Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro . In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit⁺CD45⁻ cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.     

An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea-induced Kitl(Steel) alleles

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced Kitl(Sl) alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved alpha-helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.     

An allelic series of mutations in the Kit ligand gene of mice. II. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral blood cells of Kitl(Steel) mice

The ligand for the Kit receptor tyrosine kinase is Kit ligand (Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), which is encoded at the Steel (Sl) locus of mice. Previous studies revealed that Kitl(Sl) mutations have semidominant effects; mild pigmentation defects and macrocytic, hypoplastic anemia occur in heterozygous mice, and more severe pigmentation defects and anemia occur in homozygotes. Lethality also occurs in mice homozygous for severe Kitl(Sl) mutations. We describe the effects of seven new N-ethyl-N-nitrosourea (ENU)-induced Kitl(Sl) mutations and two previously characterized severe Kitl(Sl) mutations on pigmentation, peripheral blood cells, and mouse survival. Mice heterozygous for each of the nine mutations had reduced coat pigmentation and macrocytosis of peripheral blood. In the case of some of these mutations, however, red blood cell (RBC) counts, hemoglobin concentrations, and hematocrits were normal in heterozygotes, even though homozygotes exhibited severely reduced RBC counts and lethality. In homozygous mice, the extent of anemia generally correlates with effects on viability for most Kitl(Sl) mutations; i.e., most mutations that cause lethality also cause a more severe anemia than that of mutations that allow viability. Interestingly, lethality and anemia were not directly correlated in the case of one Kitl(Sl) mutation.     

Secondary c‐Kit mutations confer acquired resistance to RTK inhibitors in c‐Kit mutant melanoma cells

Activation of the c‐Kit receptor tyrosine kinase is rare in melanoma, but occurs in 20‐40% of melanoma arising on mucosal membranes, acral skin and skin with chronic sun‐induced damage. Many activating c‐Kit mutations have been shown to be highly sensitive to imatinib mesylate, although the majority of patients with c‐Kit mutant melanoma eventually progress on this inhibitor. We examined acquired resistance to imatinib and the newer generation inhibitor nilotinib in resistant c‐kit mutant melanoma sublines. Four imatinib‐resistant and six nilotinib‐resistant sublines had acquired additional, secondary c‐Kit mutations. The secondary A829P c‐Kit mutation rendered cells resistant to imatinib, but did not suppress the activity of the tyrosine kinase inhibitors nilotinib and dasatinib. Sublines with an additional T670I c‐Kit mutation showed resistance to imatinib, nilotinib and dasatinib, but responded to sunitinib. The concurrent inhibition of the MAPK and PI3K pathways was also effective at promoting apoptosis in the parent and derived resistant sublines. Our data provide a rationale for treating patients with melanoma progressing on imatinib or nilotinib with alternative RTK inhibitors or inhibitors targeting the MAPK and PI3K signalling cascades.     

Temporal and molecular separation of the kit receptor tyrosine kinase's roles in zebrafish melanocyte migration and survival

The Kit receptor tyrosine kinase is required by vertebrate melanocytes for their migration and survival. The relationship between these developmental roles of Kit, however, remains poorly understood. Here, we use two genetic approaches to demonstrate that Kit's roles in the migration and survival of embryonic melanocytes in the zebrafish (Danio rerio) are temporally and functionally independent. We use a temperature-sensitive kit mutation to show that kit promotes melanocyte migration and survival during distinct stages of development. These experiments additionally reveal that melanocyte migration is neither necessary nor sufficient for subsequent survival. We also identify kit alleles that molecularly separate kits roles in migration and survival. These results suggest that the melanocyte changes its response to Kit receptor signaling and function during development, first to promote migration, then to promote survival through distinct Kit-dependent mechanisms.     

内皮素-2促进体外培养的绵羊皮肤黑色素细胞增殖及黑色素生成

内皮素（endothelin,ET）及其受体在黑色素细胞成熟分化时起着有效的促进作用.然而,内皮素-2（ET-2）在黑色素生成的作用方面,还处于争论中或报道不一致.在此,我们研究ET-2对体外培养的绵羊皮肤黑色素细胞增殖和黑色素生成的影响.比较实验组ET-2（1,10,100 nmol/L）与空白对照组,通过MTT法和Ando等的方法检测出黑色素细胞的增殖率和黑色素含量显著增加.荧光定量PCR和Western 印迹分别检测出内皮素受体B(Bdnrb);酪氨酸激酶受体(Kit);酪氨酸酶(Tyr);酪氨酸相关蛋白-1(Tyrp-1)基因的mRNA水平及蛋白水平的相对表达量显著增加.这些数据表明,ET-2可能促进绵羊的皮肤黑色素细胞增殖和黑色素生成.     

A specific endoplasmic reticulum export signal drives transport of stem cell factor (Kitl) to the cell surface

Stem cell factor, also known as Kit ligand (Kitl), belongs to the family of dimeric transmembrane growth factors. Efficient cell surface presentation of Kitl is essential for the migration, proliferation, and survival of melanocytes, germ cells, hemopoietic stem cells, and mastocytes. Here we demonstrate that intracellular transport of Kitl to the cell surface is driven by a motif in the cytoplasmic tail that acts independently of the previously described basolateral sorting signal. Transport of Kitl to the cell surface is controlled at the level of the endoplasmic reticulum (ER) and requires a C-terminal valine residue positioned at a distance of 19-36 amino acids from the border between the transmembrane and cytoplasmic domains. Deletion or substitution of the valine with other hydrophobic amino acids results in ER accumulation and reduced cell surface transport of Kitl at physiological expression levels. When these mutant proteins are overexpressed in the ER, they are transported by bulk flow to the cell surface albeit at lower efficiency. A fusion construct between Kitl and the green fluorescent protein-labeled extracellular domain of a temperature-sensitive mutant of vesicular stomatitis virus G protein revealed the valine-dependent recruitment into coat protein complex II-coated ER exit sites and vesicular ER to Golgi transport in living cells. Thus the C-terminal valine defines a specific ER export signal in Kitl. It is responsible for the capture of Kitl at coat protein complex II-coated ER exit sites, leading to subsequent cell surface transport under physiological conditions.     

Photodynamic therapy inhibits melanogenesis through paracrine effects by keratinocytes and fibroblasts

Photodynamic therapy (PDT) is a treatment option for skin cancer and premalignant skin diseases and exhibits rejuvenation effects, including reducing fine wrinkles and whitening, on aged skin. In this study, we investigated the mechanism underlying the whitening effects of PDT on melanocytes (MCs) in vitro and in vivo. Exposure of MCs to PDT in vitro reduced their melanin content and tyrosinase activity without, however, affecting cell survival. Interestingly, melanogenesis was also inhibited by exposing MCs to conditioned media of PDT‐treated keratinocytes or dermal fibroblasts. This paracrine effect was likely due to a decreased release of melanocyte‐stimulating cytokines such as Kit ligand and hepatocyte growth factor from these cells. Furthermore, we observed that PDT reduced mottled hyperpigmentation of photoaged patient skin in vivo, highlighting the clinical importance of skin whitening by PDT.     

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.     

The phosphatidyl inositol 3-kinase pathway is central to the pathogenesis of Kit-activated melanoma

Mouse Kit L575P, the ortholog of human KIT L576P, a common KIT mutation found in human melanoma was expressed in an immortalized but non-transformed mouse Ink4a-Arf-deficient melanocyte cell line. The resultant Ink4a-Arf-deficient Kit L575P-expressing melanocytes exhibited increased proliferation, the ability to grow in soft agar, and increased migration. When these cells were injected subcutaneously into NOD/SCID/gamma(c) mice, melanomas arose in 5 of 7 (71%) mice. One of seven mice (14%) injected with these cells developed metastatic disease. Evaluation of signal transduction pathways downstream of constitutively activated Kit L575P revealed striking activation of the phosphatidyl inositol 3-kinase (PI3K) pathway. Inhibition of the PI3K pathway pharmacologically or genetically abolished the transformation phenotypes gained by the L575P single mutant. These studies validate this Kit L575P-activated model of melanoma and establish the PI3K pathway as a dominant signaling pathway downstream of Kit in melanoma.     

A role for kit receptor signaling in Leydig cell steroidogenesis

Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo.     

Regulation of cellular function through stem cell factor and c‐Kit receptor in the cerebellar cell culture

Evidence that stem cell factor (SCF) and c‐Kit receptor tyrosine kinase expressed in the cerebellum during postnatal development, suggests a possible contribution of the SCF/Kit signaling pathway in the cerebellar development. In the present study, we prepared cerebellar cultures from C57Bl/6J mouse at postnatal day 6 to investigate the role of c‐Kit receptor and SCF in regulation of cell growth and viability in the postnatal cerebellar cells. SCF increased the number of survival cells and density of calbindin and GFAP expression in the immunoblot analysis. Treatment with c‐Kit antibody accelerated cellular loss in serum‐free media and decreased the expression of calbindin and GFAP. The recovery effects of SCF on the cellular proliferation and the expression of functional proteins in the cultures containing c‐Kit antibody suggest an involvement of SCF/Kit pathways in the control of postnatal development of cerebellar cells.     

Melanocytes in development and cancer

Melanocytes are pigment‐producing cells in the skin of humans and other vertebrates. A number of genes involved in melanocyte development and vertebrate pigmentation have been characterized, largely through studies of a diversity of pigment mutations in a variety of species. Embryonic development of the melanocyte initiates with cell fate specification in the neural crest, which is then followed by cell migration and niche localization. Many genes involved in melanocyte development have also been implicated in the development of melanoma, an aggressive and fatal form of skin cancer that originates in the melanocyte. Although early stage melanomas that have not spread to the lymph nodes can be excised with little risk of recurrence, patients diagnosed with metastatic melanoma have a high mortality rate due to the resistance of most tumors to radiotherapy and chemotherapy. Transformed melanocytes that develop into melanomas proliferate abnormally and often begin to grow radially in the skin. Vertical growth can then follow this radial growth, leading to an invasion through the basement membrane into the underlying dermis and subsequent metastasis. It is still unclear, however, how a normal melanocyte becomes a melanoma cell, and how melanoma utilizes the properties of the normal melanocyte and its progenitors in its progression. The goal of this mini‐review is to highlight the role of melanocyte developmental pathways in melanoma, and to discuss recent studies and tools being used to illuminate this connection. J. Cell. Physiol. 222:38–41, 2010. © 2009 Wiley‐Liss, Inc.     

Coupling of the radiosensitivity of melanocyte stem cells to their dormancy during the hair cycle

Current studies have revealed that stem cells are more radiosensitive than mature cells. As somatic stem cells are mostly kept in a quiescent state, this conflicts with Bergonié and Tribondeau's law that actively mitotic cells are the most radiosensitive. In this study, we focused on hair graying to understand the stress‐resistance of melanocyte stem cells (McSCs). We used Dct‐H2B‐GFP transgenic mice which enables the stable visualization of McSCs and an anti‐Kit monoclonal antibody which selectively eradicates amplifying McSCs. The results demonstrate that quiescent McSCs are rather radiosensitive, but the coexistence of non‐quiescent McSCs provides the stem cell pool with radioresistance. The irradiated quiescent McSCs prematurely differentiate in the niche upon their activation without sufficiently renewing themselves for cyclic hair pigmentation. These data indicate that tissue radiosensitivity is largely dependent on the state of somatic stem cells under their local microenvironment.