma risk factors include insecticides and occupational exposures We applaud the excellent review by Fortes and de Vries on occupational risk factors for melanoma in the April issue of the International Journal of Dermatology. We concur that the elimination of carcinogenic exposure is vitally important in the prevention of this cancer. Previously, we have reported the association of melanoma with two other occupations, specifically chemists with high exposure to insecticides and banana plantation workers in Costa Rica, where dibromochloropropane is sprayed onto the fields from the air whilst farmers tend to their crops. Indeed, there is a strong temporal relationship between the increased usage of insecticides since World War II and the enhanced incidence of melanoma. The association of pesticides with cancer is not novel, and the International Agency for Research on Cancer lists the insecticides used occupationally which are known to be carcinogens. Nevi are derived from epidermal melanocytes, which, in turn, are derived from nerve cells from the neural crest. Given that the specific target of action of most insecticides is the nervous system, such as neurotransmitter receptors and substances, it is entirely feasible that pesticides would be a common mode for the induction of melanoma. The connection with cancer also reflects the fact that melanocytes are not simply pigment-producing cells, but also produce substances with a range of biological functions, including structural strengthening by cross-linking proteins, antimicrobial defense, photon shielding, and chemoprotection. Indeed, melanoma could almost be considered to represent a defect in (auto)immunity. In insects, which do not have antibodies, melanin is the primary mode of antimicrobial defense. In humans, the links between immunity and melanization are numerous. For example, attractin potentiates the production of α-melanocyte-stimulating hormone. In response to various stimuli, melanocytes secrete a wide range of molecules, including cytokines, melanocortin peptides, catecholamines, serotonin, and nitric oxide. These secretary products, in turn, affect numerous types of cells, including keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells. In humans, melanin is not the principal form of immune response, but it maintains its capabilities to be involved in the innate immune defense system. In addition to their involvement in human inflammation, melanocytes can also function as phagocytes against microorganisms as well as foreign material. Even in humans, melanocytes and melanosomes have the phagocytic and enzymatic machinery for antigen processing, and appear to be a component of the skin immune defense system. Melanosomes are lysosomal structures containing numerous lysosomal enzymes, including αmannosidase, acid phosphatase, β-acetylglucosaminidase, βgalactosidase, and acid lipase. It has been proposed that the major function of melanocytes, melanosomes, and melanin in human skin is to inhibit the proliferation of bacterial, fungal, and parasitic infections in the epidermis and dermis. In conclusion, more epidemiologic studies and laboratory investigations on the possible association between malignant melanoma and insecticides/various occupations are warranted.

Tfe3 and Tfeb transcriptionally regulate peroxisome proliferator-activated receptor γ2 expression in adipocytes and mediate adiponectin and glucose levels in mice

ABSTRACT Members of the MiT transcription factor family are pivotal regulators of several lineage-selective differentiation programs. We show that two of these, Tfeb and Tfe3, control the regulator of adipogenesis, peroxisome proliferator-activated receptor γ2 (Pparγ2). Knockdown of Tfeb or Tfe3 expression during in vitro adipogenesis causes dramatic downregulation of Pparγ2 expression as well as adipogenesis. Additionally, we found that these factors regulate Pparγ2 in mature adipocytes. Next, we demonstrated that Tfeb and Tfe3 act directly by binding to consensus E-boxes within the Pparγ transcriptional regulatory region. This transcriptional control also exists in vivo, as we discovered that wild-type mice in the fed state increased their expression of Tfe3, Tf3b, and Pparγ in white adipose tissue. Furthermore, Tfe3 knockout (Tfe3KO) mice in the fed state failed to upregulate Pparγ and the adiponectin gene, a Pparγ-dependent gene, confirming the in vivo role for Tfe3. Lastly, we found that blood glucose is elevated and serum adiponectin levels are suppressed in the Tfe3KO mice, indicating that the Tfe3/Tfeb/Pparγ2 axis may contribute to whole-body energy balance. Thus, we offer new insights into the upstream regulation of Pparγ by Tfe3/Tf3b and propose that targeting these transcription factors may offer opportunities to complement existing approaches for the treatment of diseases that have dysregulated energy metabolism.