Ginnalin B induces differentiation markers and modulates the proliferation/differentiation balance via the upregulation of NOTCH1 in human epidermal keratinocytes

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro-d-glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 μM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G₀/G₁ phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.     

Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma

Establishing an effective epidermal barrier requires a series of coordinated molecular events involving keratinocytes (KCs) within a stratified epithelium. Epidermal maturation depends on convergence of pathways involving components of NF-kappaB and peroxisome proliferator activated receptor (PPAR) signaling systems that promote terminal differentiation and production of a stratum corneum. The Notch-1 receptor and its ligand Delta-1 have been proposed by others to participate in early events in KC differentiation. Here, we establish differential expression patterns for several Notch receptors and ligands in normal human skin. These immunolocalization findings, together with functional studies demonstrating increased levels of Notch ligand/receptors occurring during the onset of differentiation, prompted use of a soluble Notch ligand, a peptide derived from the most conspicuously expressed ligand in skin, Jagged-1. Exposing submerged KC monolayers to this peptide (JAG-1) in co-presence of elevated calcium ion concentration, produced stratification with loricrin expression. Using a living human epidermal equivalent (EE) model system, when submerged cultures were raised to an air/liquid interface to generate a fully mature epidermis, activation of Notch signaling was detected. Addition of JAG-1 peptide to submerged EEs was sufficient to induce epidermal maturation. Moreover, a soluble decoy Notch inhibitor prevented such differentiation and corneogenesis in human EEs exposed to either an air/liquid interface or to the JAG-1 peptide. In KC monolayers, addition of JAG-1 peptide induced IKKalpha mediated NF-kappaB activation, as well as increased PPARgamma expression. Immunoprecipitation/Western blot analysis revealed a physical association between the p65 subunit of NF-kappaB and PPARgamma. These results indicate that activation of Notch signaling is necessary for maturation of human epidermis, and activation by a soluble Notch ligand is sufficient to trigger complete KC differentiation including cornification.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

Annexin1 regulates the erythroid differentiation through ERK signaling pathway

K562 cells can be used as a model of erythroid differentiation on being induced by hemin. We found that the level of annexin1 gene expression was notably increased during this indicated process. To test the hypothesis that annexin1 can regulate erythropoiesis, K562 cell clones in which annexin1 was stably increased and was knocked down by RNAi were established, respectively. With analysis by hemoglobin quantification, benzidine staining, and marker gene expression profile determination, we confirmed that hemin-induced erythroid differentiation of K562 cells was modestly stimulated by overexpression of annexin1 while it was significantly blocked by knock down of annexin1. Further studies revealed that the mechanisms of annexin1 regulation of the erythroid differentiation was partially related to the increased ERK phosphorylation and expression of p21(cip/waf), since specific inhibitor of MEK blocked the function of annexin1 in erythroid differentiation. We concluded that annexin1 exerted its erythropoiesis regulating effect by ERK pathway.     

Transforming growth factor beta1 induces differentiation in human papillomavirus-positive keratinocytes.

Human papillomaviruses (HPVs) are implicated in the etiology of anogenital cancers. Expression of the HPV E6 and E7 oncoproteins is believed to contribute to the carcinogenic process. Progressive loss of the ability to differentiate and resistance to the growth-inhibitory effects of endogenous signals also appear important in multistep tumorigenesis. Transforming growth factor beta1 (TGF-beta1) is a potent growth inhibitor for a variety of cultured cells. There have been conflicting reports on the ability of TGF-beta1 to inhibit the growth of HPV-positive keratinocytes in monolayer cultures. We have employed the organotypic (raft) tissue culture system, which more accurately mimics the in vivo cellular environment and architecture. We have investigated the TGF-beta1 response of HPV-positive keratinocytes derived from neoplastic cervical biopsies. Growth of these cell lines as raft tissues showed that many were altered in the ability to stratify and synthesize differentiation-specific proteins. When the organotypic tissues were treated with TGF-beta1, a more complete differentiation of the keratinocytes was induced. Treatment with 12-0-tetradecanoylphorbol-13-acetate gave similar results. TGF-beta1 treatment of HPV-positive raft epithelia led to a dose-dependent increase in E7 RNA expression in contrast to results from previous studies with monolayer cultures. Furthermore, TGF-beta1 interfered with the proliferation of HPV-positive cell lines grown in monolayer cultures. Our results suggest that loss of the ability to express markers of differentiation, a characteristic of malignancy, is a two-step process. The first step is reversible; the second is irreversible.     

Pulsed electric current induces the differentiation of human keratinocytes

Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest.     

Cobalt chloride induces neuronal differentiation of human mesenchymal stem cells through upregulation of microRNA-124a

Human mesenchymal stem cells (hMSCs) are known to have the capacity to differentiate into various cell types, including neurons. To examine our hypothesis that miRNA was involved in neuronal differentiation of hMSCs, CoCl₂, a hypoxia-mimicking agent was used to induce neuronal differentiation, which was assessed by determining the expression of neuronal markers such as nestin and Tuj1. Treatment of hMSCs with CoCl₂ led to increased expression of miR-124a, a neuron-specific miRNA. HIF-1α silencing and JNK inhibition abolished CoCl₂-induced miR-124a expression, suggesting that JNK and HIF-1α signals were required for the miR-124a expression induced by CoCl₂ in hMSCs. Overexpression of miR-124a or CoCl₂ treatment suppressed the expression of anti-neural proteins such as SCP1 and SOX9. Silencing of both SCP1 and SOX9 induced neuronal differentiation of hMSCs, indicating that suppression of miR-124a targets is important for CoCl₂-induced neuronal differentiation of hMSCs. Knockdown of HIF-1α or inhibition of JNK restored the expression of SCP1 and SOX9 in CoCl₂-treated cells. Inhibition of miR-124a blocked CoCl₂-induced suppression of SCP1 and SOX9 and abolished CoCl₂-induced neuronal differentiation of hMSCs. Taken together, we demonstrate that miR-124a is critically regulates CoCl₂-induced neuronal differentiation of hMSCs by suppressing the expression of SCP1 and SOX9.     

Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway

The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype.     

Midkine induces epithelial-mesenchymal transition through Notch2/Jak2-Stat3 signaling in human keratinocytes

Epithelial-to-mesenchymal transition (EMT) underlies cell plasticity and embryonic development and is frequently observed in advanced tumorigenesis. We demonstrated that midkine (MK), a retinoic acid-inducible heparin-binding mitogen, promotes EMT in immortalized HaCaT keratinocytes. We showed that MK binds to the Notch2 receptor in HaCaT keratinocytes. We further found that MK activates Notch2 signaling leading to protein/protein interactions between Hes1 and Jak2/Stat3 intermediates. We thus suggest that MK-induced cross talk of Notch2/Jak2/Stat3 signaling pathways can regulate cell plasticity and motility contributing to the EMT and later stages of tumorigenesis.     

Autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway

Autophagy plays important roles in self-renewal and differentiation of stem cells. Hepatic progenitor cells (HPCs) are thought to have the ability of self-renewal as well as possess a bipotential capacity, which allows them to differentiate into both hepatocytes and bile ductular cells. However, how autophagy contributes to self-renewal and differentiation of hepatic progenitor cells is not well understood. In this study, we use a well-established rat hepatic progenitor cell lines called WB-F344, which is treated with 3.75 mM sodium butyrate (SB) to promote the differentiation of WB-F344 along the biliary phenotype. We found that autophagy was decreased in the early stage of biliary differentiation, and maintained a low level at the late stage. Activation of autophagy by rapamycin or starvation suppressed the biliary differentiation of WB-F344. Further study reported that autophagy inhibited Notch1 signaling pathway, which contributed to biliary differentiation and morphogenesis. In conclusions, autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.     

Hypoxia induces the activation of human hepatic stellate cells LX-2 through TGF-beta signaling pathway

Hypoxia is a common environmental stress factor and is also associated with various physiological and pathological conditions such as fibrogenesis. The activation of hepatic stellate cells (HSCs) is the key event in the liver fibrogenesis. In this study, the behavior of human HSCs LX-2 in low oxygen tension (1% O2) was analyzed. Upon hypoxia, the expression of HIF-1alpha and VEGF gene was induced. The result of Western blotting showed that the expression of alpha-SMA was increased by hypoxic stimulation. Furthermore, the expression of MMP-2 and TIMP-1 genes was increased. Hypoxia also elevated the protein expression of the collagen type I in LX-2 cells. The analysis of TGF-beta/Smad signaling pathway showed that hypoxia potentiated the expression of TGF-beta1 and the phosphorylation status of Smad2. Gene expression profiles of LX-2 cells induced by hypoxia were obtained by using cDNA microarray technique.     

Phosphatidylinositol 3-kinase regulates early differentiation in human laryngeal keratinocytes

Summary Epidermal growth factor receptor (EGFR) signaling regulates a variety of cellular functions, including proliferation, gene expression, and differentiation. Infection of laryngeal epithelial cells by human papillomaviruses causes recurrent respiratory papillomas, benign tumors characterized by an altered pattern of differentiation. Papilloma cells overexpress the EGFR and have constitutively active extracellular signal-regulated kinase (ERK) and enhanced phosphatidylinositol 3-kinase (PI3K) activity, but overexpression of the lipid phosphatase PTEN (Phosphatase and Tensin Homolog) reduces activation of Akt by PI3K. We hypothesized that the altered differentiation of papillomas reflects these changes in signaling from the EGFR-ERK and PI3K-Akt pathways and that one or both of these pathways is required for the normal differentiation process in mucosal epithelium. Inhibiting either the enzymatic activity or the synthesis of PI3K in uninfected laryngeal cells blocked expression of keratin-13 (K13), a protein induced during normal differentiation. In contrast, inhibiting activation of ERK had minimal effect. Using ribonucleic acid interference to reduce protein levels of integrinlinked kinase 1 or phosphoinositide-dependent protein kinase 1, intermediates in the activation of Akt by PI3K, or reducing levels of Akt-1 itself did not inhibit K13 expression by normal laryngeal keratinocytes. We conclude that PI3K activation is an important regulator of expression of K13, a marker for the normal differntiation process in human mucosal keratinocytes, that this function does not require activation of Akt-1, and that the failure to express K13 in papilloma cells is not because of reduction in activated Akt.     

Sympathetic neuron can promote osteoblast differentiation through BMP signaling pathway

Communication between sympathetic neurons and osteoblasts through the adrenergic receptor pathway has already been reported. To investigate whether the sympathetic neurons have a direct effect on osteoblast differentiation, an in vitro Transwell coculture system was established in which osteoblasts were cocultured with sympathetic neurons with no cell-to-cell contact. The expression of osteogenesis-related genes was upregulated in osteoblasts cocultured with sympathetic neurons. Meanwhile, bone morphogenetic protein (BMP) mRNA and protein expressions were detected in sympathetic neurons, and BMP secretion from sympathetic neurons was also confirmed. However, transfection with BMP-2 and/or BMP-6 siRNA in sympathetic neurons caused a down-regulation of osteogenesis-related genes in the cocultured osteoblasts. Sympathetic neurons promoted osteoblast differentiation through BMP signaling pathway, implying that the integrity of sympathetic neurons was important for optimal bone formation and remodeling.     

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.     

Tagitinin C induces ferroptosis through PERK-Nrf2-HO-1 signaling pathway in colorectal cancer cells

Rationale: Colorectal cancer (CRC) is a common malignant tumor of the digestive system. However, the efficacy of surgery and chemotherapy is limited. Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death (RCD) and plays a vital role in tumor suppression. Ferroptosis inducing agents have been studied extensively as a novel promising way to fight against therapy resistant cancers. The aim of this study is to investigate the mechanism of action of tagitinin C (TC), a natural product, as a novel ferroptosis inducer in tumor suppression.Methods: The response of CRC cells to tagitinin C was assessed by cell viability assay, clonogenic assay, transwell migration assay, cell cycle assay and apoptosis assay. Molecular approaches including Western blot, RNA sequencing, quantitative real-time PCR and immunofluorescence were employed as well.Results: Tagitinin C, a sesquiterpene lactone isolated from Tithonia diversifolia, inhibits the growth of colorectal cancer cells including HCT116 cells, and induced an oxidative cellular microenvironment resulting in ferroptosis of HCT116 cells. Tagitinin C-induced ferroptosis was accompanied with the attenuation of glutathione (GSH) levels and increased in lipid peroxidation. Mechanistically, tagitinin C induced endoplasmic reticulum (ER) stress and oxidative stress, thus activating nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). As a downstream gene (effector) of Nrf2, heme oxygenase-1 (HO-1) expression increased significantly with the treatment of tagitinin C. Upregulated HO-1 led to the increase in the labile iron pool, which promoted lipid peroxidation, meanwhile tagitinin C showed synergistic anti-tumor effect together with erastin.Conclusion: In summary, we provided the evidence that tagitinin C induces ferroptosis in colorectal cancer cells and has synergistic effect together with erastin. Mechanistically, tagitinin C induces ferroptosis through ER stress-mediated activation of PERK-Nrf2-HO-1 signaling pathway. Tagitinin C, identified as a novel ferroptosis inducer, may be effective chemosensitizer that can expand the efficacy and range of chemotherapeutic agents.