Young Kwan Sung

Glypican‐3 is overexpressed in human hepatocellular carcinoma

To identify candidate genes that could be used as diagnostic and therapeutic targets for hepatocellular carcinoma (HCC), we searched for the genes that are overexpressed in HCC by combining representational difference analysis and microarray. Genes such as glypican‐3 (GPC3), insulin‐like growth factor 2, long‐chain fatty‐acid‐coenzyme A ligase 4, farnesyl diphosphate synthase were frequently identified in our screening. Northern blot analysis with these four genes confirmed their overexpression in HCC. Among them we found that GPC3 transcript is upregulated in six out of seven cases of HCC. Immunoblot and immunohistochemical staining using polyclonal anti‐GPC3 antibodies further confirmed that GPC3 protein is indeed increased in HCC tumor samples. We also found that GPC3 is secreted into culture media from cell lines derived from HCC. We conclude that GPC3 is a good molecular marker for HCC. (Cancer Sci 2003; 94: 259–262)

Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation

The discovery of stem cells in the adult central nervous system raises questions concerning the neurotrophic factors that regulate postnatal neuronal development. Olfactory receptor neurons (ORNs) are a useful model, because they are capable of robust neurogenesis throughout adulthood. We have investigated the role of leukemia inhibitory factor (LIF) in postnatal neuronal development by using ORNs as a model. LIF is a multifunctional cytokine implicated in various aspects of neuronal development, including phenotype determination, survival, and in response to nerve injury. LIF-deficient mice display significant increases, both in the absolute amount and in the number of cells expressing olfactory marker protein, a marker of mature ORNs. The maturation of ORNs was significantly inhibited by LIF in vitro. LIF activated the STAT3 pathway in ORNs, and transfection of ORNs with a dominant negative form of STAT3 abolished the effect of LIF. These findings demonstrate that LIF negatively regulates ORN maturation via the STAT3 pathway. Thus, LIF plays a critical role in controlling the transition of ORNs to maturity. Consequently, a population of ORNs is maintained in an immature state to facilitate the rapid repopulation of the olfactory epithelium with mature neurons during normal cell turnover or after injury.

Plunc, a Member of the Secretory Gland Protein Family, Is Up-regulated in Nasal Respiratory Epithelium after Olfactory Bulbectomy

Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy. We isolated the rat homologue ofplunc, a murine gene highly expressed in lung and nasopharyngeal regions, by this method. Rat plunc encodes a 270-amino acid protein containing a putative signal peptide.plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization.plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis. Antibodies against Plunc detected a 31-kDa protein in lung, heart, and spleen. Rat nasal epithelium displayed robust immunoreactivity that was highly localized to the microvilli layer of respiratory epithelium. The expression of plunc was up-regulated after bulbectomy in respiratory epithelium. We also detected secreted plunc in rat and human mucus. Sequence and homology analyses suggest that Plunc is a member of the secretory gland protein family with putative bactericidal/bacteriostatic function. This is the first protein found in respiratory epithelium whose expression is regulated by olfactory neuronal injury and may provide protection against infection subsequent to injury.

Dickkopf 1 Promotes Regression of Hair Follicles

Recently, we suggested that Dickkopf 1 (DKK-1) is a pathogenic mediator involved in male pattern baldness. As premature catagen onset is a key characteristic of male pattern baldness, in this study, we evaluated whether DKK-1 has a role as a catagen inducer in hair cycling. Herein, we report that recombinant human DKK-1 (rhDKK-1) injection into the hypodermis of mice during anagen caused premature onset of catagen, whereas neutralizing DKK-1 antibody delayed anagen-to-catagen transition in mice. Moreover, treatment with rhDKK-1 led to a decrease in final hair follicle length, whereas DKK-1 antibody led to an increase compared with control animals. In addition, DKK-1 and DKK-1 messenger RNA expression is most upregulated in follicular keratinocytes of late anagen in depilation-induced hair cycle progression. Moreover, we observed that rhDKK-1 blocks canonical Wnt-mediated activation of β-catenin signaling and induces the proapoptotic protein Bax, resulting in apoptosis in outer root sheath keratinocytes. Taken together, our data strongly suggest that DKK-1 is involved in anagen-to-catagen transition in the hair cycle by regulating the activity of follicular keratinocytes.

A Guide to Studying Human Hair Follicle Cycling In Vivo

Hair follicles (HFs) undergo life-long cyclical transformations, progressing through stages of rapid growth (anagen), regression (catagen), and relative “quiescence” (telogen). Since HF cycling abnormalities underlie many human hair growth disorders, the accurate classification of individual cycle stages within skin biopsies is clinically important and essential for hair research. For preclinical human hair research purposes, human scalp skin can be xenografted onto immunocompromised mice to study human HF cycling and manipulate long-lasting anagen in vivo. While available for mice, a comprehensive guide on how to recognize different human hair cycle stages in vivo is lacking. Here, we present such a guide, which uses objective, well-defined, and reproducible criteria and integrates simple morphological indicators with advanced, (immuno)-histochemical markers. This guide also characterizes human HF cycling in xenografts and highlights the utility of this model for in vivo hair research. Detailed schematic drawings and representative micrographs provide examples of how best to identify human HF stages, even in sub-optimally sectioned tissue, and practical recommendations are given for designing human-on-mouse hair cycle experiments. Thus, this guide seeks to offer a benchmark for human hair cycle stage classification, for both hair research experts and newcomers to the field.

Minoxidil activates β-catenin pathway in human dermal papilla cells: A possible explanation for its anagen prolongation effect

BACKGROUND It is believed that the length of the actively growing phase of the anagen hair cycle mainly contributes to hair length. Recent studies showed that maintenance of β-catenin activity in the dermal papilla cells (DPCs) enables hair follicles to keep actively growing. Topical minoxidil treatment promotes hair growth in men with androgenetic alopecia, suggesting that minoxidil may prolong the actively growing phase of the anagen hair cycle. OBJECTIVE To investigate whether minoxidil prolongs the anagen hair cycle in mice and, if so, to investigate whether minoxidil activates β-catenin pathway in human DPCs. METHODS Dorsal skins of C57BL/6 mice were depilated to synchronize the hair cycle. After 10 days, 3% minoxidil were topically applied daily for 10 days. Sections of back skins were stained with hematoxylin and eosin. Hair follicles were graded and hair cycle score (HCS) was calculated. Cultured human DPCs were transiently transfected with the β-catenin responsive TCF reporter plasmid (pTopflash) and corresponding negative control reporter (pFopflash) to assess the activity of β-catenin signaling by minoxidil. Immunofluorescence staining and immunoblot were performed to examine the expression and localization of β-catenin in the presence or absence of minoxidil. Phosphorylation of GSK3β, PKA and PKB were also examined by immunoblot after minoxidil treatment. RT-PCR analysis and immunoblot were employed to investigate the expression of β-catenin pathway targets in DPCs, such as Axin2, Lef-1, and EP2. RESULTS Modest extension of anagen phase thereby delay of catagen progression was observed by application of minoxidil in mice. Minoxidil stimulated the transcriptional activity of pTopflash but not pFopflash. Nuclear accumulation of β-catenin was also observed after minoxidil treatment. Immunoblot further showed that minoxidil treatment increases the phosphorylation of GSK3β, PKA and PKB. Moreover, minoxidil induced Axin2, Lef-1, and EP2 expression. CONCLUSION Our results strongly suggest that minoxidil extends the anagen phase by activating β-catenin activity in the DPCs.

Dihydrotestosterone-Inducible IL-6 Inhibits Elongation of Human Hair Shafts by Suppressing Matrix Cell Proliferation and Promotes Regression of Hair Follicles in Mice

Autocrine and paracrine factors are produced by balding dermal papilla (DP) cells following dihydrotestosterone (DHT)-driven alterations and are believed to be key factors involved in male pattern baldness. Herein we report that the IL-6 is upregulated in balding DP cells compared with non-balding DP cells. IL-6 was upregulated 3  hours after 10-100  nM DHT treatment, and ELISA showed that IL-6 was secreted from balding DP cells in response to DHT. IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) were expressed in follicular keratinocytes, including matrix cells. Recombinant human IL-6 (rhIL-6) inhibited hair shaft elongation and suppressed proliferation of matrix cells in cultured human hair follicles. Moreover, rhIL-6 injection into the hypodermis of mice during anagen caused premature onset of catagen. Taken together, our data strongly suggest that DHT-inducible IL-6 inhibits hair growth as a paracrine mediator from the DP.

Fatty acid‐CoA ligase 4 is overexpressed in human hepatocellular carcinoma

Fatty acid‐CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified arachidonic acid (AA) level in cells. It has been shown that FACL4 blocks apoptosis and promotes colon carcinogenesis by lowering the cellular level of unesterified AA. Consistent with this, FACL4 is upregulated in colon adenocarcinoma. The status of FACL4 in other tumors including hepatocellular carcinoma (HCC) is not known. Here, we report that FACL4 is overexpressed in human HCC compared with adjacent normal liver tissues. FACL4 mRNA and protein were overexpressed in 5 out of 12 (41.7%) and 3 out of 8 (37.5%) cases of HCC, respectively. Immunohistochemical staining showed strong fine granular intracytoplasmic staining in tumor cells, whereas we observed occasional weak staining in normal liver tissues surrounding the tumors. We found that 14 out of 37 (37.8%) HCC expressed moderate to strong FACL4 immunostaining. Both normal adult and fetal liver tissues showed very weak to no detectable staining, whereas 3 out of 10 (30%) cirrhotic livers expressed weak staining. In addition, we found that 4 out of 8 (50%) human hepatoma cell lines expressed high levels of FACL4 by northern blot analysis. Our results show that FACL4 is a new molecular marker for HCC and suggest that the FACL4 pathway may be involved in liver carcinogenesis. (Cancer Sci 2003; 94: 421–424)

Ultraviolet B Preconditioning Enhances the Hair Growth-Promoting Effects of Adipose-Derived Stem Cells Via Generation of Reactive Oxygen Species

Hypoxia induces the survival and regenerative potential of adipose-derived stem cells (ASCs), but there are tremendous needs to find alternative methods for ASC preconditioning. Therefore, this work investigated: (1) the ability of low-dose ultraviolet B (UVB) radiation to stimulate the survival, migration, and tube-forming activity of ASCs in vitro; (2) the ability of UVB preconditioning to enhance the hair growth-promoting capacity of ASCs in vivo; and (3) the mechanism of action for ASC stimulation by UVB. Although high-dose UVB decreased the proliferation of ASCs, low-dose (10 or 20 mJ/cm(2)) treatment increased their survival, migration, and tube-forming activity. In addition, low-dose UVB upregulated the expression of ASC-derived growth factors, and a culture medium conditioned by UVB-irradiated ASCs increased the proliferation of dermal papilla and outer root sheet cells. Notably, injection of UVB-preconditioned ASCs into C(3)H/HeN mice significantly induced the telogen-to-anagen transition and increased new hair weight in vivo. UVB treatment significantly increased the generation of reactive oxygen species (ROS) in cultured ASCs, and inhibition of ROS generation by diphenyleneiodonium chloride (DPI) significantly attenuated UVB-induced ASC stimulation. Furthermore, NADPH oxidase 4 (Nox4) expression was induced in ASCs by UVB irradiation, and Nox4 silencing by small interfering RNA, like DPI, significantly reduced UVB-induced ROS generation. These results suggest that the primary involvement of ROS generation in UVB-mediated ASC stimulation occurs via the Nox4 enzyme. This is the first indication that a low dose of UVB radiation and/or the control of ROS generation could potentially be incorporated into a novel ASC preconditioning method for hair regeneration.

L-Ascorbic acid 2-phosphate promotes elongation of hair shafts via the secretion of insulin-like growth factor-1 from dermal papilla cells through phosphatidylinositol 3-kinase

Background  l‐Ascorbic acid 2‐phosphate (Asc 2‐P), a derivative of l‐ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice.