RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3beta phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.     

The effects of epidermal growth factor on gene expression in human fibroblasts

A better understanding of the molecular effects of epidermal growth factor (EGF) on target cell can help to reveal important aspects of cellular proliferation, transformation, and apoptosis, as well as embryonic and fetal development. In this study, we examined the differences in gene expression of cultured fibroblasts with EGF stimulation for 48 h by using high-density complementary deoxyribonucleic acid (cDNA) arrays. We found that EGF could cause widespread alteration in gene expression. Eight hundred and fifty-five genes, more than 20% of those assayed, showed changed expression, which are involved in various cellular processes, such as energetic metabolism, biosynthesis, the progress of cell cycle, and the signaling pathways of receptor tyrosine kinase (RTKs) and G protein-coupled receptors (GPCRs). The most striking finding is that long-term EGF treatment on cultured fibroblasts resulted in down-regulation of the genes encoding membrane receptors and ion channels and desensitized RTKs and GPCRs to their physiological and nonphysiological stimuli, which seems to be a slow-acting, but permanent, effect of EGF on RTK and GPCR signaling pathways and to play important roles in embryonic and fetal development.     

Regulation of epidermal growth factor receptor gene expression

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.     

Identification of insulin-like growth factor binding proteins from cultured human epidermal keratinocytes

The role and mechanisms of action of insulin-like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF-I receptors and are responsive to IGF-I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF-I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37–42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP-2. Radioimmunoassay of IGFBP-1, -3, and -6 revealed detectable levels of IGFBP-3 and significant levels of IGFBP-6, but not IGFBP-1. Northern analysis following culture in complete medium revealed that at early passage IGFBP-1, -2, -4, and -6 mRNAs were detectable. IGFBP-3 and -5 mRNAs were not detectable. Following culture in growth factor-free medium a 37–42 kD band, consistent with IGFBP-3, was predominant and a 24 kD band consistent with IGFBP-4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte-derived IGFBPs are likely to play a role in the transport and targeting of IGF-I from dermally derived fibroblasts to the epidermis. © 1995 Wiley-Liss, Inc.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.     

Direct modulation of epidermal growth factor binding by cholecystokinin

The effects of cholecystokinin-octapeptide (CCK8), the biologically active C-terminal moiety of cholecystokinin (CCK), on the binding of epidermal growth factor (EGF) were studied in isolated rat pancreatic acini. CCK8 inhibited 125I-EGF binding in a dose-dependent manner. One-half maximal inhibition occurred at 5 X 10(-10)M, and maximal inhibition at 10(-8)M CCK8. This inhibitory effect was detectable within 5 minutes of addition of CCK8, and was not associated with enhanced degradation of 125I-EGF in incubation media. Unlabeled EGF exerted only a slightly greater inhibitory effect than CCK8 on 125I-EGF binding at equivalent molar concentrations. In contrast to CCK8, the gastrointestinal hormone vasoactive intestinal polypeptide (VIP) did not significantly alter EGF binding. CCK8 also inhibited EGF binding in mouse pancreatic acini, but did not alter binding in A-431 human carcinoma cells. These findings suggest that physiological levels of CCK may regulate EGF binding in the pancreas and other tissues with receptors for both hormones. They thus point to a previously unrecognized mechanism for hormonal interaction.     

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012. © 2011 Wiley Periodicals, Inc.     

Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene

Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.     

Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. We have previously shown that growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. Here we show that the control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. We also show, for the first time in Drosophila, that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors.