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.

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.

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.

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.

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein.

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

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.

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.

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 at 0.1-ms pulse width induces p53 protein phosphorylation and G2 arrest in human epithelial cells.

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

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.

Insulin receptor activation through its accumulation in lipid rafts by mild electrical stress.

Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin-induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low-intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin-independent manner and improved insulin resistance in peripheral tissues of high fat-fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin-resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin-independent manner, and might be beneficial for insulin resistance in type 2 diabetes.

MEF/ELF4 transactivation by E2F1 is inhibited by p53.

Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unexplored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

Glucose uptake in rat skeletal muscle L6 cells is increased by low-intensity electrical current through the activation of the phosphatidylinositol-3-OH kinase (PI-3K) / Akt pathway.

Activation of Akt by insulin is transmitted via phosphatidylinositol-3-OH kinase (PI-3K) and enhances glucose uptake. The PI-3K/Akt signaling is diminished in insulin resistance. Thus, approaches that activate PI-3K/Akt signaling leading to improved glucose uptake may ameliorate hyperglycemia. Here we showed that low-intensity electrical current or mild electrical stimulation (MES) activated the PI-3K/Akt signaling and increased the glucose uptake in rat skeletal muscle (L6) cells. The glucose uptake enhanced by MES in muscle cells, the major cells involved in glucose disposal, suggests MES may have a possible beneficial effect on hyperglycemia.

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.

The crystal structure of the green tea polyphenol (-)-epigallocatechin gallate-transthyretin complex reveals a novel binding site distinct from the thyroxine binding site.

Amyloid fibril formation is associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Familial amyloid polyneuropathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T(4)). TTR variants contribute to the pathogenesis of amyloidosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigallocatechin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TTR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V30M TTR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy.

An acylic polyisoprenoid derivative, geranylgeranylacetone protects against visceral adiposity and insulin resistance in high-fat-fed mice.

Induction of heat shock protein (HSP)72 improves insulin resistance and obesity in diabetic animal models. Geranylgeranylacetone (GGA), known as an antiulcer drug, induces HSP72 and protects organs against several cellular stresses. This study investigated whether GGA administration would induce HSP72 in liver and render physiological protection against high-fat feeding in mice. A single and 4-wk oral administration of 200 mg/kg GGA was performed in high-fat diet (HFD)-fed mice. Metabolic parameters, cytokines, and gene expressions related to insulin signaling were evaluated. A single administration of GGA induced HSP72 in liver of normal chow-fed and HFD-fed mice. Insulin resistance after HFD was slightly ameliorated. Four weeks of GGA administration also increased HSP72 in liver and significantly improved insulin resistance and glucose homeostasis upon glucose challenge. Activation of c-jun NH2-terminal kinase (JNK) was attenuated, and insulin signaling was improved in the liver of HFD mice. Visceral adiposity was decreased in GGA-treated mice, accompanied by reduced leptin and increased adiponectin levels. GGA can be a novel therapeutic approach to treat metabolic syndrome as well as type 2 diabetes by improving insulin signaling and reducing adiposity. These beneficial effects of GGA could be mediated through HSP72 induction and JNK inactivation in the liver.

TLR3 induction by anticancer drugs potentiates poly I:C-induced tumor cell apoptosis.

Toll-like receptor 3 (TLR3) has gained recognition as a novel molecular target for cancer therapy because TLR3 activation by its synthetic ligand poly I:C directly causes tumor cell death. Recently, we reported that tumor suppressor p53 increases the expression of TLR3 in several tumor cell lines. Another study also showed that interferon-alpha (IFN-alpha) up-regulates TLR3 expression. We thus hypothesized that various anticancer drugs such as p53-activating reagents and IFNs may potentiate poly I:C-induced tumor cell death through the up-regulation of TLR3 expression. Here, we screened several anticancer drugs that, together with poly I:C, effectively cause tumor cell death in colon carcinoma HCT116 cells. We found that the DNA-damaging reagent 5-fluorouracil (5-FU) increased TLR3 mRNA expression and potentiated poly I:C-induced apoptosis in HCT116 p53(+/+) cells but had only minimal effect in p53(-/-) cells, indicating a p53-dependent pathway. On the other hand, IFN-alpha increased poly I:C-induced apoptosis and the TLR3 mRNA level in HCT116 p53(+/+) and p53(-/-) cell lines. Furthermore, the combination of poly I:C, 5-FU and IFN-alpha induced the highest apoptosis in HCT116 p53(+/+) and p53(-/-) cells. Taken together, these data suggest that the anticancer drugs increased TLR3 expression and subsequently potentiated poly I:C-induced apoptosis likely via p53-dependent and -independent pathways. Considering that the p53 status in malignant cells is heterogeneous, this combination approach may provide a highly effective tumor therapy.

Endoplasmic reticulum stress increases the expression and function of toll-like receptor-2 in epithelial cells.

Endoplasmic reticulum (ER) stress is involved in a wide range of pathological conditions including neurodegenerative disorders, diabetes mellitus, atherosclerosis, inflammation, and infection. The ability of ER stress to induce an inflammatory response is considered to play a role in the pathogenesis of these diseases. However, its role in regulating the gene expression and function of toll-like receptors (TLRs), host defense receptors that recognize invading pathogens, remains unknown. Here we showed that several well-characterized ER stress inducers (thapsigargin, tunicamycin, and dithiothreitol) increase the expression of TLR2 in epithelial cells. Ligand-responsiveness of TLR2 was also enhanced by ER stress inducers, implying a contributory role of ER stress for the regulation of TLR2-dependent inflammatory responses. Furthermore, there was significant increase of TLR2 mRNA level in the livers of tunicamycin-treated mice and high-fat diet-fed mice, suggesting an impact of ER stress in vivo on the expression of TLR2. Overexpression and knockdown experiments showed the importance of activating transcription factor 4 (ATF4), an ER stress-induced transcription factor, in the induction of TLR2 expression during ER stress. This was confirmed by the increased expression and function of TLR2 during treatment with salubrinal, an activator of ATF4 pathway. Taken together, our study provides further insights into the role of ER stress in enhancing host bacterial response or in exaggerating the inflammatory condition via up-regulating TLR2 expression.

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.

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.