TGF-α Expression Research Articles

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960–1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin γ1, connexin 43, Fas, integrin α6, TGFα, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCβII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFβ was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions.

A dual role of FGF10 in proliferation and coordinated migration of epithelial leading edge cells during mouse eyelid development

The development of the eyelid requires coordinated cellular processes of proliferation, cell shape changes, migration and cell death. Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit open-eyelids at birth. To elucidate the roles of FGF10 during eyelid formation, we examined the expression pattern of Fgf10 during eyelid formation and the phenotype of Fgf10-null eyelids in detail. Fgf10 is expressed by mesenchymal cells just beneath the protruding epidermal cells of the nascent eyelid. However, Fgf10-null epithelial cells running though the eyelid groove do not exhibit typical cuboid shape or sufficient proliferation. Furthermore, peridermal clumps are not maintained on the eyelid leading edge, and epithelial extension does not occur. At the cellular level, the accumulation of actin fibers is not observed in the mutant epithelial leading edge. The expression of activin/inhibin betaB (ActbetaB/Inhbb) and transforming growth factor alpha (Tgfa), previously reported to be crucial for eyelid development, is down-regulated in the mutant leading edge, while the onset of sonic hedgehog (Shh) expression is delayed on the mutant eyelid margin. Explant cultures of mouse eyelid primordia shows that the open-eyelid phenotype of the mutant is reduced by exogenous FGF10 protein, and that the expression of ActbetaB and Tgfa is ectopically induced in the thickened eyelid epithelium by the FGF10 protein. These results indicate a dual role of FGF10 in mouse eyelid development, for both proliferation and coordinated migration of eyelid epithelial cells by reorganization of the cytoskeleton, through the regulation of activin, TGFalpha and SHH signaling.

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Ca o 2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca 2+] o , and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Ca o 2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Ca o 2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Ca o 2+. This is consistent with the known expression of TGFα by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Ca o 2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.

TGF-α perturbs surfactant homeostasis in vivo

To determine potential relationships between transforming growth factor (TGF)-alpha and surfactant homeostasis, the metabolism, function, and composition of surfactant phospholipid and proteins were assessed in transgenic mice in which TGF-alpha was expressed in respiratory epithelial cells. Secretion of saturated phosphatidylcholine was decreased 40-60% by expression of TGF-alpha. Although SP-A, SP-B, and SP-C mRNA levels were unchanged by expression of TGF-alpha, SP-A and SP-B content in bronchoalveolar lavage fluid was decreased. The minimum surface tension of surfactant isolated from the transgenic mice was significantly increased. Incubation of cultured normal mice type II cells with TGF-alpha in vitro did not change secretion of surfactant phosphatidylcholine and SP-B, indicating that TGF-alpha does not directly influence surfactant secretion. Expression of a dominant negative (mutant) EGF receptor in the respiratory epithelium blocked the TGF-alpha-induced changes in lung morphology and surfactant secretion, indicating that EGF receptor signaling in distal epithelial cells was required for TGF-alpha effects on surfactant homeostasis. Because many epithelial cells were embedded in fibrotic lesions caused by TGF-alpha, changes in surfactant homeostasis may at least in part be influenced by tissue remodeling that results in decreased surfactant secretion. The number of nonembedded type II cells was decreased 30% when TGF-alpha was expressed during development and was increased threefold by TGF-alpha expression in adulthood, suggesting possible alteration of type II cells on surfactant metabolism in the adult lung. Abnormalities in surfactant function and decreased surfactant level in the airways may contribute to the pathophysiology induced by TGF-alpha in both the developing and adult lung.

306. Testicular growth factor expression after hemicastration in the neonatal boar

The molecular mechanisms associated with testicular hypertrophy after hemicastration are poorly understood. Sertoli cells in culture underwent increased proliferation when exposed to fibroblast growth factor-2 (FGF2)1 and transforming growth factor-α (TGFα).2 To determine whether FGF2 and TGFα expression is upregulated during accelerated testicular growth after hemicastration in the boar animals were assigned to (a) control (n = 12), no treatment; (b) hemicastrated (n=14), left testis removed on Day 10 after birth. The right testis was removed from half the control and hemicastrated boars, respectively, on Days 15 and 20 after birth. RNA was extracted from sections of frozen testis, cDNA synthesised using TaqMan® Reverse Transcription, and real-time PCR performed. FGF2 expression was determined using forward (5′GTG TTA CAG ACG AGT GTT TCT TTT TTG3′), internal (5′acg act gga atc taa t3′) and reverse (5′TTC CTC GAC CGG TAA GTA TTG TAG T3′) primers. TGFα expression was similarly determined using forward (5′GGC TGT CCT CAT CAT CAC ATG T3′), internal (5′tgc tga tac act gct gc3′) and reverse (5′CGG CAC CAC TCA CAG TGT TT3′) primers. Data were analysed by ANOVA and LSD test (testis weight) and unpaired two-tailed t-tests assuming equal variance (FGF2, TGFα). There was no difference (P > 0.05) in testis weight between hemicastrated (3.9 ± 0.3 g; mean ± SEM) and control (3.6 ± 0.5 g) boars on Day 5 but on Day 10 hemicastrated boars had a greater (P = 0.01) testis weight (6.2 ± 0.8 g) than controls (4.3 ± 0.4 g). There were no differences (P > 0.05) between control and hemicastrated boars in TGFα or FGF2 expression on Days 5 and 10. It is concluded from the findings that upregulation of TGFα or FGF2 gene expression is not required for testicular hypertrophy subsequent to hemicastration in the neonatal boar. (1)Biol Reprod (1987); 37: 665–674.(2)Mol Cell Endocrinol (2001); 181: 221–227.

Sex differences in expression of transforming growth factor-α and epidermal growth factor receptor mRNA in waved-1 and C57Bl6 mice

A reduction of transforming growth factor-α (TGFα) expression in the spontaneous Waved-1 ( Wa-1) mutant mouse causes specific behavioral and anatomical changes, including reduced fear learning and stress response and enlarged lateral ventricles. These alterations are observed predominantly in male Wa-1 mice after puberty. We hypothesized that regional differences in the expression of TGFα and its receptor, epidermal growth factor receptor (EGFR), may regulate the sexual dimorphism of the brain structures and functions during postnatal development. In general, fear learning-associated structures, including hippocampus and amygdala, showed maximum expression before puberty, regardless of genotype. In contrast, an overall temporal delay in the rise of both transcript levels, which peaked around or after puberty onset, was observed for the major stress regulatory hypothalamo-pituitary-adrenal axis. This pattern of expression was reversed for amygdala EGFR and hypothalamus TGFα and EGFR transcripts in males. When regional TGFα expression was compared between control and Wa-1 mice, far more complex patterns than expected were observed that revealed sex- and structure-dependent differences. In fact, the amygdala, hypothalamus, and pituitary TGFα expression pattern in Wa-1 exhibited a clear sex dependency across various age groups. Surprisingly, there was no compensatory up-regulation of the EGFR transcript in Wa-1 mice. The observed expression patterns of the TGFα signaling system during normal development and in the Wa-1 mutant mouse suggest complex sex- and age-dependent transcription regulatory mechanisms.

Reduced Nuclear Translocation of Nuclear Factor (NF)-κB p65 in the Footpad Epidermis of Dogs Infected with Distemper Virus

Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

Proteolytic processing of TGFα redirects its mitogenic activity: the membrane-anchored form is autocrine, the secreted form is paracrine

Wild-type transforming growth factor α (TGFα) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFα-mediated phenotype, we bred TGFα mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor. To further characterize this observation, we generated two lineages of transgenic mice that overexpress mutant forms of TGFα: a processed soluble form (s TGFα) and a cytoplasmic-deleted form (TGFαΔC). While sTGFα expression in lactotrope cells failed to induce autocrine lactotrope hyperplasia, the pituitary became very enlarged due to proliferation of neighboring interstitial cells. In contrast, the TGFαΔC mice did not develop a phenotype, although the mRNA and protein were present in the pituitary and this form of TGFα was confirmed to be biologically active and targeted properly to the plasma membrane of cultured CHO cells. The results suggest that the cytoplasmic domain of TGFα is required for autocrine parenchymal tumor formation in the pituitary gland. This signal cannot be inhibited by the EGFR-tr. Conversely, the released form of TGFα appears to have primarily paracrine activity.

Expression Levels of EGF, TGF-α, and EGF-R Are Significantly Reduced in Pre-Implantation Cloned Mouse Embryos

Altered expression of genes encoding mitogenic growth factors thought to play key regulatory roles in preimplantation development may contribute to poor cloning efficiency. In this study, we compared the pattern, timing, and level of expression of epidermal growth factor (EGF) and transforming growth factor–alpha (TGF-α) genes and their cognate receptor EGFR in mouse embryos derived by cloning with in vivo–(naturally fertilized) and in vitro–fertilized embryos at four preimplantation stages of development. In cloned embryos, EGF, TGF- α, and EGF-R expression levels were nearly undetectable, while expression of a housekeeping gene, transcription factor SP1, was normal. In contrast, growth factor genes were expressed in in vitro–fertilized embryos, although significantly less than in in vivo embryos (p < 0.05). In all embryos examined, the timing and pattern of gene expression were similar. Expression in the gravid uterus paralleled that in preimplantation embryos, while no expression was measured in non-gravid uteri. Our results lead us to speculate that altered levels of expression of EGF, TGF-α, and EGF-R impair mammalian development, which could account for the poor survival potential of cloned mouse embryos.

A retinoid X receptor (RXR)-selective agonist bexarotene produces synergistic growth inhibitory activity with gefitinib in non-small-cell lung cancer (NSCLC) cell lines

7157 Background: Gefitinib (Iressa) is an orally active epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of advanced non-small cell lung cancer (NSCLC). Bexarotene (Targretin) is a selective RXR agonist approved for the treatment of CTCL, and with clinical activity enhancing survival and delaying progression in NSCLC patients. Our previously published data (Cancer Res. 2001) has shown that bexarotene can suppress the growth of epithelial tumors by modulating the expression and function of EGFR and its ligand, TGFα. Methods: The expression of EGFR, Her-2, EGF, and TGFα were evaluated in a panel of 10 NSCLC cell lines, representing different histomorphologies. Cells were treated with bexarotene either alone or in combination with Iressa and growth and viability were examined. Combination treatments were either concomitant or sequential. Results: Exposure of NSCLC cell lines to bexarotene as a single agent had modest growth inhibitory effects (at > 1 μM). However, exposure of several NSCLC lines to bexarotene (1–3 μM) produced significant decreases in the expression of EGFR, Her-2 and TGFα mRNA and protein. EGF levels were also decreased to a much lesser degree. Bexarotene combined with Iressa produced an enhanced growth inhibitory activity (a left-ward shift of 3–5 fold in the IC50) when compared to Iressa alone. Analysis of the growth inhibition of the bexarotene /gefitinib combination demonstrated a synergistic (CI<1) concentration-dependent growth inhibition in several of the NSCLC cell lines. The extent of synergy between bexarotene and gefitinib was greater at modest drug effect (≤IC50). Conclusions: These results suggest that bexarotene can inhibit the expression of several components of the EGFR-Her-2 signaling pathways, and can cooperate with EGFR-targeted agents to decrease proliferation of NSCLC cells in vitro. These data support the clinical observation, also presented at this meeting (Dragnev, K. et al) suggesting that Targretin has beneficial effects when used with EGFR-TKIs in advanced NSCLC. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Pharmaceuticals Ligand Pharmaceuticals

A retinoid X receptor (RXR)-selective agonist bexarotene produces synergistic growth inhibitory activity with gefitinib in non-small-cell lung cancer (NSCLC) cell lines

7157 Background: Gefitinib (Iressa) is an orally active epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of advanced non-small cell lung cancer (NSCLC). Bexarotene (Targretin) is a selective RXR agonist approved for the treatment of CTCL, and with clinical activity enhancing survival and delaying progression in NSCLC patients. Our previously published data (Cancer Res. 2001) has shown that bexarotene can suppress the growth of epithelial tumors by modulating the expression and function of EGFR and its ligand, TGFα. Methods: The expression of EGFR, Her-2, EGF, and TGFα were evaluated in a panel of 10 NSCLC cell lines, representing different histomorphologies. Cells were treated with bexarotene either alone or in combination with Iressa and growth and viability were examined. Combination treatments were either concomitant or sequential. Results: Exposure of NSCLC cell lines to bexarotene as a single agent had modest growth inhibitory effects (at > 1 μM). However, exposure of several NSCLC lines to bexarotene (1–3 μM) produced significant decreases in the expression of EGFR, Her-2 and TGFα mRNA and protein. EGF levels were also decreased to a much lesser degree. Bexarotene combined with Iressa produced an enhanced growth inhibitory activity (a left-ward shift of 3–5 fold in the IC50) when compared to Iressa alone. Analysis of the growth inhibition of the bexarotene /gefitinib combination demonstrated a synergistic (CI<1) concentration-dependent growth inhibition in several of the NSCLC cell lines. The extent of synergy between bexarotene and gefitinib was greater at modest drug effect (≤IC50). Conclusions: These results suggest that bexarotene can inhibit the expression of several components of the EGFR-Her-2 signaling pathways, and can cooperate with EGFR-targeted agents to decrease proliferation of NSCLC cells in vitro. These data support the clinical observation, also presented at this meeting (Dragnev, K. et al) suggesting that Targretin has beneficial effects when used with EGFR-TKIs in advanced NSCLC. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Ligand Pharmaceuticals Ligand Pharmaceuticals

Maternal separation suppresses TGFα mRNA expression in the prefrontal cortex of male and female neonatal C57BL/6 mice

Male C57BL/6 mice that undergo maternal separation (MS) early in life demonstrate higher levels of anxiety upon reaching adulthood compared to normally reared offspring. This study reports that neonatal males and females that undergo MS have reduced mRNA levels of transforming growth factor-α (TGFα) in the prefrontal cortex, an area of the brain implicated in emotionality, compared to normally reared animals. TGFα expression was unaffected by MS in the hippocampus. These data indicate that MS leads to a brain region-specific suppression of TGFα expression early in development.

Growth, Differentiation and Senescence of Normal Human Urothelium in an Organ-Like Culture

Objective: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model. Materials and Methods: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous β-galactosidase activity and p16INK4a mRNA expression. Results: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor α (TGFα) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16INK4a mRNA expression and β-galactosidase activity increased on day 25, giving signs of senescence. Conclusions: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.

Future of Family Medicine Recommendations Confirm Need for Increased Research from Family Physicians

<h3>Background</h3> Transforming growth factor α (TGFα) is a peptide growth factor known to be expressed in normal haemopoiesis. It is also expressed in a range of epithelial neoplasms but has not been assessed in haemopoietic malignancies. We have performed an immunohistochemical evaluation of TGFα in acute and chronic myeloid malignancies. <h3>Methods</h3> TGFα expression was semiquantitatively assessed in 69 normal bone marrow trephines and 157 cases of myeloid malignancy using an immunohistochemical approach. <h3>Results</h3> Blast cells of myeloid origin in acute myeloid leukaemia (AML), myelodysplasia and accelerated and blast phases of chronic myeloid leukaemia (CML) were TGFα positive. In acute promyelocytic leukaemia the neoplastic cells had significantly weaker TGFα expression than seen in other forms of AML. The blast cells in CML-accelerated and blast phases were positive with similar expression to AML. <h3>Conclusions</h3> TGFα is expressed in neoplastic myeloblasts and could, therefore, be used as blast cell biomarker in diagnostic haematopathology. In addition, TGFα immunohistochemistry may be of use in identifying a therapeutic target.

Transient induction of TGF-alpha disrupts lung morphogenesis, causing pulmonary disease in adulthood.

Clinical studies have associated increased transforming growth factor (TGF)-alpha and EGF receptor with lung remodeling in diseases including bronchopulmonary dysplasia (BPD). BPD is characterized by disrupted alveolar and vascular morphogenesis, inflammation, and remodeling. To determine whether transient increases in TGF-alpha are sufficient to disrupt postnatal lung morphogenesis, we utilized neonatal transgenic mice conditionally expressing TGF-alpha. Expression of TGF-alpha from postnatal days 3 to 5 disrupted postnatal alveologenesis, causing permanent enlargement of distal air spaces in neonatal and adult mice. Lung volume-to-body weight ratios and lung compliance were increased in adult TGF-alpha transgenic mice, whereas tissue and airway elastance were reduced. Elastin fibers in the alveolar septae were fragmented and disorganized. Pulmonary vascular morphogenesis was abnormal in TGF-alpha mice, with attenuated and occasionally tortuous arterial branching. The ratios of right ventricle weight to left ventricle plus septal weight were increased in TGF-alpha mice, indicating pulmonary hypertension. Electron microscopy showed gaps in the capillary endothelium and extravasation of erythrocytes into the alveolar space of TGF-alpha mice. Hemorrhage and inflammatory cells were seen in distal air spaces at 1 mo of age. In adult TGF-alpha mice, alveolar remodeling, nodules, proteinaceous deposits, and inflammatory cells were seen. Immunostaining for pro-surfactant protein C showed that type II cells were abundant in the nodules, as well as neutrophils and macrophages. Trichrome staining showed that pulmonary fibrosis was minimal, apart from areas of nodular remodeling in adult TGF-alpha mice. Transient induction of TGF-alpha during early alveologenesis permanently disrupted lung structure and function and caused chronic lung disease.

Myristoylated Naked2 escorts transforming growth factor alpha to the basolateral plasma membrane of polarized epithelial cells.

The epidermal growth factor receptor ligands transforming growth factor alpha (TGF alpha) and amphiregulin are delivered to the basolateral surface of polarized epithelial cells where they are cleaved by TACE/ADAM17. Basolateral sorting information resides in their cytoplasmic tail domains, but tail-interacting proteins required for basolateral trafficking have not been identified. Naked (NKD)1 and NKD2 are mammalian homologs of Drosophila Naked Cuticle, which negatively regulates canonical Wnt signaling by binding Dishevelled. We present evidence that NKD2, but not NKD1, binds to basolateral sorting motifs in the cytoplasmic tail of TGF alpha. Processing and cell-surface delivery of TGF alpha are accelerated in NKD2-overexpressing Madin-Darby canine kidney cells. NKD2 is myristoylated on glycine, the second residue. On expression of myristoylation-defective (G2A) NKD2, neither NKD2 nor TGF alpha appears at the basolateral plasma membrane of polarized Madin-Darby canine kidney cells; however, membrane staining for TGF alpha is restored on silencing expression of this mutant NKD2. Amphiregulin does not interact with NKD2 and retains its basolateral localization in G2A-NKD2-expressing cells, as do Na(+), K(+) ATPase alpha 1 and E-cadherin. These data identify an unexpected function for NKD2, i.e., myristoylation-dependent escort of TGF alpha to the basolateral plasma membrane of polarized epithelial cells.

135 Induction of Gene Expression for EGF‐like Ligands in Porcine Wounds Following Receptor Overexpession

A rationale to explain the role of the large and redundant family of EGF cytokines with multiple receptor combinations has proven elusive. We examined endogenous gene expression levels during wound repair for four EGF‐like cytokines (Transforming growth factor‐alpha (TGFα), Heparin‐binding epidermal growth factor (HB‐EGF), Epiregulin (EPR) and Betacellulin (BC) using a quantitative real‐time PCR technique. In addition, cytokine gene expression was evaluated after overexpression of different of EGF receptor forms (erbB‐1, erbB‐3 and erbB‐4) or a control B‐galactosidase gene using an adenoviral transfection strategy to introduce either homo or heterodimer receptor combinations. At baseline, endogenous gene expression levels of TGFα and HB‐EGF were 10‐fold and 100‐fold higher than EPR and BC, respectively. Expression of TGFα was increased by 3‐fold in the receptor combination of erbB‐1/erbB‐4 expressing wounds. Recent studies on the EGF‐related polypeptides HB‐EGF, EPR and BC have shown preferential binding to erbB‐4. Our study suggests that wounds predominantly expressing erbB‐4 showed a 2.5‐fold increase in gene expression of HB‐EGF. A synergistic 4‐fold increase in gene expression was noted when erbB‐4 overexpression was treated with HB‐EGF ligand. In contrast, wounds transfected with erbB‐4 alone showed no change in EPR gene expression compared to controls. However, wounds treated with TGFα showed 3‐fold increase in EPR gene expression and EPR potentiated itself. Similarly, wounds transfected with erbB‐4 and treated with growth factors TGFα and EGF upregulated the gene expression of BC by 4‐fold as compared to controls. Interestingly, wounds overexpressing erbB‐3 with topica application of EGF accentuated expression of TGFα by 7‐fold. In histologic sections, these wounds showed a 2‐fold increase in the dermal region compared to control. Thus, the EGF‐like family of ligands and its homo‐or heterodimer receptor pairs have the potential in vivo to evoke differing profiles or intensities of gene expression patterns as they mediate wound repair.

Raf and RhoA Cooperate to Transform Intestinal Epithelial Cells and Induce Growth Resistance to Transforming Growth Factor β

Abstract Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) β. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E2 levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGFα expression, or TGFβ sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFβ resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFβ sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.

Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: role of AF-1 and AF-2

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection. To examine the role of the transcriptional activation function (AF) domains of ERα in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGFα) in MDA-MB-231 cells stably transfected with wild-type (wt) ERα or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtERα showed a superagonistic response to resveratrol. Deletion of AF-1 or mutation of AF-2 attenuated the effect of resveratrol disproportionately compared to that of E2, while deletion of AF-2 abrogated the response to both ligands. In TGFα expression assays, resveratrol acted as a full agonist in cells expressing wtERα. Deletion of AF-1 attenuated stimulation by E2 more severely than that by resveratrol, as did deletion of AF-2. In contrast, mutation of AF-2 left both ligands with a limited ability to induced TGFα expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene.

Transforming growth factor α and epidermal growth factor receptor in reactive and malignant mesothelial proliferations

Abstract Background.—Growth factors such as transforming growth factor α (TGF-α) and epidermal growth factor receptor (EGFR) play an important role in cell proliferation. The immunohistochemical expression of these factors has been extensively studied in malignant tumors including mesothelioma. However, the comparative expression of these growth factors in mesothelioma and reactive mesothelial proliferations has been less well studied. Objective.—To evaluate the possible role of TGF-α and EGFR in the clinically important distinction between reactive mesothelial proliferations and malignant mesothelioma. Methods.—The expression of TGF-α and EGFR was studied in 39 cases of mesothelioma and 30 cases of reactive mesothelial proliferations by means of immunohistochemistry. Results.—Fourteen (70%) of 20 reactive mesothelial proliferations tested and 29 (76%) of 38 mesotheliomas tested expressed TGF-α. One (3%) of 30 reactive mesothelial proliferations and 17 (45%) of 39 mesotheliomas expressed EGFR. Conclusions...