Tomohisa Hirobe

Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.

How are proliferation and differentiation of melanocytes regulated

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte‐macrophage colony‐stimulating factor. Melanocyte differentiation is also stimulated by alpha‐melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.

Short- and long-term effects of low-dose prenatal X-irradiation in mouse cerebral cortex, with special reference to neuronal migration

Abstract To elucidate the short- and long-term effects of ionizing radiation on cell migration in the developing cerebral cortex, we labeled proliferating cells on day 14 of gestation of mice with bromodeoxyuridine (BrdU) followed by a single exposure to 0.1–1 Gy of X-rays. The brains of embryos on day 17 and offspring at 2, 3 and 8 weeks after birth were processed for BrdU immunohistochemistry to trace the movements of BrdU-labeled cells. The location of BrdU-labeled neurons in the cerebral cortex was quantitatively analyzed between irradiated animals and non-irradiated controls. We have demonstrated that the initial migration of BrdU-labeled cells from the matrix cell zone towards the cortical plate during embryonic periods was decelerated when exposed to X-rays of 0.25, 0.5 and 1 Gy on embryonic day 14, and that aberrantly placed neurons in the cerebral neocortex were noted in younger animals that were irradiated prenatally, whereas such derangement was less pronounced in mature animals. These observations suggest that some modification process might have occurred during the postnatal period.

A cytogenetic approach to the effects of low levels of ionizing radiations on occupationally exposed individuals

The aim of the present study was to assess occupationally induced chromosomal damage in hospital workers exposed to low levels of ionizing radiation. Thirty-two interventional cardiologists, 36 nuclear medicine physicians and 33 conventional radiologists were included in this study, along with 35 healthy age- and sex-matched individuals as the control group. We used conventional metaphase chromosome aberration (CA) analysis, cytokinesis-block micronucleus (MN) assay as important biological indicators of ionizing radiation exposure. Occupational dosimetry records were collected over the last year (ranged from 0.25 to 48mSv) and their whole life exposure (ranged from 1.5 to 147mSv). The results showed significantly higher frequencies of dicentric and acentric CAs (p<0.001) and MN (p<0.01) in all exposed groups than in the controls. Taking all the confounding factors into account, no obvious trend of increased chromosomal damages as a function of either duration of employment, exposed dose, sex or age was observed. Interventional cardiologists had the highest rates of CA and MN frequencies between the worker groups, though the differences were not significant. These results indicate that long term exposure to low dose ionizing radiation could result in DNA damage. Hence, the personnel who work in the hospitals should carefully apply the radiation protection procedures.

Stimulation of the proliferation and differentiation of mouse pink-eyed dilution epidermal melanocytes by excess tyrosine in serum-free primary culture

The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink‐eyed dilution (p) locus (black, C57BL/10JHir‐P/P) and their congenic mutant mouse (pink‐eyed dilution, C57BL/10JHir‐p/p) were cultured with a serum‐free melanocyte growth medium supplemented with additional L‐tyrosine (Tyr) from initiation of the primary culture. L‐Tyr inhibited the proliferation of P/P melanocytes in a dose‐dependent manner, whereas L‐Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L‐Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose‐dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L‐Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L‐Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L‐Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5‐S‐cysteinyldopa (5‐S‐CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/P melanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L‐Tyr, but difficult to accumulate them in melanosomes. J. Cell. Physiol. 191: 162–172, 2002.

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum‐free melanoblast/melanocyte‐proliferation medium supplemented with dibutyryl adenosine 3′:5′‐cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti‐LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.

Effects of fibroblast-derived factors on the proliferation and differentiation of human melanocytes in culture

BACKGROUND Although keratinocyte-derived factors are known to promote the proliferation and differentiation of human epidermal melanocytes, it is not fully understood whether fibroblast-derived factors work in a similar way. OBJECTIVE The aim of this study is to clarify whether fibroblast-derived factors are involved in regulating the proliferation and differentiation of human melanocytes with or without keratinocytes using serum-free culture system. METHODS Human epidermal melanoblasts and melanocytes were cultured in a serum-free growth medium supplemented with fibroblast-derived factors such as keratinocyte growth factor (KGF) with or without keratinocytes, and the effects of KGF on the proliferation and differentiation of melanocytes were studied. RESULTS KGF stimulated the proliferation of melanoblasts in the presence of dibutyryl cAMP (DBcAMP), basic fibroblast growth factor (bFGF), transferrin (Tf), and endothelin-1 (ET-1). Although KGF stimulated the differentiation, melanogenesis, and dendritogenesis in the presence of DBcAMP, Tf, and ET-1 without keratinocytes, KGF required the presence of keratinocytes for the stimulation of melanocyte proliferation. CONCLUSION These results suggest that fibroblast-derived KGF stimulates the proliferation of human melanoblasts in synergy with cAMP, bFGF, Tf, and ET-1, the differentiation of melanocytes in synergy with cAMP, Tf, and ET-1, and the proliferation of melanocytes in synergy with cAMP, Tf, ET-1, and undefined keratinocyte-derived factors.

Neural crest cells retain their capability for multipotential differentiation even after lineage-restricted stages.

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage‐restricted precursors. We analyzed the differentiation of early‐stage NC‐like cells derived from Sox10‐IRES‐Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit− cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)‐glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10‐IRES‐Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit− cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage‐restricted stages. Developmental Dynamics 240:1681–1693, 2011.

The effect of chalone on the cell cycle in the epidermis during wound healing.

Abstract A cut was made on the ear conch of mouse and an extract containing epidermal chalone was injected subcutaneously 2 days later. The time changes after the chalone administration in the number of cells labeled with 3 H-thymidine, in the number of grains on labeled cells and in the number of mitoses within the regenerating epidermis surrounding the wound were investigated by means of autoradiography (ARG). Grain counts decreased temporarily in early phase (0–2 h) after chalone injection. This decrease in grain count resulted in a decrease in the number of labeled cells on the ARG of a short exposure but not in that on the ARG of a long exposure. A decrease in the number of labeled cells on the ARG of a long exposure was evident at 6 h when the grain counts reverted to a level similar to the control without chalone. The number of mitoses reached a minimum at 2 h and then recovered quickly, indicating a rapid disappearance of the inhibition of cells in G 2 from entering M phase. Mitoses decreased again thereafter, presumably as a result caused by inhibition of cells in the preceding S phase from completing DNA synthesis. The extract made similarly from liver or kidney affected neither the mitotic nor the DNA synthetic activities. These results indicate that the epidermal chalone or chalones inhibit the epidermal cell proliferation in, at least, 3 different processes of the cell cycle; the DNA synthesis in S phase, the transition from G 1 to S phase and the transition from G 2 to M phase.

ACTH4–12 is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum‐free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.