Eriodictyon angustifolium extract, but not Eriodictyon californicum extract, reduces human hair greying.

OBJECTIVE: Yerba Santa (Eriodictyon angustifolium and Eriodictyon californicum) has been used for many years in traditional medicine. However, the effect of Yerba Santa on melanogenesis has not yet been investigated. We aimed to assess the biological effects of Yerba Santa on hair pigmentation. METHODS: Yerba Santa extracts were assessed for their cytological effects following X-ray irradiation treatment and then tested directly for the prevention of human hair greying. Ultra-performance liquid chromatography (UPLC) was utilized to identify the individual extract components. RESULTS: Eriodictyon angustifolium extract significantly increased melanin synthesis in the melanoma cell line through activation of the WNT/MITF/tyrosinase-signalling pathway. In contrast, E. californicum had no effect on melanin synthesis. E. angustifolium extract also demonstrated a protective effect against the damage induced by X-ray irradiation in human keratinocytes. Application of the extracts to subjects who had grey beards demonstrated a reduced number of grey beard hair per year specifically with the E. angustifolium extract. A significant decrease in grey head hair was also observed after application of E. angustifolium extract. Upregulation of gene expression related to melanin production and WNT signalling was observed after the application of E. angustifolium extract. Sterubin was the most abundant flavonoid detected by UPLC in E. angustifolium extract. In addition, sterubin showed the highest difference in terms of quantity, between E. angustifolium and E. californicum extract. CONCLUSION: Eriodictyon angustifolium extract, which is abundant in sterubin, may be suitable as a potential cosmetic and medical agent for the prevention and improvement of hair greying.

OBJECTIF: Yerba Santa (Eriodictyon angustifolium et Eriodictyon californicum) est utilisé depuis de nombreuses années en médecine traditionnelle. Cependant, l'effet de Yerba Santa sur la mélanogenèse n'a pas encore été étudié. Notre objectif était d'évaluer les effets biologiques de Yerba Santa sur la pigmentation des cheveux. MÉTHODES: Les extraits de Yerba Santa ont été évalués pour leurs effets cytologiques après un traitement d'irradiation aux rayons X, puis testés directement pour la prévention du grisonnement des cheveux humains. La chromatographie liquide ultra-performante (UPLC) a été utilisée pour identifier les composants d'extrait individuels. RÉSULTATS: L'extrait d'E. angustifolium a augmenté de manière significative la synthèse de mélanine dans la lignée cellulaire du mélanome par l'activation de la voie de signalisation WNT/MITF/tyrosinase. En revanche, E. californicum n'a eu aucun effet sur la synthèse de mélanine. L'extrait d'E. angustifolium a également démontré un effet protecteur contre les dommages induits par l'irradiation aux rayons X dans les kératinocytes humains. L'application des extraits à des sujets qui avaient une barbe grise a démontré un nombre réduit de poils gris par an spécifiquement avec l'extrait d'E. angustifolium. Une diminution significative des cheveux gris a également été observée après l'application d'extrait d'E. angustifolium. Une régulation à la hausse de l'expression des gènes liée à la production de mélanine et à la signalisation WNT a été observée après l'application d'extrait d'E. angustifolium. La stérubine était le flavonoïde le plus abondant détecté par UPLC dans l'extrait d'E. angustifolium. De plus, la stérubine a montré la plus grande différence en termes de quantité entre E. angustifolium et E. californicum. CONCLUSION: L'extrait d'E. angustifolium, qui est abondant en stérubine, peut convenir comme agent cosmétique et médical potentiel pour la prévention et l'amélioration du grisonnement des cheveux.

Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

OBJECTIVE: In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. METHODS: Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. RESULTS: High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. CONCLUSIONS: ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma.

A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus.

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunohistochemistry. Here, we show a simple system for detection and assessment of functional CAR expression in human tumor cells, using the green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 h after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy.

Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium.

An important goal for the investigation of the proliferation of mammalian cells is to establish a fully defined condition for culturing them in vitro. Here, we report establishment of a fully defined culture condition that supports the primary culture of normal c-kit+IL-7 receptor (IL-7R)+ B precursor cells without the aid of stromal cell lines. This defined culture condition contains IL-7, the ligand for c-kit, transferrin, insulin, and bovine serum albumin as protein components. By using the cell lines derived from RAG2(-/-) mice, which do not differentiate into c-kit- stage, we have evaluated the role of each protein in the cell cycle progression of c-kit+IL-7R+ B precursor cells. Since B precursor cells can grow without insulin, c-kit remains a sole functional receptor tyrosine kinase for their growth. While both c-kit ligand (KL) and IL-7 are the requisite molecules for sustained proliferation of B precursor cells, each molecule plays distinct roles. IL-7 starvation results in prompt arrest of the cells at G1. An accumulation of the cells in the mitotic phase was also detected. Thus, the major role of IL-7 is to regulate the G1/S transition and the process of cytokinesis of B precursor cells. Although prolonged KL starvation over 48 h resulted in accumulation of G1 cells, its effect could not be detected within 24 h, which is long enough for all the cells to complete one cell cycle. This suggests that KL might be involved in the cell cycle progression of B precursor cells in a manner that its signal could still be effective in the one or two cell cycles that follow. Although molecular nature of the signals underlying the present observation awaits future investigation, the method described in this report would provide a useful model system for investigating the signaling pathways that are involved in the cell cycle progression of B precursor cells.

Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

Stepwise progression of B lineage differentiation supported by interleukin 7 and other stromal cell molecules.

Growth of early B precursor cells was investigated in vitro by using rIL-7 and IL-7-defective stromal cell line PA6 as separate growth signals. B cell development proceeds through three sequential stages different from the growth signal requirement. The cells in the first stage require PA6 alone for the proliferation, and differentiate into the second stage, which requires both PA6 and IL-7 for its growth. When IL-7 is available for the cells in the second stage, they proliferate extensively on the PA6 layer, and some acquire the ability to proliferate in response to IL-7 alone. This sequential change of growth signal requirement, however, does not proceed autonomously along the time schedule. The possibility that it is primarily directed by the result of Ig gene rearrangement is considered. This mode of growth control may explain why only functional B cells are selected in the error-prone process of Ig gene rearrangement during B lineage differentiation.

Interleukin 7 production and function in stromal cell-dependent B cell development.

The role of IL-7 in the stromal cell-dependent B cell development was investigated using two stromal cell clones, ST2 and PA6; the former supports B lymphopoiesis while the latter can not. We demonstrate here that: (a) the ability of the stromal cell clone to produce IL-7 correlates well with the stromal cell activity to support B lymphopoiesis; (b) IL-7 production by ST2 is inducible rather than constitutive; (c) the IL-7-dependent B cell itself is a potent inducer of IL-7 production by ST2; (d) addition of rIL-7 to the PA6 layer renders this in vitro environment B lymphopoietic; and (e) the differentiation from early B progenitor to pre-B cell requires both IL-7 and other stromal cell molecule(s) yet to be identified.

Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor.

The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.

Characterization of dental pulp stem cells of human tooth germs.

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs.

Overexpression of retinoic acid receptor alpha suppresses myeloid cell differentiation at the promyelocyte stage.

Retinoic acid receptor (RAR) alpha is required to heterodimerize with retinoid X receptor (RXRs) in order to regulate myeloid differentiation. If so, it is expected that overexpression of normal RAR alpha may perturb the RAR alpha/RXR heterodimer formation and also the differentiation of myeloid cells. We have described here the morphology and the RA response of human RAR alpha cDNA transduced murine bone marrow cells using a retroviral vector. Most of RAR alpha transduced cells displayed promyelocyte like morphology and their proportion of c-kit expressing population was increased remarkably compared with the control (Neor gene transduced cells). Furthermore, this morphology was observed even after these cells were brought into the semisolid culture containing IL-3 alone. Interestingly, immature RAR alpha transduced cells differentiated into mature granulocytes under the condition of the high concentration of RA(10(-6) M). We did not observe any effect of RAR alpha on monocytes. These results indicate that overexpression of normal RAR alpha is sufficient for inducing maturation arrest of myeloid cell lineage that is similar to the phenotype found in the acute promyelocytic leukemia bearing PML-RAR alpha translocation.

Promotion of early osteoclastogenesis and B lymphopoiesis in the bone marrow of transgenic rats with the env-pX gene of human T-cell lymphotropic virus type I.

Human T-cell lymphotropic virus type I (HTLV-I) is associated with various clinical disorders including adult T cell leukemia, myelopathy, arthropathy. Hypercalcemia resulting from osteoclast activation and a variety of hematopoietic abnormalities have been also observed in HTLV-I infected patients, however, precise mechanism about initial trigger(s) prior to presenting symptoms is still unknown. In this study, to assess effects of HTLV-I on hematopoiesis, we analysed characteristics of early hematopoietic precursors in HTLV-I env-pX transgenic rats. Progenitor cells for osteoclasts were significantly increased even in the marrow of asymptomatic env-pX rats. Progenitors for B cells were also highly enriched, while colony forming cells (CFC) elicited by GM-CSF(CFU-GM) and M-CSF(CFU-M) were comparable to normal littermates. Following arthritis in env-pX transgenic rats, osteoclastogenesis was further augmented and the CFCs were increased. Bone marrow cells carrying adjuvant-induced arthritis retained a constant number of progenitors for osteoclast and B lymphocytes, whereas the number of CFU-GM and CFU-M increased. These results indicate that the env-pX transgene affect early stages of osteoclast and B-cell lineages prior to developing diseases, in contrast, an increase of the CFCs was caused indirectly by arthritis. This study provides a novel standpoint for the mechanisms of pathogenesis by HTLV-I.

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.

Sequence organization of repetitive sequences enriched in small polydisperse circular DNAs from HeLa cells.

A total of 36 clones were randomly selected from a recombinant DNA library of small polydisperse circular DNA (spcDNA) molecules from HeLa cells and were shown to contain repetitive sequences of different reiteration frequencies that ranged from several hundred to several hundred thousand per genome. Sequencing of representative clones revealed tandem repeats of alphoid (alpha) satellite DNA, clustered repeats of the Alu family, KpnI family sequences, tandem repeats of an alpha satellite DNA specific to the X chromosome (alpha X), and A + T-rich segments carrying short stretches of poly(A) or poly(T). DNA rearrangement was frequently found in the repetitive sequences enriched in these spcDNA clones. Short regions of homology that were patchy and inverted were often found, especially at the novel joint where spcDNA sequences are circularized. The presence of these inverted repeats suggests that HeLa spcDNAs are formed by a mechanism that involves looping out of the spcDNA region and joining of the flanking DNA by illegitimate recombination.

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.

CD9 molecule expressed on stromal cells is involved in osteoclastogenesis.

Osteoclasts are derived from hematopoietic stem cells and their development is dependent on the products of stromal cells. CD9, a member of the tetraspan transmembrane-superfamily, is expressed on both hematopoietic cells and stromal cells. Addition of antagonistic rat anti-mouse CD9 antibody (KMC8.8) to cultures inhibited osteoclastogenesis on established stromal cell layers. When rat bone marrow cells depleted of adherent stromal cells were cultured on mouse stromal cells, numerous tartrate-resistant acid phosphatase-positive multinuclear cells were observed, and KMC8.8, which recognizes mouse but not rat CD9, completely prevented the generation of osteoclasts, suggesting that the CD9 expressed on the stromal cell is essential for osteoclastogenesis. Possibly for the same reason, KMC8.8 pretreatment of the mouse macrophage-like cell line C7, which is able to differentiate into mature osteoclasts, did not inhibit subsequent C7 cell differentiation, whereas the addition of KMC8.8 to cocultures of C7 cells with stromal cells inhibited the differentiation of C7 cells into osteoclasts. Moreover, we found that blockage of a signal via CD9 on stromal cells reduced transcription of the osteoclast differentiation factor (Odf) gene, which, together with macrophage colony-stimulating factor, is essential for osteoclastogenesis. These results revealed that CD9 molecules on stromal cells play a critical role in osteoclast development, possibly by modulating the expression of Odf.

Presence of osteoclast precursors in colonies cloned in the presence of hematopoietic colony-stimulating factors.

Osteoclasts are derived from hematopoietic stem cells, but the relationship between osteoclast precursors (OCPs) and hematopoietic colony-forming cells (CFCs) has not yet been clarified. Although osteoclasts share certain cell surface markers and growth factor requirements with their macrophage and monocyte cell lineages, osteoclasts are a different lineage with regard to the requirement for signaling via c-Kit. To investigate whether CFCs are able to differentiate into osteoclasts, we performed in vitro studies of osteoclastogenesis. We performed progenitor assays in the presence of hematopoietic colony-stimulating factors. Primary colonies were plucked and examined for their potential to differentiate into osteoclasts. We found that osteoclasts are present in colonies elicited by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kB ligand (RANKL) in semisolid cultures. Moreover, a part of the cells composing the colonies elicited by granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF alone possessed the potential to differentiate into osteoclasts. These OCPs in the colonies were enriched in the c-Fms+ large-sized cell fraction and had a foamy cell morphology, like mature macrophages. A small number of cells in M-CSF-promoted and GM-CSF-promoted colonies formed secondary colonies in the semisolid medium containing these factors. The frequency of OCPs in these secondary colonies elicited by M-CSF was 10 times higher than that elicited by GM-CSF. Multiple origins of OCPs that differentiate into mature osteoclasts are proposed based on the observation that osteoclasts could be generated from OCPs that emerged from CFCs induced under different conditions or developmental stages.

Sequential requirements for SCL/tal-1, GATA-2, macrophage colony-stimulating factor, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development.

OBJECTIVE: Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells. MATERIALS AND METHODS: We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development. RESULTS: The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors. CONCLUSION: These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units.

Flt3/Flk-2 and c-Kit are not essential for the proliferation of B lymphoid progenitor cells in the bone marrow of the adult mouse.

The receptor tyrosine kinase Flk-2/Flt3 was originally cloned from hematopoietic stem cell-enriched fetal liver and placenta and is believed by some investigators to play a role in the regulation of the hematopoietic stem cell. However, targeted disruption of the flt3 gene results in a specific deficiency in early B cell progenitors. Using an antagonistic monoclonal antibody developed against the extracellular domain of Flt3, we investigated the expression and function of the molecule on B lymphoid lineage cells in the bone marrow (BM) of adult mice. Approximately 10% of B220+ cells in the BM expressed Flt3 on the cell surface, and most of the cells belonged to a pro-B cell fraction when judged by an expression pattern of CD43, heat-stable antigen, and BP-1. However, B lymphoid precursor cells that are clonable in vitro could not be enriched in the B220+/Flt3+ cell fraction sorted by flow cytometry. Furthermore, proliferation of B lymphoid precursor cells in the adult BM was not blocked by administration of the antagonistic monoclonal antibodies against Flt3 and c-Kit, suggesting that signalings mediated by Flt3 and c-Kit receptors are not essential for the proliferation of B cell progenitors in adult mouse BM.

Involvement of platelet-derived growth factor receptor-alpha in hair canal formation.

Hair follicles develop and are maintained by multiple rounds of inductive events involving interactions among various cell types within the follicles and the adjacent mesenchyme. Although evidence suggests that several growth factors, cell adhesion molecules, and transcriptional regulators are involved in those cell-cell interactions, the molecular mechanisms regulating each pivotal step of hair follicle development, such as formation of the hair germ, root sheath, sebaceous gland, and hair canal, remain largely unknown. In this study, we established the antagonistic monoclonal antibody APA5 against platelet-derived growth factor (PDGF) receptor-alpha (PDGFR-alpha) and used it to investigate the role of PDGFR-alpha in neonatal skin development. In addition to the dermal mesenchyme, a known site of PDGFR-alpha expression, immunohistologic staining of neonatal skin detected transient expression of PDGFR-alpha in the perinatal epidermis for several days. On the other hand, ligands for PDGFR-alpha were detected in epithelial cells and sebaceous glands of hair follicles. To determine whether this contiguous expression of PDGF and PDGFR-alpha in neonatal skin plays a functional role, we injected APA5 into neonates to block the function of PDGFR-alpha. Consistent with the PDGF/PDGFR-alpha expression in the neonatal skin, two defects were induced by this procedure. First, hair canal formation in the epidermis was severely suppressed. Second, the growth of dermal connective tissues and of hair follicles of pelage hairs was suppressed. These results indicate that PDGF signals are involved in both the epidermis-follicle interaction and the dermal mesenchyme-follicle interaction required for hair canal formation and the growth of the dermal mesenchyme, respectively.

Identification of the control regions for mouse c-kit gene transcription induced by retinoic acid.

The c-kit gene encodes a receptor tyrosine kinase that reveals the pleiotropic effects both in the developing embryo and in the adult animal. In this study, we characterized transcriptional control of the c-kit gene. F9 teratocarcinoma cells differentiated and expressed the c-kit gene upon exposure to retinoic acid (RA). We isolated c-kit genomic DNA from approximately 20 kb upstream to approximately 10 kb downstream of the transcribed region. We identified two control regions for c-kit gene transcription by fusing genomic sequences to bacterial chloramphenicol acetyl transferase reporter genes and introducing the resultant constructs into differentiated F9 cells. The fragments, Sac I-Apa I in the 9th intron and Xho I-Kpn I in the 13th intron induced reporter gene activities. Nucleotide sequencing of the regions showed in that there were no consensus sequences of the RA receptor or cyclic AMP-responsive elements. Cyclic AMP augmented the expression of the c-kit gene induced by RA; when combined with RA, it was less effective on the reporter gene activity. In contrast to other reports, continuous exposure to the reagents was not required for c-kit expression. These findings suggested that transcription of the c-kit gene is regulated by complex controls and that there are different control regions responsive to RA and cyclic AMP.