Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Independent origins of fetal liver haematopoietic stem and progenitor cells.

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors-previously regarded as descendants of HSCs5-from the HLF+ precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hi cells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

Hematopoietic Stem Cell-Independent Differentiation of Mast Cells From Mouse Intraembryonic VE-Cadherin+ Cells.

Hematopoietic stem cell (HSC)-independent hematopoiesis from hemogenic endothelial cells (HECs) in the mouse embryo has been recognized as a source of tissue-resident hematopoietic cells in adult mice. Connective tissue mast cells (MCs) have been reported to originate from VE-cadherin (VE-cad)-expressing HECs in the yolk sac and embryo proper (EP) by a VE-cad-Cre-mediated lineage-tracing analysis. However, it remains unclear whether MCs are generated via a conventional HSC-dependent hematopoietic differentiation pathway, or whether through a fast-track pathway bypassing the emergence of HSCs. Here, we investigated whether EP-derived VE-cad+ cells differentiate into MCs independently of HSCs. VE-cad+ cells isolated from the embryonic day (E) 9.5-10.5 EP robustly formed connective tissue-type MCs in a newly established co-culture system using PA6 stromal cells. In contrast, bone marrow (BM) reconstitution assays of cultured cells indicated that E9.5 VE-cad+ cells did not differentiate into transplantable HSCs in this culture condition. Lymphoid-biased HSCs with a limited self-renewal capacity were occasionally detected in some cultures of E10.5 VE-cad+ cells, while MC growth was constantly observed in all cultures examined. HSCs purified from adult BM required a more extended culture period to form MCs in the PA6 co-culture than the embryonic VE-cad+ cells. Furthermore, E9.5-E10.5 VE-cad+ cells contributed to tissue-resident MCs in postnatal mice when transplanted into the peritoneal cavity of newborn mice. These results suggest that EP-derived VE-cad+ cells generate MCs independently of HSC development in vitro and possess the potential of generating connective tissue MCs in vivo, although the exact differentiation program remains unsolved.

The canonical smooth muscle cell marker TAGLN is present in endothelial cells and is involved in angiogenesis.

Elongation of vascular endothelial cells (ECs) is an important process in angiogenesis; however, the molecular mechanisms remain unknown. The actin-crosslinking protein TAGLN (transgelin, also known as SM22 or SM22α) is abundantly expressed in smooth muscle cells (SMCs) and is widely used as a canonical marker for this cell type. In the course of studies using mouse embryonic stem cells (ESCs) carrying an Tagln promoter-driven fluorescence marker, we noticed activation of the Tagln promoter during EC elongation. Tagln promoter activation co-occurred with EC elongation in response to vascular endothelial growth factor A (VEGF-A). Inhibition of phosphoinositide 3-kinase (PI3K)-Akt signaling and mTORC1 also induced EC elongation and Tagln promoter activation. Human umbilical vein endothelial cells (HUVECs) elongated, activated the TAGLN promoter and increased TAGLN transcripts in an angiogenesis model. Genetic disruption of TAGLN augmented angiogenic behaviors of HUVECs, as did the disruption of TAGLN2 and TAGLN3 genes. Tagln expression was found in ECs in mouse embryos. Our results identify TAGLN as a putative regulator of angiogenesis whose expression is activated in elongating ECs. This finding provides insight into the cytoskeletal regulation of EC elongation and an improved understanding of the molecular mechanisms underlying the regulation of angiogenesis.

Bone morphogenetic protein 4 differently promotes distinct VE-cadherin+ precursor stages during the definitive hematopoietic development from embryonic stem cell-derived mesodermal cells.

Definitive hematopoietic cells develop from fetal liver kinase 1 (Flk1)+ mesodermal cells during the in vitro differentiation of mouse embryonic stem cells (ESCs). VE-cadherin+CD41-CD45-(V+41-45-) hemogenic endothelial cells (HECs) and VE-cadherin+CD41+CD45- (V+41+45-) cells mediate the definitive hematopoietic development from Flk1+ cells. Bone morphogenetic protein 4 (BMP4) is known to be essential for the formation of mesoderm. However, the role of BMP4 in differentiation of the VE-cadherin+ definitive hematopoietic precursors from the mesoderm has been elusive. We addressed this issue using a co-aggregation culture of ESC-derived Flk1+ cells with OP9 stromal cells. This culture method induced V+41-45- cells, V+41+45- cells, and CD45+ cells from Flk1+ cells. V+41+45- cells possessed potential for erythromyeloid and T-lymphoid differentiation. When Flk1+ cells were cultured in the presence of a high concentration of BMP4, the generation of V+41-45- cells was enhanced. The increase in V+41-45- cells led to the subsequent increase in V+41+45- and CD45+ cells. The addition of BMP4 also increased hematopoietic colony-forming cells of various lineages. Furthermore, BMP4 promoted the expansion of V+41+45- cells independently of the preceding V+41-45- cell stage. These results suggest that BMP4 has promotive effects on the differentiation of V+41-45- HECs from Flk1+ mesodermal cells and the subsequent proliferation of V+41+45- hematopoietic precursors. These findings may provide insights for establishing a culture system to induce definitive hematopoietic stem cells from ESCs.

Glucagon-like peptide-1 analogue liraglutide facilitates wound healing by activating PI3K/Akt pathway in keratinocytes.

AIMS: Diabetes induces various skin troubles including foot ulcer. This type of skin ulcer is refractory but the pathogenesis is not so certain. Recent study show that glucagon-like peptide-1 (GLP-1) analogues reduce foot complications with diabetes (Pérez et al., 2015), however, the role of GLP-1/GLP-1R axis is not fully understood, and clear evidence of GLP-1 to facilitate wound closure is still lacking. In this study, we investigated whether a potent GLP-1R agonist liraglutide affects wound healing process. METHODS: The expression of GLP-1R in HaCaT cells were indentified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunoblotting analysis. To assess the effect on wound closure in keratinocytes, we performed in vitro scratch assay using the IncuCyte system (Essen BioSciences, Ann Arborm MI). We applied ointment containing liraglutide on full-thickness wounds in the dorsum of female balb/c mice (n = 6) until healing. To investigate the effect on PI3K/Akt pathway, we used IncuCyte system in HaCaT treated with PI3K inhibitor and Akt inhibitor. RESULTS: Keratinocytes expressed GLP-1R and liraglutide induced their migration. Liraglutide facilitated the wound healing in mice. Liraglutide upregulated keratinocyte migration via PI3K/Akt activation. CONCLUSIONS: Our study suggests that liraglutide may be a potential target drug to improve skin ulcer with diabetes through its specific receptor GLP-1.

Corrigendum to "Perillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes".

[This corrects the article DOI: 10.1155/2018/9524657.].

Perillaldehyde Inhibits AHR Signaling and Activates NRF2 Antioxidant Pathway in Human Keratinocytes.

The skin covers the outer surface of the body, so the epidermal keratinocytes within it are susceptible to reactive oxygen species (ROS) generated by environmental pollutants such as benzo(a)pyrene (BaP), a potent activator of aryl hydrocarbon receptor (AHR). Antioxidant activity is generally mediated by the nuclear factor-erythroid 2-related factor-2 (NRF2) and heme oxygenase-1 (HO1) axis in human keratinocytes. Perillaldehyde is the main component of Perilla frutescens, which is a medicinal antioxidant herb traditionally consumed in East Asia. However, the effect of perillaldehyde on the AHR/ROS and/or NRF2/HO1 pathways remains unknown. In human keratinocytes, we found that perillaldehyde (1) inhibited BaP-induced AHR activation and ROS production, (2) inhibited BaP/AHR-mediated release of the CCL2 chemokine, and (3) activated the NRF2/HO1 antioxidant pathway. Perillaldehyde is thus potentially useful for managing inflammatory skin diseases or disorders related to oxidative stress.

Protective role of 6-formylindolo[3,2-b]carbazole (FICZ), an endogenous ligand for arylhydrocarbon receptor, in chronic mite-induced dermatitis.

BACKGROUND: Chronic eczema such as atopic dermatitis imposes significant socio-econo-psychologic burdens on the affected individuals. In addition to conventional topical treatments, phototherapy is recommended for patients with extensive lesions. Although immunosuppression is believed to explain its primary effectiveness, the underlying mechanisms of phototherapy remain unsolved. Ultraviolet irradiation generates various tryptophan photoproducts including 6-formylindolo[3,2-b]-carbazole (FICZ). FICZ is known to be a potent endogenous agonist for aryl hydrocarbon receptor (AHR); however, the biological role of FICZ in chronic eczema is unknown. OBJECTIVE: To investigate the effect of FICZ on chronic eczema such as atopic dermatitis. METHODS: We stimulated HaCaT cells and normal human epidermal keratinocytes (NHEKs) with or without FICZ and then performed quantitative reverse transcriptase polymerase chain reaction, immunofluorescence, and siRNA treatment. We used the atopic dermatitis-like NC/Nga murine model and treated the mice for 2 weeks with either Vaseline® as a control, FICZ ointment, or betamethasone 17-valerate ointment. The dermatitis score, transepidermal water loss, histology, and expression of skin barrier genes and proteins were evaluated. RESULTS: FICZ significantly upregulated the gene expression of filaggrin in both HaCaT cells and NHEKs in an AHR-dependent manner, but did not affect the gene expression of other barrier-related proteins. In addition, FICZ improved the atopic dermatitis-like skin inflammation, clinical scores, and transepidermal water loss in NC/Nga mice compared with those of control mice. On histology, FICZ significantly reduced the epidermal and dermal thickness as well as the number of mast cells. Topical FICZ also significantly reduced the gene expression of Il22. CONCLUSION: These findings highlight the beneficial role of FICZ-AHR and provide a new strategic basis for developing new drugs for chronic eczema.

Aryl hydrocarbon receptor activation restores filaggrin expression via OVOL1 in atopic dermatitis.

Filaggrin (FLG) mutation is a well-confirmed genetic aberration in atopic dermatitis (AD). Genome-wide association studies on AD have revealed other susceptibility genes, for example, Ovo-like 1 (OVOL1). Nonetheless, the relation between FLG and OVOL1 is unclear. Because aryl hydrocarbon receptor (AHR; a ligand-activated transcription factor), plays a role in FLG expression in keratinocytes, we hypothesized that AHR regulates FLG expression via OVOL1. To demonstrate this mechanism, we analyzed FLG expression in OVOL1-overexpressing or OVOL1-knockdown normal human epidermal keratinocytes (NHEKs). Furthermore, we tested whether AHR activation by 6-formylindolo(3,2-b)carbazole (FICZ), an endogenous AHR ligand, or Glyteer, clinically used soybean tar, upregulates FLG and OVOL1 expression in NHEKs. We found that (1) OVOL1 regulates FLG expression; (2) AHR activation upregulates OVOL1; and (3) AHR activation upregulates FLG via OVOL1. Moreover, nuclear translocation of OVOL1 was less pronounced in AD skin compared with normal skin. IL-4-treated NHEKs, an in vitro AD skin model, also showed inhibition of the OVOL1 nuclear translocation, which was restored by FICZ and Glyteer. Thus, targeting the AHR-OVOL1-FLG axis may provide new therapeutics for AD.

6-Formylindolo[3,2-b]Carbazole Accelerates Skin Wound Healing via Activation of ERK, but Not Aryl Hydrocarbon Receptor.

Wound healing is an elaborate process composed of overlapping phases, such as proliferation and remodeling, and is delayed in several circumstances, including diabetes. Although several treatment strategies for chronic wounds, such as growth factors, have been applied, further alternatives are required. The skin, especially keratinocytes, is continually exposed to UV rays, which impairs wound healing. 6-Formylindolo[3,2-b]carbazole (FICZ) is a tryptophan photoproduct formed by UV exposure, indicating that FICZ might be one of the effectors of UV radiation. In contrast, treatment with tryptophan, the precursor for FICZ, promoted wound closure in keratinocytes. Therefore, the aim of our study was to determine the role of FICZ in wound healing. Here we showed that FICZ enhanced keratinocyte migration through mitogen-activated protein kinase/extracellular signal-regulated kinase activation, and promoted wound healing in various mouse models, including db/db mice, which exhibit wound healing impairments because of type 2 diabetes. Moreover, FICZ, the endogenous ligand of an aryl hydrocarbon receptor, accelerated migration even in the aryl hydrocarbon receptor knockdown condition and also promoted wound healing in DBA/2 mice, bearing a low-affinity aryl hydrocarbon receptor, suggesting that FICZ enhanced keratinocyte migration in a mitogen-activated protein kinase/extracellular signal-regulated kinase-dependent, but aryl hydrocarbon receptor-independent, manner. The function of FICZ might indicate the possibility of its clinical use for intractable chronic wounds.

Mild electrical stimulation with heat shock guides differentiation of embryonic stem cells into Pdx1-expressing cells within the definitive endoderm.

BACKGROUND: Because of the increasing number of diabetic patients, it is important to generate pancreatic and duodenal homeobox gene 1 (Pdx1)-expressing cells, which are capable of differentiating into pancreatic endocrine ß cells. Mild electrical stimulation was reported to modulate the differentiation of ES cells into ectoderm-derived neuronal cells or mesoderm-derived cardiac cells. RESULTS: In this study, we report that mild electrical stimulation with heat shock (MET) potentiates the differentiation of ES cells into definitive endoderm-derived Pdx1-expressing cells. MET has no effect when applied to early definitive endoderm on differentiation day 5. A 1.87-fold increase in the proportion of Pdx1-expressing cells was observed when stimulation was applied to the late definitive endoderm one day prior to the immergence of Pdx1/GFP-expressing cells on differentiation day 7. Pdx1 mRNA was also up-regulated by MET. The potentiating effect of MET synergized with activin and basic fibroblast growth factor into Pdx1-expressing cells. Moreover, MET stimulation on late definitive endoderm up-regulated heat shock protein 72 and activated various kinases including Akt, extracellular signal-regulated kinase, p38, and c-jun NH2-terminal kinase in ES cells. CONCLUSIONS: Our findings indicate that MET induces the differentiation of Pdx1-expressing cells within the definitive endoderm in a time-dependent manner, and suggest useful application for regenerative medicine.

Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes.

BACKGROUND: Dioxins and other environmental pollutants are toxic and remain in biological tissues for a long time leading to various levels of oxidative stress. Although the toxicity of these agents has been linked to activation of the aryl hydrocarbon receptor (AHR), no effective treatment has been developed. OBJECTIVE: To explore novel phytochemicals that inhibit AHR activation in keratinocytes. METHODS: Keratinocytes were used in this study because the skin is one of the organs most affected by dioxin and other environmental pollutants. HaCaT cells, which are a human keratinocyte cell line, and normal human epidermal keratinocytes were stimulated with benzo[a]pyrene to induce AHR activation, and the effects of traditional Japanese Kampo herbal formulae were analyzed. Quantification of mRNA, western blotting, immunofluorescence localization of molecules, siRNA silencing, and visualization of oxidative stress were performed. RESULTS: Cinnamomum cassia extract and its major constituent cinnamaldehyde significantly inhibited the activation of AHR. Cinnamaldehyde also activated the NRF2/HO1 pathway and significantly alleviated the production of reactive oxygen species in keratinocytes. The inhibition of AHR signaling and the activation of antioxidant activity by cinnamaldehyde operated in a mutually independent manner as assessed by siRNA methods In addition, AHR signaling was effectively inhibited by traditional Kampo formulae containing C. cassia. CONCLUSION: Cinnamaldehyde has two independent biological activities; namely, an inhibitory action on AHR activation and an antioxidant effect mediated by NRF2/HO1 signaling. Through these dual functions, cinnamaldehyde may be beneficial for the treatment of disorders related to oxidative stress such as dioxin intoxication, acne, and vitiligo.

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3ß Signaling Pathway and Promotes Hepatocellular Injury.

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3ß signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Gene regulation of filaggrin and other skin barrier proteins via aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds to structurally diverse chemicals including dioxins, coal tar, flavonoids and tryptophan photoproducts. Upon ligation, cytoplasmic AHR translocates to the nucleus, heterodimerizes with aryl hydrocarbon receptor nuclear translocator and mediates numerous biological effects by inducing the transcription of various AHR-responsive genes such as epidermal barrier proteins. The activation of AHR usually generates oxidative stress. However, AHR also mediates antioxidant signaling by a plethora of ligands via nuclear factor-erythroid 2-related factor-2. Both oxidative and antioxidant ligands upregulate the expression of the filaggrin gene. We review the role of AHR signaling in the gene regulation of epidermal barrier proteins.

Z-ligustilide ameliorated ultraviolet B-induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO-1 and suppression of NF-κB pathway.

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z-ligustilide (Z-lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB-induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z-lig significantly rescued UVB-induced NHEKs damage in a dosage-dependent manner. Pretreatment of NHEKs with Z-lig inhibited UVB-induced ROS production in NHEKs. Both silencing the nuclear factor E2-related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase-1 (HO-1) inhibitor, cancelled the inhibitory effect of Z-lig on UVB-induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z-lig reduced UVB-induced nuclear factor kappa B (NF-κB)-dependent inflammatory mediators (IL-6, IL-8 and MCP-1) production at both mRNA and protein level. In the presence of Z-lig, UVB-induced NF-κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z-lig can suppress UVB-induced ROS generation through Nrf2/HO-1 upregulation and inflammation by suppressing the NF-κB pathway, suggesting that Z-lig may be beneficial in protecting skin from UVB exposure.

Mild electrical stimulation increases stress resistance and suppresses fat accumulation via activation of LKB1-AMPK signaling pathway in C. elegans.

Electrical current at physiological strength has been applied as a therapeutic approach for various diseases. Several of our works showed that mild electrical stimulation (MES) at 0.1-ms pulse width has positive impact on organisms. But despite the growing evidence of the beneficial effects of MES, its effects on individual animals and the molecular underpinnings are poorly understood and rarely studied. Here, we examined the effects of MES on individual animal and its mechanisms by mainly using Caenorhabditis elegans, a powerful genetic model organism. Interestingly, MES increased stress resistance and suppressed excess fat accumulation in wild-type N2 worms but not in AMPK/AAK-2 and LKB1/PAR-4 mutant worms. MES promoted the nuclear localization of transcription factors DAF-16 and SKN-1 and consequently increased the expression of anti-stress genes, whereas MES inhibited the nuclear localization of SBP-1 and suppressed the expression of lipogenic genes. Moreover, we found that MES induced the activation of LKB1/PAR4-AMPK/AAK2 pathway in C. elegans and in several mammalian cell lines. The mitochondrial membrane potential and cellular ATP level were slightly and transiently decreased by MES leading to the activation of LKB1-AMPK signaling pathway. Together, we firstly and genetically demonstrated that MES exerts beneficial effects such as stress resistance and suppression of excess fat accumulation, via activation of LKB1-AMPK signaling pathway.

Resveratrol inhibition of human keratinocyte proliferation via SIRT1/ARNT/ERK dependent downregulation of aquaporin 3.

BACKGROUND: Aquaporin 3 (AQP3) is the predominant aquaporin in the skin and is overexpressed in hyperplastic epidermal disorders. Upregulation of AQP3 contributes to keratinocyte proliferation and epidermal hyperplasia. Resveratrol, a natural polyphenol, has an anti-proliferative effect on normal human epidermal keratinocytes (NHEKs), but its exact mechanism remains largely unknown. OBJECTIVE: To investigate the ability and mechanism of resveratrol to affect the proliferation and the AQP3 expression in NHEKs. METHODS: NHEKs treated with resveratrol were analyzed. BrdU incorporation assay, real-time PCR, Western blotting and RNA interference using small interfering RNA were employed. RESULTS: At non-toxic concentrations (less than 40µM), resveratrol inhibited the proliferation of NHEKs. Resveratrol inhibited the ERK phosphorylation and the AQP3 expression with reciprocal upregulation of ARNT expression in a concentration-dependent manner. The inhibitory effects of resveratrol on the ERK phosphorylation and the AQP3 expression were canceled by transfection of siRNA for ARNT, but not by that for AhR. Furthermore, the induction effect of resveratrol on ARNT expression was canceled after SIRT1 was knocked down in NHEKs. CONCLUSION: Resveratrol inhibited NHEK proliferation by downregulating the expression of AQP3 in an SIRT1/ARNT/ERK dependent fashion. This novel mechanism may facilitate drug innovation for hyperplastic skin disorders.

Z-Ligustilide inhibits benzo(a)pyrene-induced CYP1A1 upregulation in cultured human keratinocytes via ROS-dependent Nrf2 activation.

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is an environmental contaminant that can induce cytochrome P4501A1 (CYP1A1) upregulation via aryl hydrocarbon receptor (AhR) activation and provoke inflammation. Here, we investigated the effect of Z-Ligustilide, an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on BaP-induced CYP1A1 upregulation in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z-Ligustilide significantly inhibited BaP-induced CYP1A1 upregulation in NHEKs. Treatment of NHEKs with Z-Ligustilide induced Nuclear factor-E2-related factor 2 (Nrf2) nuclear translocation and expression of the Nrf2-regulated genes for haeme oxygenase-1 (HO-1) and NAD(P)H: quinine oxidoreductase-1 (NQO1). AhR silencing, SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor) and LY294002 (a PI3K inhibitor) did not suppress Z-Ligustilide-induced Nrf2 activation. Moreover, treatment of NHEKs with Z-Ligustilide increased reactive oxygen species (ROS) and L-N-acetylcysteine (L-NAC, an antioxidant) attenuated Z-ligustilide-induced Nrf2 nuclear translocation and HO-1 expression. L-NAC or knock-down of Nrf2 significantly attenuated the inhibitory effects of Z-Ligustilide on BaP-induced CYP1A1 upregulation in NHEKs. Taken together, these findings suggest that Z-Ligustilide can suppress BaP-induced CYP1A1 upregulation through ROS-dependent Nrf2 pathway activation and may be beneficial in preventing or treating BaP-induced skin damage.

Reduction of CC-chemokine ligand 5 by aryl hydrocarbon receptor ligands.

BACKGROUND: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that recognizes a large number of xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), dioxins, and some endogenous ligands. Despite numerous investigations targeting AhR ligands, the precise physiological role of AhR remains unknown. OBJECTIVE: We explored novel AhR target genes, especially focused on inflammatory chemokine. METHODS: We treated (1) HaCaT, a human keratinocyte cell line, (2) normal human epidermal keratinocytes (NHEKs), and (3) mouse primary keratinocytes with AhR ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ; endogenous ligand) and benzo[a]pyrene (BaP; exogenous ligand). Then, we detected mRNA and protein of chemokine using quantitative RT-PCR and ELISA. We next clarified the relationship between AhR and chemokine expression using AhR siRNA. In addition, we measured serum chemokine levels in patients with Yusho disease (oil disease), who were accidentally exposed to dioxins in the past. RESULTS: We identified CC-chemokine ligand 5 (CCL5), a key mediator in the development of inflammatory responses, as the AhR target gene. AhR ligands (FICZ and BaP) significantly reduced CCL5 mRNA and protein expression in HaCaT cells. These effects were observed in NHEKs and mouse primary keratinocytes. AhR knockdown with siRNA restored CCL5 inhibition by AhR ligands. In addition, AhR ligands exhibited a dose-dependent suppression of CCL5 production induced by Th1-derived cytokines. Finally, serum levels of CCL5 in patients with Yusho disease, were significantly lower than in controls. CONCLUSION: Our findings indicate that CCL5 is a target gene for AhR, and might be associated with the pathology of dioxin exposure.