ABSTRACT
Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.
Subject(s)
Freezing , Melanins , Melanocytes , Wnt Signaling Pathway , beta Catenin , Animals , Guinea Pigs , Mice , Apoptosis , beta Catenin/metabolism , Cell Survival , Glycogen Synthase Kinase 3 beta/metabolism , Hyperpigmentation/therapy , Interferon Type I , Intramolecular Oxidoreductases/metabolism , Melanins/biosynthesis , Melanocytes/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Monophenol Monooxygenase/metabolism , Oxidoreductases/metabolism , Ultraviolet RaysABSTRACT
The 1064-nm Q-switched Nd:YAG laser is demonstrated to be effective for non-ablative skin rejuvenation, but the molecular mechanism by which dermis responses to laser-induced damage and initiates skin remodeling is still unclear. HaCaT cells and 3T3 skin fibroblasts were irradiated with the 1064-nm Q-switched Nd:YAG laser at the different doses. Then, cells were collected and lysed for PCR and Western blot analysis. Cell viability was detected by Cell Counting Kit-8 (CCK-8) before and after laser irradiation. The expressions of S100A8, advanced glycosylation end product-specific receptor (RAGE) and inflammatory cytokines in two cell lines were markedly upregulated after laser treatments. The PCR, Western blot, and ELISA analysis showed the significant increase of type I and III procollagen in the 3T3 cells treated with the 1064-nm laser. Interestingly, si S100A8 effectively inhibited the expression of cytokines and collagen, while S100A8 treatments significantly increased them. P-p38 and p-p65 levels were also elevated after the 1064-nm laser irradiation, which is positively related with S100A8. Cell viability and reactive oxygen species (ROS) levels were not changed, while the content of superoxidase dismutase (SOD) in two cells was increased after laser irradiation. Our results demonstrated that the overexpression of S100A8 induced by the 1064-nm laser irradiation triggered inflammatory reactions in skin cells. The inflammatory microenvironment and improvement of skin antioxidant capacity contribute to new collagen synthesis in the skin cells. Thus, S100A8 was required for laser-induced new collagen synthesis in skin cells. p38/MAPK and NF-κB signal pathways were involved in S100A8-mediated inflammatory reactions in response to laser irradiation.
Subject(s)
Calgranulin A/metabolism , Lasers, Solid-State/therapeutic use , Skin/metabolism , Skin/radiation effects , Animals , Calgranulin A/genetics , Cell Line , Cell Shape/radiation effects , Cell Survival/radiation effects , Collagen/biosynthesis , Collagen/metabolism , Female , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , Inflammation/pathology , Keratinocytes/metabolism , Keratinocytes/radiation effects , Mice , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , Rejuvenation , Signal Transduction , Superoxide Dismutase/metabolism , Up-Regulation/genetics , Up-Regulation/radiation effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
It has been reported that the proteasome activator REGγ is associated with multiple oncogenic pathways in human cancers. However, the role of REGγ in the development of melanoma and the underlying mechanisms remain unclear. In this study, we attempted to investigate the effects of REGγ on human melanoma cell proliferation in vitro and in vivo. We demonstrated that knockdown of REGγ inhibited melanoma cell growth and arrested melanoma cell at G1 phase. Furthermore, depletion of REGγ also inhibited the xenograft growth of human melanoma. Mechanistically, REGγ activates Wnt/ß-catenin signal pathway by degrading GSK-3ß in melanoma cell lines and mouse models. Transient knockdown of ß-catenin effectively blocked cell proliferation in REGγ wild-type melanoma cells. In human melanoma samples, REGγ was overexpressed and positively correlated with ß-catenin levels. This study demonstrates that REGγ is a central molecule in the development of melanoma by regulating Wnt/ß-catenin pathway. This suggests that targeting REGγ could be an alternative therapeutic approach for melanoma.
Subject(s)
Autoantigens/genetics , Cell Proliferation/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Melanoma/genetics , Proteasome Endopeptidase Complex/genetics , Wnt Signaling Pathway/genetics , beta Catenin/metabolism , Animals , Autoantigens/metabolism , Cell Line, Tumor , Female , G1 Phase Cell Cycle Checkpoints , Gene Knockdown Techniques , Glycogen Synthase Kinase 3 beta/genetics , Humans , Melanoma/metabolism , Mice , Neoplasm Transplantation , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering , beta Catenin/geneticsABSTRACT
Our recent studies suggest a role for the proteasome activator REG (11S regulatory particles, 28-kDa proteasome activator)γ in the regulation of tumor protein 53 (p53). However, the molecular details and in vivo biological significance of REGγ-p53 interplay remain elusive. Here, we demonstrate that REGγ-deficient mice develop premature aging phenotypes that are associated with abnormal accumulation of casein kinase (CK) 1δ and p53. Antibody array analysis led us to identify CK1δ as a direct target of REGγ. Silencing CK1δ or inhibition of CK1δ activity prevented decay of murine double minute (Mdm)2. Interestingly, a massive increase of p53 in REGγ(-/-) tissues is associated with reduced Mdm2 protein levels despite that Mdm2 transcription is enhanced. Allelic p53 haplodeficiency in REGγ-deficient mice attenuated premature aging features. Furthermore, introducing exogenous Mdm2 to REGγ(-/-) MEFs significantly rescues the phenotype of cellular senescence, thereby establishing a REGγ-CK1-Mdm2-p53 regulatory pathway. Given the conflicting evidence regarding the "antiaging" and "proaging" effects of p53, our results indicate a key role for CK1δ-Mdm2-p53 regulation in the cellular aging process. These findings reveal a unique model that mimics acquired aging in mammals and indicates that modulating the activity of the REGγ-proteasome may be an approach for intervention in aging-associated disorders.
Subject(s)
Aging, Premature/etiology , Aging, Premature/metabolism , Casein Kinase Idelta/metabolism , Proteasome Endopeptidase Complex/deficiency , Aging, Premature/pathology , Animals , Autoantigens/genetics , Female , Genes, p53 , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Proteasome Endopeptidase Complex/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Skin/metabolism , Skin/pathology , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The 800-nm diode laser is widely used for hair removal and also promotes collagen synthesis, but the molecular mechanism by which dermis responses to the thermal damage induced by the 800-nm diode laser is still unclear. Ten 2-month-old mice were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for PCR, Western blot analysis, and histological study at day 3 or 30 after laser irradiation. The expression of S100a8 and its two receptors (advanced glycosylation end product-specific receptor, RAGE and toll-like receptor 4, TRL4) was upregulated at day 3 after laser treatments. P-p65 levels were also elevated, causing the increase of cytokine (tumor necrosis factor, TNF-α and interleukin 6, IL-6) and MMPs (MMP1a, MMP9). At day 30, PCR and Western blot analysis showed significant increase of type I and III procollagen in the dermis treated with laser. Importantly, skin structure was markedly improved in the laser-irradiated skin compared with the control. Thus, it seemed that S100a8 upregulation triggered NF-κB signal pathway through RAGE and TLR4, responding to laser-induced dermis wound healing. The involvement of the NF-κB pathway in MMP gene transcription promoted the turnover of collagen in the skin, accelerating new collagen synthesis.
Subject(s)
Collagen/metabolism , Lasers, Semiconductor/therapeutic use , Low-Level Light Therapy/methods , Signal Transduction , Skin/radiation effects , Animals , Calgranulin A/metabolism , Collagen/genetics , Cosmetic Techniques , Female , Gene Expression/radiation effects , Interleukin-6/metabolism , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , NF-kappa B/metabolism , Skin/metabolism , Skin Aging/radiation effects , Tumor Necrosis Factor-alpha/metabolism , Up-RegulationABSTRACT
Phlorizin is well known to inhibit sodium/glucose cotransporters in the kidney and intestine for the treatment of diabetes, obesity and stress hyperglycaemia. However, the effects of phlorizin against ultraviolet B (UVB) irradiation and its molecular mechanism are still unknown. We examined the effects of phlorizin on skin keratinocyte apoptosis, reactive oxygen species (ROS) production, pro-inflammatory responses after UVB irradiation and the changes of some signal molecules by in vitro and in vivo assay. We observed that phlorizin pretreatments inhibited HaCaT cell apoptosis and overproduction of ROS induced by UVB. Phlorizin also decreased the expression of UVB-induced pro-inflammatory cytokines, such as interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and interleukin-8 (IL-8) at the mRNA level. Topical application of phlorizin on UVB-exposed skin of nude mice prevented the formation of scaly skin and erythema, inhibited the increase of epidermal thickness and reduced acute inflammation infiltration in skin. Additionally, PCR, Western blot and immunohistochemical data showed that phlorizin reversed the overexpression of cyclooxygenase-2 (Cox-2) induced by UVB irradiation both in vitro and in vivo. The activation of p38 and JNK mitogen-activated protein kinases (MAPK) after UVB irradiation was also inhibited by phlorizin. These findings suggest that phlorizin is effective in protecting skin against UVB-induced skin damage by decreasing ROS overproduction, Cox-2 expression and the subsequent excessive inflammation reactions. It seemed that p38 and JNK MAPK signal pathways are involved in the regulation of the protective function of phlorizin.
Subject(s)
MAP Kinase Signaling System , Phlorhizin/pharmacology , Skin Aging/drug effects , Skin Aging/radiation effects , Skin/drug effects , Skin/metabolism , Ultraviolet Rays/adverse effects , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Line , Cyclooxygenase 2 Inhibitors/pharmacology , Humans , Inflammation/etiology , Inflammation/pathology , Inflammation/prevention & control , Mice , Mice, Inbred BALB C , Mice, Nude , Reactive Oxygen Species/metabolism , Skin Aging/physiologyABSTRACT
BACKGROUND/OBJECTIVES: The objective of this study was to investigate the effects of daidzein on collagen metabolism and its underlying mechanism in cultured skin fibroblast and nude mouse skin. METHODS: Skin fibroblasts were exposed to different concentrations of daidzein (0.5-50 µg/mL) for 24 h or 48 h, respectively. Female nude mice were treated topically with 200 µg/mL daidzein once a day for 6 weeks. Cell viability and cell cycle were determined by MTT and flow cytometer. The transcriptional activity of collagen type I was evaluated and the expression of procollagen, matrix metalloproteinase-1 (MMP1) and MMP2 were measured by real-time polymerase chain reaction. A Western blot analysis was applied to detect the levels of phosphorylated-Smad2 and Smad3. RESULTS: In the daidzein-treated cells the expression of type I procollagen increased markedly while the expressions of MMP1, and MMP2 was significantly inhibited. Additionally, the mouse skin showed more collagen deposition after daidzein treatment. The levels of transforming growth factor (TGF)-ß, phosphorylated-smad2 and smad3 were also higher in the daidzein treated skin fibroblasts than in the controls. CONCLUSIONS: The results showed that daidzein treatment can increase skin collagen synthesis and inhibit collagen degradation in vitro and in vivo. It seems that TGF-ß/smad signalling pathways play an important role in daidzein-induced collagen accumulation.
Subject(s)
Collagen Type I/drug effects , Fibroblasts/drug effects , Isoflavones/pharmacology , Phytoestrogens/pharmacology , Signal Transduction/drug effects , Animals , Cell Line , Cell Survival/drug effects , Collagen Type I/genetics , Collagen Type I/metabolism , Female , Fibroblasts/metabolism , Humans , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 2/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation/drug effects , Procollagen/genetics , Smad2 Protein/drug effects , Smad2 Protein/metabolism , Smad5 Protein/drug effects , Smad5 Protein/metabolism , Transcription, Genetic/drug effects , Transforming Growth Factor beta/drug effects , Transforming Growth Factor beta/metabolismABSTRACT
Hordenine is effective in treating hyperpigmentation, fighting diabetes and resisting fibrosis and acute inflammation. However, the role of Hordenine on hair growth has not been elucidated. Here, we found that Hordenine treatments significantly enhance proliferation of primary mouse dermal-papilla cells (DPCs) and increase the activity of DPCs in a dose-dependent manner. Additionally, Hordenine markedly promoted the elongation of the hair shaft in the model of in vitro-cultured mouse vibrissa follicle and accelerated hair regrowth in a mouse model of depilation-induced hair regeneration. Real-time PCR, Western Blot and immunofluorescent assays showed that nuclear ß-catenin and its downstream gene expression such as Lef1, Axin2, Cyclin D1 and ALP were greatly upregulated in DPCs and mouse hair follicles after Hordenine treatments. Moreover, the increased DPCs' proliferation and hair shaft elongation of cultured mouse vibrissa follicles induced by Hordenine treatments were rescued by a Wnt/ß-catenin signaling inhibitor, FH535. These data indicate that Hordenine can effectively enhance DPCs' activity and accelerate hair regrowth through activating the Wnt/ß-catenin signaling pathway. Therefore, these findings suggest Hordenine/its derivatives may be potentially used for preventing and treating alopecia in the future.
Subject(s)
Hair Follicle , Wnt Signaling Pathway , Mice , Animals , beta Catenin/genetics , beta Catenin/metabolism , Cells, Cultured , Hair/metabolism , Cell ProliferationABSTRACT
The proteasome activator REGγ mediates a shortcut for the destruction of intact mammalian proteins. The biological roles of REGγ and the underlying mechanisms are not fully understood. Here we provide evidence that REGγ regulates cellular distribution of p53 by facilitating its multiple monoubiquitylation and subsequent nuclear export and degradation. We also show that inhibition of p53 tetramerization by REGγ might further enhance cytoplasmic relocation of p53 and reduce active p53 in the nucleus. Furthermore, multiple monoubiquitylation of p53 enhances its physical interaction with HDM2 and probably facilitates subsequent polyubiquitylation of p53, suggesting that monoubiquitylation can act as a signal for p53 degradation. Depletion of REGγ sensitizes cells to stress-induced apoptosis, validating its crucial role in the control of apoptosis, probably through regulation of p53 function. Using a mouse xenograft model, we show that REGγ knockdown results in a significant reduction of tumor growth, suggesting an important role for REGγ in tumor development. Our study therefore demonstrates that REGγ-mediated inactivation of p53 is one of the mechanisms involved in cancer progression.
Subject(s)
Autoantigens/metabolism , Proteasome Endopeptidase Complex/metabolism , Tumor Suppressor Protein p53/metabolism , Active Transport, Cell Nucleus , Animals , Autoantigens/genetics , Cell Line, Tumor , Cell Nucleus/chemistry , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/chemistry , Cytoplasm/genetics , Cytoplasm/metabolism , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Proteasome Endopeptidase Complex/genetics , Protein Binding , Protein Multimerization , Protein Transport , Random Allocation , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , UbiquitinationABSTRACT
BACKGROUND: Yohimbine hydrochloride (YH) is a prescription drug to treat erectile dysfunction. It also had potential in fighting high blood pressure and diabetic neuropathy as well as promoting weight loss. OBJECTIVE: The aim of the study is to investigate the anti-melanogenic function of yohimbine hydrochloride and reveal its underlying molecular mechanism. METHODS: B16F10 mouse melanoma cells, Melan-A murine melanocyte, Zebrafish embryos and C57BL/6 mouse ear skins were treated with different concentrations of YH. The extracellular and cellular melanin content was detected by spectrometry. The expression of microphthalmia-associated transcription factor (MITF), tyrosinase and the activities of Wnt/ß-catenin and p38/MAPK signal pathways were determined by RT-qPCR, Western blot analysis and immunofluorescent staining. RESULTS: Melanin production could be effectively inhibited by YH at the safe concentration in vitro and in vivo. Q-PCR and WB results showed that the expression of MITF and tyrosinase were strongly downregulated after YH treatments along with the reduction of tyrosinase activity. YH markedly inhibited ß-catenin nuclear accumulation and p38 phosphorylation in B16F10 cells compared with the untreated controls. Importantly, the increase of MITF expression induced by ß-catenin activator BIO and p38 activator anisomycin could be fully reversed by YH treatments. CONCLUSIONS: These results indicate that YH can function as an anti-melanogenic agent, at least in part, by inhibiting Wnt/ß-catenin and p38/MAPK signal pathways. Therefore, YH may be potentially used as a skin-whitening compound for preventing hyperpigmentation disorders in the future.
Subject(s)
Melanins , Melanoma, Experimental , Animals , Cell Line, Tumor , Mice , Mice, Inbred C57BL , Microphthalmia-Associated Transcription Factor , Monophenol Monooxygenase , Signal Transduction , Yohimbine , Zebrafish , beta Catenin , p38 Mitogen-Activated Protein KinasesABSTRACT
P2RY6 is highly expressed in skin keratinocytes, but its function in skin diseases is unclear. We use a two-step chemical induction method to induce mouse skin tumor formation. Multiple in vitro and in vivo assays were used to explore the role of P2RY6 in skin tumors. We report that P2ry6-deficient mice exhibit marked resistance to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papilloma formation compared with wild-type mice. Consistent with these findings, epidermal hyperplasia in response to TPA was suppressed in the P2ry6-knockout or MRS2578 (P2RY6 antagonist)-treated mice. The dramatic decrease in hyperplasia and tumorigenesis due to P2ry6 disruption was associated with the suppression of TPA-induced keratinocyte proliferation and inflammatory reactions. Notably, P2ry6 deletion prevented the TPA-induced increase in YAP nuclear accumulation and its downstream gene expression in an MST/LATS1-dependent manner. On TPA stimulation, enhanced activation of MAPK/extracellular signalâregulated kinase kinase 1 and ß-catenin were also impaired in P2ry6-knockout primary keratinocytes, tumor tissues, or MRS2578-treated HaCaT cells. Moreover, mutual promotion of the YAP and ß-catenin signaling pathways was observed in normal skin cells treated with TPA, whereas P2ry6 deletion could inhibit their crosstalk by regulating MAPK/extracellular signalâregulated kinase kinase 1. Thus, P2RY6 is a critical positive regulator of skin tumorigenesis through the modulation of the Hippo/YAP and Wnt/ß-catenin signaling pathways.
Subject(s)
Receptors, Purinergic P2 , Skin Neoplasms , Wnt Signaling Pathway , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Carcinogenesis/pathology , Hyperplasia/pathology , Keratinocytes/metabolism , Mice , Receptors, Purinergic P2/metabolism , Skin/pathology , Skin Neoplasms/chemically induced , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tetradecanoylphorbol Acetate/toxicity , YAP-Signaling Proteins/metabolism , beta Catenin/metabolismABSTRACT
The 800-nm diode laser is used clinically for hair removal and leg vein clearance. However, the effects of the laser on skin collagen synthesis have not been established. This study aims to research whether the 800-nm laser can be used for non-ablative rejuvenation and its possible mechanism by using an animal model. Eight 2-month-old rats were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for histological study and dermal thickness measurement at day 30 after laser irradiation. The expression of procollagen type I, III, IV, transforming growth factor-ß (TGF-ß), Smad2, 3, 4, and phosphorylated-Smad2, 3 in the rat skin was analyzed 24 h after completing all laser treatments by using RT-PCR and Western blot. Immunohistochemistry was performed to evaluate the content of type I collagen in the skin at day 30 after laser irradiation. The 800-nm diode laser treatments markedly improved the histological structure and increased dermal thickness compared to the non-irradiated controls. Laser irradiation at 40 J/cm(2) significantly up-regulated the expression of procollagen type I and IV, TGF-ß and Smad2, 3, 4. The p-Smad2 and p-Smad3 levels were also enhanced in the laser-irradiated skin. The 800-nm laser is effective in improving skin structure and inducing skin new collagen expression. New collagen synthesis induced by the 800-nm laser was mediated by TGF-ß/Smad signaling pathway. Thus, it seemed that the 800-nm laser could be used for non-ablative rejuvenation in the future.
Subject(s)
Collagen/biosynthesis , Lasers, Semiconductor/therapeutic use , Low-Level Light Therapy , Skin/metabolism , Skin/radiation effects , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Base Sequence , Collagen/genetics , DNA Primers/genetics , Female , Rats , Rats, Sprague-Dawley , Signal Transduction/radiation effects , Skin Aging/genetics , Skin Aging/physiology , Skin Aging/radiation effects , Smad Proteins/genetics , Transforming Growth Factor beta/genetics , Up-Regulation/radiation effectsABSTRACT
BACKGROUND: REGγ acts as a proteasome activating factor mediating proteasome degradation of substrate proteins in an ATP and ubiquitination independent manner and also as an important regulator of cell cycle, proliferation and apoptosis. Hair cycle involves dynamic, continuous morphological changes of three stages (anagen, catagen and telogen). OBJECTIVE: The function of REGγ in hair cycling is still unclear. METHODS: Here, we used REGγ knockout 293 T cells, inducible 293WT and 293N151Y cell, REGγ knockout mice to identify the novel molecular mechanism of REGγ in regulating hair follicle stem cells. RESULTS: In the present study, we found that REGγ deletion markedly delayed the transition of hair follicles from telogen to anagen and hair regeneration in mice. We also observed significant decrease of hair follicle stem cell number, stem-like property and proliferation ability. Interestingly, the results from real-time PCR, FACS, Western Blot and immunofluorescent analysis showed that REGγ deletion could greatly downregulate Lgr5 expression in the hair follicles. Meanwhile, REGγ was demonstrated to directly interact with LHX2 and promotes its degradation. Importantly, REGγ specific deletion in Lgr5+ stem cells induced the marked delay of hair regeneration after depilation. CONCLUSION: These data together indicate that REGγ was a new mediator of Lgr5 expression in hair follicle at least partly by promoting the degradation of its suppressive transcription factor LHX2. It seemed that REGγ regulated hair anagen entry and hair regrowth by activating Lgr5 positive hair follicle stem cells.
Subject(s)
Autoantigens/metabolism , Hair Follicle/growth & development , LIM-Homeodomain Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Stem Cells/physiology , Transcription Factors/metabolism , Animals , Apoptosis/physiology , Autoantigens/genetics , Cell Proliferation/physiology , Gene Knockdown Techniques , HEK293 Cells , Hair Follicle/metabolism , Humans , Keratinocytes , Male , Mice , Mice, Knockout , Primary Cell Culture , Proteasome Endopeptidase Complex/genetics , Proteolysis , Receptors, G-Protein-Coupled/metabolism , Regeneration/physiology , Up-Regulation/physiologyABSTRACT
Cultured human skin fibroblasts were irradiated twice successively with the 1.5 J/cm(2) of 532-nm and 1,064-nm lasers, respectively. The mRNA of procollagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), heat-shock protein 70 (Hsp70), interleukin-6 (IL-6) and transforming growth factor beta (TGF-beta) were analyzed at 24 and 48 h post-irradiation by using RT-PCR. Both lasers significantly increased the expression of type I and III procollagen, TIMP1, and TIMP2, but decreased MMP1 and MMP2 expression. The 1,064-nm laser initiated TGF-beta expression while the 532-nm laser elicited the increase of Hsp70 and IL-6. The increase/decrease rates of procollagen, TIMPs and MMPs for the 1,064-nm laser were higher than that of the 532-nm laser. Thus, both lasers effectively accelerated collagen synthesis and inhibited collagen degradation. Collagen synthesis induced by the 1,064-nm laser might be partly due to the upregulation of TGF-beta expression, while the increase of Hsp70 and IL-6 might be partly responsible for collagen synthesis stimulated by the 532-nm laser. With the parameters used in this study, the 1,064-nm infrared laser is more effective in promoting the beneficial molecular activities than the 532-nm visible laser.
Subject(s)
Collagen/metabolism , Collagen/radiation effects , Lasers, Solid-State/therapeutic use , Low-Level Light Therapy , Skin/metabolism , Skin/radiation effects , Base Sequence , Cells, Cultured , Collagen/genetics , DNA Primers/genetics , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Expression/radiation effects , HSP70 Heat-Shock Proteins/genetics , Humans , Interleukin-6/genetics , Matrix Metalloproteinases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Skin Aging/radiation effects , Tissue Inhibitor of Metalloproteinases/genetics , Transforming Growth Factor beta/geneticsABSTRACT
It is known that LGR4 plays an important role in hair follicle (HF) development, but the impact of LGR4 on the hair cycle is still unclear. In this study, we have found that K14-Cre-mediated skin epithelia-specific deletion of Lgr4 results in delayed anagen entry during the physiological hair cycle and compromised HF regeneration upon transplantation. We show that, although Lgr4 deletion does not appear to affect the number of quiescent HF stem cells, it leads to reduced numbers of LGR5+ and actively proliferating stem cells in the HFs. Moreover, LGR4-deficient HFs show molecular changes consistent with decreased mTOR and Wnt signaling but upregulated BMP signaling. Importantly, the reactivation of the protein kinase B pathway by injecting the protein kinase B activator SC79 in Lgr4-/- mice can effectively reverse the hair cycle delay. Together, these data suggest that LGR4 promotes the normal hair cycle by activating HF stem cells and by influencing the activities of multiple signaling pathways that are known to regulate HF stem cells. Our study also implicates LGR4 as a potential target for treating hair disorder in the future.
Subject(s)
Adult Stem Cells/physiology , Hair Follicle/growth & development , Receptors, G-Protein-Coupled/metabolism , Acetates/administration & dosage , Adult Stem Cells/drug effects , Animals , Benzopyrans/administration & dosage , Bone Morphogenetic Proteins/metabolism , Cell Proliferation/drug effects , Female , Hair Follicle/cytology , Hair Follicle/drug effects , Male , Mice , Mice, Knockout , Models, Animal , Proto-Oncogene Proteins c-akt/agonists , Proto-Oncogene Proteins c-akt/metabolism , Receptors, G-Protein-Coupled/genetics , Regeneration/drug effects , Skin/cytology , Skin/metabolism , TOR Serine-Threonine Kinases/metabolism , Up-Regulation , Wnt Signaling Pathway/drug effectsABSTRACT
Anaplastic thyroid cancer (ATC) is the most aggressive human thyroid malignancy, characterized by dedifferentiation and resistance to radioiodine therapy. The underlying mechanisms regulating ATC dedifferentiation are largely unknown. Here, we show that REGγ, a noncanonical proteasome activator highly expressed in ATC, is an important regulator of differentiation in ATC cells. Ablation of REGγ significantly restored expression of thyroid-specific genes, enhanced iodine uptake, and improved the efficacy of 131I therapy in ATC xenograft models. Mechanistically, REGγ directly binds to the TGF-ß signaling antagonist Smad7 and promotes its degradation, leading to the activation of the TGF-ß signal pathway. With gain- and loss-of-function studies, we demonstrate that Smad7 is an important mediator for the REGγ function in ATC cell dedifferentiation, which is supported by expression profiles in human ATC tissues. It seems that REGγ impinges on repression of thyroid-specific genes and promotion of tumor malignancy in ATC cells by activating the TGF-ß signal pathway via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for allowing radioiodine therapy in anaplastic thyroid cancer patients with poor prognosis.
Subject(s)
Autoantigens/metabolism , Proteasome Endopeptidase Complex/metabolism , Smad7 Protein/metabolism , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Neoplasms/metabolism , Transforming Growth Factor beta/metabolism , Cell Differentiation , Cell Line , Humans , Signal Transduction , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Carcinoma, Anaplastic/radiotherapy , Thyroid Neoplasms/pathology , Thyroid Neoplasms/radiotherapyABSTRACT
BACKGROUND: Fargesin is commonly used in the treatment of allergic rhinitis, inflammation, sinusitis and headache. OBJECTIVE: The aim of the study is to investigate a new function of fargesin against melanin production and its underlying molecular mechanism. METHODS: B16F10 mouse melanoma cells, Melan-a and human epidermal melanocytes were treated with different concentrations of fargesin for the indicated time. The extracellular and cellular melanin content was detected by spectrometry at 490 nm and 405 nm, respectively. RT-qPCR and Western blot analysis were used to exam the expression of melanogenic enzymes and the activities of PKA/CREB and p38 MAPK pathway components. Zebrafish was used as an in vivo model for studying the function of fargesin in regulating melanogenesis. RESULTS: Fargesin effectively inhibited melanin production at moderate dose in mouse B16F10 melanoma cells, normal melanocyte cell lines and zebrafish. The expression of microphthalmia-associated transcription factor (MITF), its downstream melanogenic enzymes and tyrosinase activity were also strongly reduced by fargesin. Moreover, the increase of melanin production induced by UVB and forskolin could be fully reversed by fargesin treatment. Fargesin also effectively inhibited the activation of PKA/CREB and p38 MAPK as well as their interactions, which in turn is responsible for the expression of MITF and melanogenic enzymes. CONCLUSIONS: These results show that fargesin can function as an anti-melanogenic agent, at least in part, by inhibiting PKA/CREB and p38/MAPK signaling pathways. Therefore, fargesin and its derivatives may potentially be used for preventing hyperpigmentation disorders in the future.
Subject(s)
Benzodioxoles/pharmacology , Hyperpigmentation/drug therapy , Lignans/pharmacology , MAP Kinase Signaling System/drug effects , Melanins/biosynthesis , Melanocytes/drug effects , Animals , Benzodioxoles/therapeutic use , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Drug Evaluation, Preclinical , Embryo, Nonmammalian , Humans , Lignans/therapeutic use , Melanocytes/metabolism , Mice , Models, Animal , ZebrafishABSTRACT
Purpose: Colorectal cancer is one of the most commonly diagnosed cancers closely associated with inflammation and hyperactive growth. We previously demonstrated a regulatory circuit between the proteasome activator REGγ and NF-kappaB (NF-κB) during colon inflammation, known to be important in the development of colitis-associated cancer as well as sporadic colorectal cancer. How the inflammatory microenvironment affects the Hippo pathway during colorectal cancer development is largely unknown.Experimental Design: Here, we used REGγ-deficient colon cancer cell lines, REGγ knockout mice, and human colorectal cancer samples to identify the novel molecular mechanism by which REGγ functions as an oncoprotein in the development of colorectal cancer.Results: REGγ can directly interact with Lats1 and promote its degradation, which facilitates Yes-associated protein (YAP) activation in colon cancer cells. REGγ deficiency significantly attenuated colon cancer growth, associated with decreased YAP activity. Suppression of tumor growth due to REGγ depletion was overcome by constitutively active YAP. Surprisingly, reciprocal activation of the YAP and NF-κB pathways was observed in human colon cancer cells. REGγ overexpression was found in over 60% of 172 colorectal cancer specimens, highly correlating with the elevation of YAP and p65. Postoperative follow-up revealed a significantly lower survival rate in patients with concomitantly high expression of REGγ, YAP, and p-p65.Conclusions: REGγ could be a master regulator during colorectal cancer development to promote YAP signaling and reinforce cross-talks between inflammation and growth pathways, and REGγ might be a new marker for prognosis of colorectal cancer patients. Clin Cancer Res; 24(8); 2015-25. ©2018 AACR.
Subject(s)
Autoantigens/metabolism , Cell Cycle Proteins/metabolism , Colonic Neoplasms/metabolism , NF-kappa B/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Transcription Factors/metabolism , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/mortality , Colonic Neoplasms/pathology , Disease Models, Animal , Heterografts , Hippo Signaling Pathway , Humans , Mice , Prognosis , Protein Binding , Proteolysis , Survival AnalysisABSTRACT
Forkhead box D3 (FOXD3), as a transcriptional repressor, is well known to be involved in the regulation of development. Although FoxD3 is associated with several cancers, its role in colon cancer and the underlying mechanism are still unclear. Here, we first showed that FOXD3 knockdown dramatically increased the proliferation of human colon cancer cells, enhanced cell invasive ability and inhibited cell apoptosis. In vivo xenograft studies confirmed that the FOXD3-knockdown cells were more tumorigenic than the controls. Silencing FOXD3 markedly activated EGFR/Ras/Raf/MEK/ERK pathway in human colon cancer cells. In addition, blocking EGFR effectively decreased the activity of MAPK induced by FOXD3 knockdown. In human cancer tissue, the expression of FOXD3 was reduced, however, the EGFR/Ras/Raf/MEK/ERK pathway was activated. Our study indicates that FOXD3 may play a protective role in human colon formation by regulating EGFR/Ras/Raf/MEK/ERK signal pathway. It is proposed that FOXD3 may have potential as a new therapeutic target in human colon cancer treatment.
Subject(s)
Colonic Neoplasms/pathology , Down-Regulation , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Animals , Caco-2 Cells , Cell Proliferation , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , MAP Kinase Signaling System , Mice , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Transplantation , raf Kinases/metabolism , ras Proteins/metabolismABSTRACT
BACKGROUND: Naringin is a bioflavonoid and has free radical scavenging and anti-inflammatory properties. METHODS: We examined the effects of naringin on skin after ultraviolet radiation B (UVB) irradiation and the signal pathways by in vitro and in vivo assay. RESULTS: HaCaT cells pretreated with naringin significantly inhibited UVB induced-cell apoptosis and production of intracellular reactive oxygen species (ROS). The expressions of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) in HaCaT cells pretreated with naringin were decreased compared with the only UVB group. Also, the activation of p38 induced by UVB in HaCaT cells was reversed by naringin treatments. The inhibition function of naringin on p38 activity was more obvious than JNK. In vivo, topical treatments with naringin prevented the increase of epidermal thickness, IL-6 production, cell apoptosis and the overexpression of COX-2 in BALB/c mice skin irradiated with UVB. Naringin treatment also markedly blocked the activation of p38 in response to UVB stimulation in the mouse skin. CONCLUSION: Naringin can effectively protect against UVB-induced keratinocyte apoptosis and skin damage by inhibiting ROS production, COX-2 overexpression and strong inflammation reactions. It seemed that naringin played its role against UVB-induced skin damage through inhibition of mitogen-activated protein kinase (MAPK)/p38 activation.