Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics.

Activation of ErbB receptors by epidermal growth factor (EGF) or heregulin (HRG) determines distinct cell-fate decisions, although signals propagate through shared pathways. Using mathematical modeling and experimental approaches, we unravel how HRG and EGF generate distinct, all-or-none responses of the phosphorylated transcription factor c-Fos. In the cytosol, EGF induces transient and HRG induces sustained ERK activation. In the nucleus, however, ERK activity and c-fos mRNA expression are transient for both ligands. Knockdown of dual-specificity phosphatases extends HRG-stimulated nuclear ERK activation, but not c-fos mRNA expression, implying the existence of a HRG-induced repressor of c-fos transcription. Further experiments confirmed that this repressor is mainly induced by HRG, but not EGF, and requires new protein synthesis. We show how a spatially distributed, signaling-transcription cascade robustly discriminates between transient and sustained ERK activities at the c-Fos system level. The proposed control mechanisms are general and operate in different cell types, stimulated by various ligands.

A Consideration on Infinite and Finite Dosing in Skin Permeation using Reconstructed Models.

INTRODUCTION: When vitamin derivatives penetrate the epidermis, they release active compound such as ascorbic acids and tocopherols via enzymatic digestion of chemical modifiers. To determine the transdermal penetration of the derivatives, the total permeation of both the derivatives and their active compounds that released from the derivatives should be considered. In this study, we established a skin penetration test method using a cultured, reconstructed skin model with active epidermal enzymes. And we analyzed two vitamin derivatives with different chemical properties; magnesium ascorbyl phosphate (APM) and sodium tocopheryl phosphate (TPNa), both of which has been confirmed their skin permeation in the reconstructed models and the digestion to ascorbic acid (AsA) and α-tocopherol (Toc) by the epidermal enzymes, respectively. METHODS: We prepared the 1% of water solution containing either APM or TPNa. Then, we tested the cumulative permeation of the derivatives at two application volumes, 25µL/cm2 (finite dosing) and 85 µL/cm2 (infinite dosing), on cultured reconstructed skin and observed the permeation of the permeants every 2 h up to 24 h. RESULTS: When the applied formula was used to assess the evaporation rate to determine an end point of the test system, all the water evaporated in 6 h in finite model and in 8 h in infinite model. Both models showed that the cumulative permeation of the active compounds increased and a constant flux until 8 h after application, however, the flux decreased thereafter, indicating that the decreased flux depended on an end point of the test system. This indicated that our test system can analyze the permeation of the vitamin derivatives within 8 h before reaching the end point. CONCLUSION: Using an infinite model of this system, we assessed the cumulative permeation of vitamin derivatives within 8 h using a reconstructed skin model.

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells.

Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) EMBO J 25:4215-4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low- and high-affinity sites of HRG association on the cell surface.

An ErbB receptor-mediated AP-1 regulatory network is modulated by STAT3 and c-MYC during calcium-dependent keratinocyte differentiation.

A calcium gradient in skin epidermis is known to regulate epidermal differentiation. In cultures of human epidermal keratinocytes (NHEK), induction of calcium-dependent differentiation is associated with phosphorylation of ErbB receptors, including the epidermal growth factor receptor (EGFR). The activation of EGFR triggers the induction of activator protein 1 (AP-1) proteins necessary for keratinocyte terminal differentiation. Interestingly, an in vitro long-term calcium treatment revealed the activation of different ErbB receptors with different timings, which is consistent with the differential localization of each receptor in the skin layers in vivo. In the current study, the regulatory relationship between ErbB receptor activation and induction of AP-1 proteins in calcium-dependent keratinocyte differentiation was analysed. For this purpose, we used a mathematical method to predict molecular regulations from time-course proteomic data of 30 target components. The predicted network showed that the ErbB receptor might regulate AP-1 protein expression via two pathways: positive regulation by c-MYC and negative regulation by signal transducer and activator of transcription 3 (STAT3) pathways. Experimental validation analysis revealed that ErbB receptor inhibition resulted in defective induction of AP-1 proteins and suppressed terminal differentiation of keratinocytes. Inhibition of STAT3, however, affected expression of a partial set of AP-1 proteins and resulted in premature differentiation. Studies using RNAi to diminish expression of each ErbB receptor and the AP-1 proteins strongly suggested that STAT3 established a balance that maintains the appropriate set of AP-1 proteins and participates in a complex network for regulating keratinocyte differentiation.

Morphologic changes in the cytoskeleton and adhesion apparatus during the conversion from pseudostratified single columnar to stratified squamous epithelium in the developing mouse esophagus.

The apico-basal (AB) polarity of epithelial cells is maintained by organized arrays of the cytoskeleton and adhesion apparatus. We previously reported that mouse embryonic esophageal epithelium exhibits interkinetic nuclear migration (INM), an AB-polarity-based regulatory mechanism of stem-cell proliferation, and suggested that the pseudostratified single columnar epithelium, a hallmark of INM, is converted to stratified squamous epithelium via rearrangement of the cytoskeleton and cell-adhesion apparatus. Here, we chronologically examined morphological changes in the cytoskeleton and adhesion apparatus in the mouse esophageal epithelium at embryonic day (E) 11.5, E13.5, E14.5, and E15.5, during which epithelial conversion has been suggested to occur. We used phalloidin to examine the apical terminal web (ATW), immunofluorescent anti-zonula occludens protein (ZO-1) antibody to reveal ZO-1, and anti-gamma tubulin antibody to detect primary cilia (PC). At E11.5, a thick ATW, apically oriented ZO-1 and apical PC were observed, indicating a pseudostratified single columnar structure. At E13.5 and E14.5, the phalloidin-staining, ZO-1, and PC distribution patterns were not apically localized, and the epithelial cells appeared to have lost the AB polarity, suggesting conversion of the epithelial structure and cessation of INM. At E15.5, light and transmission electron microscope observations revealed the ATW, ZO-1, PC, and tight junction which were localized into two-1ayers: the apical and subapical layers of the epithelium. These findings suggest that dynamic remodeling of the cytoskeleton and adhesion apparatus is involved in the conversion from pseudostratified single columnar to stratified squamous morphology and is closely related with temporal perturbation of the AB-polarity and cessation of INM.

Ligand-specific sequential regulation of transcription factors for differentiation of MCF-7 cells.

BACKGROUND: Sharing a common ErbB/HER receptor signaling pathway, heregulin (HRG) induces differentiation of MCF-7 human breast cancer cells while epidermal growth factor (EGF) elicits proliferation. Although cell fates resulting from action of the aforementioned ligands completely different, the respective gene expression profiles in early transcription are qualitatively similar, suggesting that gene expression during late transcription, but not early transcription, may reflect ligand specificity. In this study, based on both the data from time-course quantitative real-time PCR on over 2,000 human transcription factors and microarray of all human genes, we identified a series of transcription factors which may control HRG-specific late transcription in MCF-7 cells. RESULTS: We predicted that four transcription factors including EGR4, FRA-1, FHL2, and DIPA should have responsibility of regulation in MCF-7 cell differentiation. Validation analysis suggested that one member of the activator protein 1 (AP-1) family, FOSL-1 (FRA-1 gene), appeared immediately following c-FOS expression, might be responsible for expression of transcription factor FHL2 through activation of the AP-1 complex. Furthermore, RNAi gene silencing of FOSL-1 and FHL2 resulted in increase of extracellular signal-regulated kinase (ERK) phosphorylation of which duration was sustained by HRG stimulation. CONCLUSION: Our analysis indicated that a time-dependent transcriptional regulatory network including c-FOS, FRA-1, and FHL2 is vital in controlling the ERK signaling pathway through a negative feedback loop for MCF-7 cell differentiation.

Interkinetic nuclear migration in the tracheal and esophageal epithelia of the mouse embryo: Possible implications for tracheo-esophageal anomalies.

Interkinetic nuclear migration (INM) is a cell polarity-based phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium in synchrony with the cell cycle. INM is suggested to be at least partially cytoskeleton-dependent and to regulate not only the proliferation/differentiation of stem/progenitor cells but also the localized/overall size and shape of organs/tissues. INM occurs in all three of the germ-layer derived epithelia, including the endoderm-derived gut. However, INM has not been documented in the esophagus and respiratory tube arising from the anterior foregut. Esophageal atresia with or without trachea-esophageal fistula (EA/TEF) is a relatively common developmental defect. Transcription factors and signaling molecules have been implicated in EA/TEF, but the etiology of EA/TEF-which has been suggested to involve cell polarity-related mechanisms-remains highly controversial. In the present study, we first examined whether INM exists in the trachea and esophagus of mouse embryos at embryonic day 11.5 (E11.5), just after separation of the two tubes from the anterior foregut. By labeling the DNA-synthesizing stem cell nuclei with 5-ethynyl-2'-deoxyuridine, a nucleotide analogue, and statistically analyzing chronological changes in the distribution pattern of the labeled nuclei by using multidimensional scaling, we showed the existence of INM in both the esophagus and trachea, with differences in the INM magnitude and cycle pattern. We further showed morphological changes from the INM-based pseudostratified single layer to the stratified multilayer in the esophageal epithelium in association with a temporal loss/perturbation of AB polarity, suggesting a possible relation with the pathogenesis of EA/TEF.

Feedforward regulation of mRNA stability by prolonged extracellular signal-regulated kinase activity.

Extracellular signal-regulated kinase (ERK) plays a central role in signal transduction networks and cell fate decisions. Sustained ERK activation induces cell differentiation, whereas transient ERK results in the proliferation of several types of cells. Sustained ERK activity stabilizes the proteins of early-response gene products. However, the effect of ERK activity duration on mRNA stability is unknown. We analyzed the quantitative relationship between the duration of four ERK activity kinetics and the mRNA expression profile in growth factor-treated cells. Time-course transcriptome analysis revealed that the cells with prolonged ERK activity generally showed sustained mRNA expression of late response genes but not early or mid genes. Selected late response genes decayed more rapidly in the presence of a specific ERK inhibitor than a general transcription inhibitor and the decay rate was not related to the number of AU-rich elements. Our results suggest that sustained ERK activity plays an important role in the lifespan of the mRNA encoded by late response genes, in addition to the previously demonstrated role in protein stabilization of early-response genes, including transcription factors regulating the transcription of mid and late genes. This double-positive regulation of ligand-induced genes, also termed feedforward regulation, is critical in cell fate decisions.

Epidermal growth factor receptor mutation in combination with expression of MIG6 alters gefitinib sensitivity.

BACKGROUND: Epidermal growth factor receptor (EGFR) signaling plays an important role in the regulation of cell proliferation, survival, metastasis, and invasion in various tumors. Earlier studies showed that the EGFR is frequently overexpressed in non-small-cell lung cancer (NSCLC) and EGFR mutations at specific amino acid residues in the kinase domain induce altered responsiveness to gefitinib, a small molecule EGFR tyrosine kinase inhibitor. However, the mechanism underlying the drug response modulated by EGFR mutation is still largely unknown. To elucidate drug response in EGFR signal transduction pathway in which complex dynamics of multiple molecules involved, a systematic approach is necessary. In this paper, we performed experimental and computational analyses to clarify the underlying mechanism of EGFR signaling and cell-specific gefitinib responsiveness in three H1299-derived NSCLC cell lines; H1299 wild type (H1299WT), H1299 with an overexpressed wild type EGFR (H1299EGFR-WT), and H1299 with an overexpressed mutant EGFR L858R (H1299L858R; gefitinib sensitive mutant). RESULTS: We predicted and experimentally verified that Mig6, which is a known negative regulator of EGFR and specifically expressed in H1299L858R cells, synergized with gefitinib to suppress cellular growth. Computational analyses indicated that this inhibitory effect is amplified at the phosphorylation/dephosphorylation steps of MEK and ERK. CONCLUSIONS: Thus, we showed that L858R receptor mutation in combination with expression of its negative regulator, Mig6, alters signaling outcomes and results in variable drug sensitivity.

Mutation of epidermal growth factor receptor is associated with MIG6 expression.

Controlled activation of epidermal growth factor receptor (EGFR) is systematically guaranteed at the molecular level; however, aberrant activation of EGFR is frequently found in cancer. Transcription induced by EGFR activation often involves the coordinated expression of genes that positively and negatively regulate the original signaling pathway; therefore, alterations in EGFR kinase activity may reflect changes in gene expression associated with the pathway. In the present study, we investigated transcriptional changes after EGF stimulation with or without the EGFR kinase inhibitor Iressa in H1299 human non-small-cell lung cancer cells [parental H1299, H1299 cells that overexpress wild-type EGFR (EGFR-WT) and mutant H1299 cells that overexpress EGFR where Leu858 is substituted with Arg (L858R)]. The results obtained clearly demonstrate differences in transcriptional activity in the absence or presence of EGFR kinase activity, with genes sharing the same molecular functions showing distinct expression dynamics. The results show the particular enrichment of EGFR/ErbB signaling-related genes in a differentially expressed gene set, and significant protein expression of MIG6/RALT(ERRFI1), an EGFR negative regulator, was confirmed in L858R. High MIG6 protein expression was correlated with basal EGFR phosphorylation and inversely correlated with EGF-induced extracellular signal-regulated protein kinase phosphorylation levels. Investigation of the NCI-60 cell lines showed that ERRFI1 expression was correlated with EGFR expression, regardless of tissue type. These results suggest that cells accumulate MIG6 as an inherent negative regulator to suppress excess EGFR activity when basal EGFR kinase activity is considerably high. Taking all the above together, an EGFR mutation can cause transcriptional changes to accommodate the activation potency of the original signaling pathway at the cellular level.

Effect of RNA interference of tight junction-related molecules on intercellular barrier function in cultured human keratinocytes.

Accumulating evidence shows that tight junctions (TJs) in the granular layer contribute to the epidermal barrier, suggesting that the regulation of TJ assembly in keratinocytes may provide a clue to understanding the role of barrier formation in epidermis. In this study, we investigated the behavior of TJ-related molecules in cultured human keratinocytes during keratinization induced by transfer to high-calcium medium, and the effect of RNA interference of TJ-related molecules on intercellular permeability and morphological features. The expression of TJ-related molecules and transepithelial electrical resistance were increased by transfer to high-calcium medium. In cells under the same conditions, we observed by freeze-fracture electron microscopy that TJ strands developed on the apposing cell membranes. In contrast, the transepithelial electrical resistance was clearly suppressed when the expression of claudin-1 or occludin was blocked by RNA interference. The morphological features of these knock-down cells were the same as those of MOCK cells, except for a marked decrease in the number of TJ strands. Furthermore, claudin-1 suppression inhibited occludin localization at the cell membrane, whereas suppression of occludin did not influence the localization of claudin-1. These results suggest that claudin-1 plays a crucial role in recruiting occludin to TJs, and that occludin is involved in intercellular barrier function.