Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.     

Proteomics demonstration that histone H4 is a colchicine-induced retro-modulator of growth and alkaline phosphatase activity in hair follicle dermal papilla culture

Dermal papilla cells (DPCs) control the development of hair follicles via cell-cell interactions and extracellular molecules. Colchicine affected active anagen DPCs to result in hair loss in the clinical setting. The purpose of this study was to identify the retro-modulator released by DPCs exposed to sub-toxic dose of colchicine and elucidate its effect on dermal papilla culture. The molecular-weight cutoff ultrafiltration and HPLC were used to purify the components of colchicine-treated DPC secretomes and examined their ability to down-regulate the growth and alkaline phosphatase (ALP) activity of DPCs. The active product was identified by in-gel trypsin digestion, nano-LC-ESI-MS/MS and validated by Western blot to be histone H4 (P62804), which inhibited the proliferation and diminished the ALP activity of cultured DPCs. Treating DPCs with recombinant histone H4 reproduced the growth inhibition effect whereas adding antibody to immunoneutralize histone H4 abolished this growth inhibitory consequence. DPCs with high ALP activity can induce the neogenesis of hair follicles and support the hair fiber growth in vivo. Our results indicated that sub-lethal colchicine can inactivate DPCs through releasing histone H4. Through the investigation of the retro-modulation of histone H4 on dermal papillae may give implications for understanding the mechanism of colchicine-induced hair disorder.     

Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation.

Fibroblast growth factor (FGF) 5 inhibits hair growth and induces catagen in mouse hair follicles, in vivo. Given that FGF-5 receptor (FGFR1) is expressed in dermal papilla cells (DPCs), which are known to stimulate outer root sheath cell (ORSC) proliferation, we hypothesized that FGF-5 attenuates DPC-mediated ORSC proliferation. In the present study, DPCs and ORSCs were isolated from rat vibrissae, after which the effects of FGF-5 on proliferation of ORSCs cultured in DPC-conditioned medium were assessed. We first confirmed that FGFR1 was expressed in cultured DPCs and detected FGFR2-4 as well. ORSC proliferation was increased approximately twofold when the cells were cultured in DPC-conditioned medium, and the effect was unaltered by FGF-5. In addition, FGF-5 did not directly inhibit ORSC proliferation; indeed, it actually promoted proliferation of both DPCs and ORSCs. When DPCs were first activated by exposure to FGF-1 and FGF-2, which are expressed in hair follicles during anagen, ORSC proliferation observed in the resultant conditioned medium was substantially greater than in medium conditioned by unstimulated DPCs. The FGF-1-induced enhancement was reversed by FGF-5, diminishing ORSC proliferation to control levels. By contrast, the enhancement of DPC-mediated ORSC proliferation by FGF-2 was not suppressed by FGF-5. Proliferation of ORSCs did not depend on DPC proliferation, nor did FGF-1 directly promote ORSC proliferation. Dermal papillae thus appear to require activation before they will efficiently stimulate hair growth, and FGF-5 appears to inhibit hair growth and induce catagen by blocking that activation.     

Human hair growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG)

Green tea is a popular worldwide beverage, and its potential beneficial effects such as anti-cancer and anti-oxidant properties are believed to be mediated by epigallocatechin-3-gallate (EGCG), a major constituent of polyphenols. Recently, it was reported that EGCG might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5alpha-reductase activity. However, no report has been issued to date on the effect of EGCG on human hair growth. This study was undertaken to measure the effect of EGCG on hair growth in vitro and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. EGCG promoted hair growth in hair follicles ex vivo culture and the proliferation of cultured DPCs. The growth stimulation of DPCs by EGCG in vitro may be mediated through the upregulations of phosphorylated Erk and Akt and by an increase in the ratio of Bcl-2/Bax ratio. Similar results were also obtained in in vivo dermal papillae of human scalps. Thus, we suggest that EGCG stimulates human hair growth through these dual proliferative and anti-apoptotic effects on DPCs.     

6-Gingerol Inhibits Hair Shaft Growth in Cultured Human Hair Follicles and Modulates Hair Growth in Mice

Ginger (Zingiber officinale) has been traditionally used to check hair loss and stimulate hair growth in East Asia. Several companies produce shampoo containing an extract of ginger claimed to have anti-hair loss and hair growth promotion properties. However, there is no scientific evidence to back up these claims. This study was undertaken to measure 6-gingerol, the main active component of ginger, on hair shaft elongation in vitro and hair growth in vivo, and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. 6-Gingerol suppressed hair growth in hair follicles in culture and the proliferation of cultured DPCs. The growth inhibition of DPCs by 6-gingerol in vitro may reflect a decrease in the Bcl-2/Bax ratio. Similar results were obtained in vivo. The results of this study showed that 6-gingerol does not have the ability to promote hair growth, on the contrary, can suppress human hair growth via its inhibitory and pro-apoptotic effects on DPCs in vitro, and can cause prolongation of telogen phase in vivo. Thus, 6-gingerol rather than being a hair growth stimulating drug, it is a potential hair growth suppressive drug; i.e. for hair removal.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells

The progression of androgenetic alopecia is closely related to androgen-inducible transforming growth factor (TGF)-β1 secretion by hair follicle dermal papilla cells (DPCs) in bald scalp. Physiological levels of androgen exposure were reported to increase reactive oxygen species (ROS) generation. In this study, rat vibrissae dermal papilla cells (DP-6) transfected with androgen receptor showed increased ROS production following androgen treatment. We confirmed that TGF-β1 secretion is increased by androgen treatment in DP-6, whereas androgeninducible TGF-β1 was significantly suppressed by the ROSscavenger, N-acetyl cysteine. Therefore, we suggest that induction of TGF-β1 by androgen is mediated by ROS in hair follicle DPCs. [BMB Reports 2013; 46(9): 460-464]     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Differential expression, function and response to inflammatory stimuli of 11beta-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Expression, activity and function of 11beta-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11beta-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-alpha or IL-1beta (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-gamma was without effect, and there was no difference in 11beta-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production--a characteristic feature of synovial derived fibroblasts--was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11beta-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11beta-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

Hair multiplication with dermal papilla like tissue containing human dermal papilla cells

Alopecia is not a critical disease; however it is a disease that can affect the quality of life. Many remedies have been developed to cure alopecia, but only two have been approved by the FDA. Due to the steadily increasing number of young alopecia patients, the need for new therapies for curing alopecia is very high. Recent studies on cell therapy have reported using technique to treat various diseases. We introduce upgraded hair cell therapy which tested hair structure inducing activity with bioartificial dermal papilla tissue. Hair follicles contain two types of stem cells: Outer root sheath cells (ORSCs) derived epithelial cells, and dermal cells (DPCs). In this study, we reconstructed DP-like tissues (DPLTs) using cultured dermal papilla cells (DPCs) from human hair follicles. The DPLTs were produced special media (Dermal Papilla Forming Media: DPFM) conditions in vitro, which can induce epithelial stands from implanted healthy hair without DP. We tested in vivo hair-inducing with a modified hair sandwich model. Two to three weeks DPLT injection into the mouse scalp skin, we observed new hair in the injected site and detected injected human cells from DPLTs and Outer Root Sheath Cells (ORSCs) in the new hair via human Alu-DNA-specific probe. In the future, reconstructed DPLTs may be used in in vitro studies of hair development and the morphogenesis mechanism, as well as in vitro studies of the efficacy and toxicity of drugs for baldness. These tissues will be used as an alternative medicine product for hair transplantation     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Discovery of novel 5α-reductase type II inhibitors by pharmacophore modelling,virtual screening,molecular docking and molecular dynamics simulations

Benign prostatic hyperplasia (BPH) is caused by augmented levels of androgen dihydrotestosterone (DHT) which is involved in the growth of the prostate in humans. 5α-Reductase type II (5αR2) is an intracellular enzyme that catalyses the formation of DHT from testosterone; hence, the inhibition of 5αR2 has emerged as one of the most promising strategies for the treatment of BPH. In this study, a computational approach that integrates ligand-based pharmacophore modelling, virtual screening, molecular docking and molecular dynamics (MD) simulations was adopted to discover novel 5αR2 inhibitors with less side effects. After validating by Fischer's randomisation and Güner–Henry test, the best quantitative pharmacophore model (Hypo1), consisting of two hydrogen-bond acceptors and three hydrophobic features, was subsequently used as a three-dimensional-query in virtual screening to identify potential hits from Maybridge and National Cancer Institute databases. These hits were further filtered by ADMET (absorption, distribution, metabolism, elimination and toxicology) and molecular docking experiments, and their binding stabilities were validated by 10-ns MD simulations. Finally, only one hit was identified as a potential lead based on higher predicted inhibitory activity to 5αR2 compared with the most active inhibitor (finasteride). Our results further suggest that this potential lead could easily be synthesised and has structural novelty, making it a promising candidate for treating BPH.     

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.     

Oncostatin M (OSM) primes IL-13- and IL-4-induced eotaxin responses in fibroblasts: Regulation of the type-II IL-4 receptor chains IL-4Rα and IL-13Rα1

Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in regulation of various chronic inflammatory processes. Previous work has shown that OSM induces eosinophil accumulation in mouse lungs in vivo and stimulates the eosinophil-selective chemokine eotaxin-1 synergistically with IL-4 in vitro. To examine the role of receptor regulation by OSM in synergistic eotaxin-1 responses, we here examine the modulation of the type-II IL-4 receptor (IL-4Rα and IL-13Rα1) by OSM and other gp130/IL-6 cytokine family members using NIH3T3 fibroblasts and primary mouse lung fibroblasts. We first show that OSM with either IL-13 or IL-4 synergistically induces eotaxin-1 expression in a dose-dependent fashion. Analysis of IL-4Rα expression at the protein (Western blot and FACS) and RNA (TAQMAN) levels showed that OSM markedly elevates expression by 3 h. OSM enhanced IL-13Rα1 mRNA and induced a smaller but detectable increase in total IL-13Rα1 protein. Priming fibroblasts with OSM for 6 h markedly enhanced subsequent IL-13 and IL-4-induced eotaxin-1 responses and STAT6 tyrosine-641 phosphorylation. Regulation of IL-4Rα by OSM was sensitive to inhibition of the PI3′K pathway by LY294002. These studies provide novel mechanistic insights in OSM role in regulation of synergistic eotaxin-1 responses and IL-4Rα expression in fibroblasts.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.