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The most common, and usually the only, endocrine disturbance in patients with hypothalamic hamartoma (HH) and epilepsy is central precocious puberty (CPP). The mechanism for CPP associated with HH may relate to ectopic generation and pulsatile release of gonadotropin-releasing hormone (GnRH) from the HH, but this remains an unproven hypothesis. Possible regulators of GnRH release that are intrinsic to HH tissue include the following: (1) glial factors (such as transforming growth factor α[TGFα) and (2) γ-aminobutyric acid (GABA)-mediated excitation. Both are known to be present in surgically-resected HH tissue, but are present in patients with and without a history of CPP, suggesting the possibility that symptoms related to HH are directly associated with the region of anatomic attachment of the HH to the hypothalamus, which determines functional network connections, rather than to differences in HH tissue expression or pathophysiology. CPP associated with HH presents with isosexual development prior to the age of 8 years in girls and 9 years in boys. It is not uncommon for CPP with HH to present in children at an earlier age in comparison to other causes of CPP, including in infancy. Surgical resection of the HH can be effective for treating CPP, but is reserved for patients with intractable epilepsy, since GnRH agonists are widely available and effective treatment. Other endocrine disturbances with HH are rare, but can include growth hormone deficiency, hypothyroidism, and adrenal insufficiency. Diabetes insipidus is commonly encountered postoperatively, but is not observed with HH prior to surgical intervention., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Transforming growth factor-alpha (TGF-α) is known to play multifunctional roles in the central nervous system (CNS), including the provision of neurotropic properties that protect neurons against various neurotoxic insults. Previously, we reported that TGF-α mediates estrogen-induced enhancement of glutamate transporter GLT-1 function in astrocytes. However, the regulatory mechanism of TGF-α at the transcriptional level remains to be established. Our findings revealed that the human TGF-α promoter contains consensus sites for several transcription factors, such as NF-κB and yin yang 1 (YY1). NF-κB served as a positive regulator of TGF-α promoter activity, corroborated by observations that overexpression of NF-κB p65 increased, while mutation in the NF-κB binding sites in the TGF-α promoter reduced the promoter activity in rat primary astrocytes. Pharmacological inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC; 50 μM) or quinazoline (QNZ; 10 μM) also abolished TGF-α promoter activity, and NF-κB directly bound to its consensus site in the TGF-α promoter as evidenced by electrophoretic mobility shift assay (EMSA). Dexamethasone (DX) increased TGF-α promoter activity by activation of NF-κB. Treatment of astrocytes with 100 nM of DX for 24 h activated its glucocorticoid receptor and signaling proteins, including MAPK, PI3K/Akt, and PKA, via non-genomic pathways, to enhance TGF-α promoter activity and expression. YY1 served as a critical negative regulator of the TGF-α promoter as overexpression of YY1 decreased, while mutation of YY1 binding site in the promoter increased TGF-α promoter activity. Treatment for 3 h with 250 μM of manganese (Mn), an environmental neurotoxin, decreased astrocytic TGF-α expression by activation of YY1. Taken together, our results suggest that NF-κB is a critical positive regulator, whereas YY1 is a negative regulator of the TGF-α promoter. These findings identify potential molecular targets for neurotherapeutics that may modulate TGF-α regulation and afford neuroprotection.

Epithelial-to-mesenchymal transition (EMT) endows cancer cells with enhanced invasive and metastatic potential during cancer progression. Fractalkine, also known as chemokine (C-X3-C motif) ligand 1 (CX3CL1), the only member recognized so far that belongs to the CX3C chemokine subfamily, was reported to participate in the molecular events that regulate cell adhesion, migration and survival of human prostate cancer cells. However, the relationship between CX3CL1 and EMT remains unknown. We treated DU145 and PC-3 cells with CX3CL1 under hypoxic conditions. The migration and invasion abilities of DU145 and PC-3 cells were detected by Transwell assays. Induction of EMT was verified by morphological changes in the DU145 and PC-3 cells and analysis of protein expression of EMT markers such as E-cadherin and vimentin. To identify the involved signaling pathway in CX3CL1-induced EMT, activation of epidermal growth factor receptor (EGFR) was measured using western blot analysis, and Slug expression was detected with or without an EGFR inhibitor prior to CX3CL1 treatment. Concentrations of soluble and total TGF-α in the CX3CL‑treated DU145 cells were detected by ELISA. Additionally, we determined the involvement of the TACE/TGF-α/EGFR pathway in CX3CL1‑induced EMT using RNA interference and specific inhibitors. CX3CL1 increased the migration and invasiveness of the DU145 and PC-3 cells, and resulted in characteristic alterations of EMT. Our results demonstrated that TACE/TGF-α/EGFR pathway activation and subsequent upregulation of Slug expression were responsible for CX3CL1‑induced EMT, and contributed to the migration and inva-sion of prostate cancer cells. Inhibition of TACE/TGF-α/EGFR signaling reversed EMT and led to reduced migration and invasion abilities of the prostate cancer cells. We provide initial evidence that CX3CL1 exposure resulted in EMT occurrence and enhancement of cell migration and invasion through a mechanism involving activation of TACE/TGF-α/EGFR signaling. These findings revealed that CX3CL1 may serve as a new target for the treatment of prostate cancer.

Objective: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA., Methods: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C)., Results: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage., Conclusion: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo., (© 2015, American College of Rheumatology.)

Fibronectin (FN), an extracellular matrix ligand, plays a pivotal role in cell adhesion, migration, and oncogenic transformation. Aberrant FN expression is associated with poor prognoses in various types of cancer, including breast cancer. In the current study, we investigated the relationship between FN induction and HER2 expression in breast cancer cells. Our results showed that the level of FN expression increased in response to HER family ligands, EGF and TGF-α in a time- and dose-dependent manner. On the other hand, EGF-induced FN expression decreased in response to trastuzumab, which is a HER2-targeted monoclonal antibody. However, EGF-induced FN expression was not affected by trastuzumab in JIMT-1 breast cancer cells, which are trastuzumab insensitive cells. Next, we introduced the HER2 gene into MDA-MB231 cells to verify the relationship between FN and HER2. The level of FN expression significantly increased in HER2-overexpressed MDA-MB231 cells. In contrast, the induction of FN by HER2 was significantly decreased in response to trastuzumab treatment. In addition, the induction of FN by HER2 was down-regulated by the MEK 1/2 specific inhibitor, U0126. Using conditioned culture media of vec- and HER2-overexpressed MDA-MB231 cells, we observed the cell morphology, adhesion, and invasion of MDA-MB231 cells. Interestingly, in conditioned culture media of HER2-overexpressed MDA-MB231 cells, the cell morphology was altered, and adhesion and invasion of MDA-MB231 cells significantly increased. In addition, our results showed that recombinant human FN augmented cell adhesion and invasion of MDA-MB231 cells while these inductions decreased in response to an FN inhibitor. Therefore, we demonstrated that the induction of FN by HER2 triggers cell adhesion and invasion capacities., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti-TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2-dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti-TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α-dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis.

In Drosophila melanogaster, the anteroposterior (AP) and dorsoventral (DV) axes of the oocyte and future embryo are established through the localization and translational regulation of gurken (grk) mRNA. This process involves binding of specific factors to the RNA during transport and a dynamic remodeling of the grk-containing ribonucleoprotein (RNP) complexes once they have reached their destination within the oocyte. In ovaries of spindle-class females, an activated DNA damage checkpoint causes inefficient Grk translation and ventralization of the oocyte. In a screen for modifiers of the oocyte DV patterning defects, we identified a mutation in the eIF1A gene as a dominant suppressor. We show that reducing the function of eIF1A in spnB ovaries suppresses the ventralized eggshell phenotype by restoring Grk expression. This suppression is not the result of more efficient DNA damage repair or of disrupted checkpoint activation, but is coupled to an increase in the amount of grk mRNA associated with polysomes. In spnB ovaries, the activated meiotic checkpoint blocks Grk translation by disrupting the accumulation of grk mRNA in a translationally competent RNP complex that contains the translational activator Oo18 RNA-binding protein (Orb); this regulation involves the translational repressor Squid (Sqd). We further propose that reduction of eIF1A allows more efficient Grk translation possibly because of the presence of specific structural features in the grk 5'UTR., (© 2014. Published by The Company of Biologists Ltd.)

Polycystic kidney disease (PKD) is a common genetic disorder leading to cyst formation in the kidneys and other organs that ultimately results in kidney failure and death. Currently, there is no therapy for slowing down or stopping the progression of PKD. In this study, we identified the disintegrin metalloenzyme 17 (ADAM17) as a key regulator of cell proliferation in kidney tissues of conditional knockout Ift88(-/-) mice and collecting duct epithelial cells from Ift88°(rpk) mice, animal models of autosomal recessive polycystic kidney disease (ARPKD). Using Western blotting, an enzyme activity assay, and a growth factor-shedding assay in the presence or absence of the specific ADAM17 inhibitor TMI-005, we show that increased expression and activation of ADAM17 in the cystic kidney and in collecting duct epithelial cells originating from the Ift88°(rpk) mice (designated as PKD cells) lead to constitutive shedding of several growth factors, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and transforming growth factor-α (TGF-α). Increased growth factor shedding induces activation of the EGFR/MAPK/ERK pathway and maintains higher cell proliferation rate in PKD cells compared with control cells. PKD cells also displayed increased lactate formation and extracellular acidification indicative of aerobic glycolysis (Warburg effect), which was blocked by ADAM17 inhibition. We propose that ADAM17 is a key promoter of cellular proliferation in PKD cells by activating the EGFR/ERK axis and a proproliferative glycolytic phenotype.

Radiation fibrosis of the lung is a late toxicity of thoracic irradiation. Epidermal growth factor (EGF) signaling has previously been implicated in radiation lung injury. We hypothesized that TGF-α, an EGF receptor ligand, plays a key role in radiation-induced fibrosis in lung. Mice deficient in transforming growth factor (TGF-α(-/-)) and control C57Bl/6J (C57-WT) mice were exposed to thoracic irradiation in 5 daily fractions of 6 Gy. Cohorts of mice were followed for survival (n ≥ 5 per group) and tissue collection (n = 3 per strain and time point). Collagen accumulation in irradiated lungs was assessed by Masson's trichrome staining and analysis of hydroxyproline content. Cytokine levels in lung tissue were assessed with ELISA. The effects of TGF-α on pneumocyte and fibroblast proliferation and collagen production were analyzed in vitro. Lysyl oxidase (LOX) expression and activity were measured in vitro and in vivo. Irradiated C57-WT mice had a median survival of 24.4 weeks compared to 48.2 weeks for irradiated TGF-α(-/-) mice (P = 0.001). At 20 weeks after irradiation, hydroxyproline content was markedly increased in C57-WT mice exposed to radiation compared to TGF-α(-/-) mice exposed to radiation or unirradiated C57-WT mice (63.0, 30.5 and 37.6 μg/lung, respectively, P = 0.01). C57-WT mice exposed to radiation had dense foci of subpleural fibrosis at 20 weeks after exposure, whereas the lungs of irradiated TGF-α (-/-) mice were largely devoid of fibrotic foci. Lung tissue concentrations of IL-1β, IL-4, TNF-α, TGF-β and EGF at multiple time points after irradiation were similar in C57-WT and TGF-α(-/-) mice. TGF-α in lung tissue of C57-WT mice rose rapidly after irradiation and remained elevated through 20 weeks. TGF-α(-/-) mice had lower basal LOX expression than C57-WT mice. Both LOX expression and LOX activity were increased after irradiation in all mice but to a lesser degree in TGF-α(-/-) mice. Treatment of NIH-3T3 fibroblasts with TGF-α resulted in increases in proliferation, collagen production and LOX activity. These studies identify TGF-α as a critical mediator of radiation-induced lung injury and a novel therapeutic target in this setting. Further, these data implicate TGF-α as a mediator of collagen maturation through a TGF-β independent activation of lysyl oxidase.

Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and-epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Secondary bile acids (BA) such as deoxycholic acid (DCA) promote the development of several gastrointestinal malignancies, but how they mediate this effect is unclear. In this study, we offer evidence of a mechanism involving ectodomain shedding of the EGFR ligands amphiregulin (AREG) and TGF-α, which rely upon the cell surface protease TACE/ADAM-17. Specifically, we show that AREG participates in DCA-induced EGFR and STAT3 signaling, cell-cycle progression, and tumorigenicity in human colorectal cancer and pancreatic ductal adenocarcinoma (PDAC). TACE and AREG, but not TGF-α, were overexpressed in both colorectal cancer and PDAC tissues compared with normal tissues. Exposure of colorectal cancer and PDAC cells to DCA resulted in colocalization of Src and TACE to the cell membrane, resulting in AREG-dependent activation of EGFR, mitogen-activated protein kinase (MAPK), and STAT3 signaling. Src or TACE inhibition was sufficient to attenuate DCA-induced AREG, but not TGF-α shedding. We also examined a role for the BA transporter TGR5 in DCA-mediated EGFR and STAT3 signaling. RNA interference-mediated silencing of TGR5 or AREG inhibited DCA-induced EGFR, MAPK, and STAT3 signaling, blunted cyclin D1 expression and cell-cycle progression, and attenuated DCA-induced colorectal cancer or PDAC tumorigenicity. Together, our findings define an AREG-dependent signaling pathway that mediates the oncogenic effects of secondary BAs in gastrointestinal cancers, the targeting of which may enhance therapeutic responses in their treatment., (©2014 AACR.)

Aims: The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated., Main Methods: The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1., Key Findings: Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells., Significance: These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism., (Published by Elsevier Inc.)

Reactive astrogliosis is beneficial in many aspects; however, it is also detrimental in some pathological states such as the development of lethal brain tumors. It is therefore crucial to understand the mechanisms regulating astrocyte proliferation. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor family, was shown to stimulate astrocyte proliferation in vitro. Herein, we further characterize the mitogenic potential of TWEAK on central nervous system cells. Among these cells, astrocytes express the highest level of TWEAK and Fn14 transcripts, suggesting that they are particularly sensitive to TWEAK stimulation. Using in vitro model systems, we found that TWEAK was as potent as epidermal growth factor (EGF) (a prototypical astrocyte mitogen) in mediating astrocyte proliferation. However, its mitogenic activity was delayed compared with that of EGF, suggesting distinct mechanisms of action. Using cell signaling pathway inhibitors, neutralizing antibodies, and protein assays, we further show that the mitogenic activity of TWEAK on primary astrocytes requires stimulation of the transforming growth factor-α (TGF-α) and of the epidermal growth factor receptor (EGFR) signaling pathway through extracellular signal-regulated kinase and p38 mitogen-activated protein kinase activation. In aggregates, our data demonstrate that TWEAK acts as a potent astrocyte mitogen through the induction of a TGF-α/EGFR signaling pathway. We anticipate that description of such a mechanism may allow novel approaches to human pathologies associated with astrocyte proliferation.

A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G(1) phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. We describe a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. We demonstrate that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the postsynaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand., (Copyright © 2011 Wiley Periodicals, Inc.)

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In 1888, Pierre Ménétrier first described the disease that bears his name. Many of the findings he reported then remain accepted features of the disease. Based on studies performed in our laboratory over the past 20 years, we have implicated increased transforming growth factor-α (TGFα) expression and heightened epidermal growth factor receptor (EGFR) activity in the pathogenesis of Ménétrier's disease. Herein, we provide a historical perspective of this rare disorder, review our experience with Ménétrier's disease, and discuss future challenges and opportunities posed by this disorder.

Airway hyperreactivity (AHR) and remodeling are cardinal features of asthma and chronic obstructive pulmonary disease. New therapeutic targets are needed as some patients are refractory to current therapies and develop progressive airway remodeling and worsening AHR. The mammalian target of rapamycin (mTOR) is a key regulator of cellular proliferation and survival. Treatment with the mTOR inhibitor rapamycin inhibits inflammation and AHR in allergic asthma models, but it is unclear if rapamycin can directly inhibit airway remodeling and AHR, or whether its therapeutic effects are entirely mediated through immunosuppression. To address this question, we utilized transforming growth factor-α (TGF-α) transgenic mice null for the transcription factor early growth response-1 (Egr-1) (TGF-α Tg/Egr-1(ko/ko) mice). These mice develop airway smooth muscle thickening and AHR in the absence of altered lung inflammation, as previously reported. In this study, TGF-α Tg/Egr-1(ko/ko) mice lost body weight and developed severe AHR after 3 wk of lung-specific TGF-α induction. Rapamycin treatment prevented body weight loss, airway wall thickening, abnormal lung mechanics, and increases in airway resistance to methacholine after 3 wk of TGF-α induction. Increases in tissue damping and airway elastance were also attenuated in transgenic mice treated with rapamycin. TGF-α/Egr-1(ko/ko) mice on doxycycline for 8 wk developed severe airway remodeling. Immunostaining for α-smooth muscle actin and morphometric analysis showed that rapamycin treatment prevented airway smooth muscle thickening around small airways. Pentachrome staining, assessments of lung collagen and fibronectin mRNA levels, indicated that rapamycin also attenuated fibrotic pathways induced by TGF-α expression for 8 wk. Thus rapamycin reduced airway remodeling and AHR, demonstrating an important role for mTOR signaling in TGF-α-induced/EGF receptor-mediated reactive airway disease.

Airborne particulate matter (PM) may induce or exacerbate neutrophilic airway disease by triggering the release of inflammatory mediators, such as CXC chemokine ligand (CXCL)8, from the airway epithelium. It is still unclear which PM components are driving CXCL8 responses, as most candidates occur at low concentrations in the dusts. We therefore hypothesised that different PM constituents may contribute through common mechanisms to induce CXCL8. Human bronchial epithelial cells (BEAS-2B) were exposed to different PM components (Zn²⁺/Fe²⁺ salts, 1-nitropyrene, lipopolysaccharide and diesel exhaust/mineral particles). Gene expression patterns were detected by real-time PCR array. CXCL8 responses were measured by real-time PCR and ELISA. CXCL8 regulation was assessed with a broad inhibitor panel and neutralising antibodies. Epidermal growth factor receptor (EGFR) phosphorylation was examined by immunoprecipitation and Western blotting. Component-induced gene expression was mainly linked to nuclear factor-κB, Ca²⁺/protein kinase C, phospholipase C, low-density lipoprotein and mitogenic signalling. Many inhibitors attenuated CXCL8 release induced by all PM components, but to varying extents. However, EGFR inhibition strongly reduced CXCL8 release induced by all test compounds and selected compounds increased EGFR phosphorylation. Interference with transforming growth factor (TGF)-α or tumour necrosis factor-α-converting enzyme (TACE), which mediates TGF-α ectodomain shedding, also attenuated CXCL8 release. Different PM constituents induced CXCL8 partly through similar signalling pathways but the relative importance of the different pathways varied. However, TACE/TGF-α/EGFR signalling appears to be a convergent pathway regulating innate immune responses of airway epithelial cells upon exposure to multiple airborne pollutants.

A comprehensive understanding of molecular mechanisms driving cancer onset and progression should provide a basis for improving early diagnosis, biomarker discovery and treatment options. A key value of genetically engineered mice for modeling human cancer is the possibility to analyze the entire process of tumor development. Here, we applied functional genomics approach to study step-by-step development of hepatocellular carcinoma (HCC) in the c-Myc/Tgfα transgenic mouse model of aggressive human liver cancer. We report that coexpression of c-Myc and Tgfα induces progressive and cumulative transcriptional alterations in the course of liver oncogenesis. Functional analysis of deregulated genes at the early stage of HCC disease supports a model of active hepatocyte proliferation on the background of chronic oxidative stress generated by a general metabolic disorder. In addition, early and persistent deregulation of numerous immune-related genes suggested that disruption of immune microenvironment may contribute to oncogenic process in this model of accelerated liver carcinogenesis. In particularly, by flow cytometry analysis, we found loss of the major histocompatibility complex class I expression in dysplastic hepatocytes followed by upregulation of numerous activating ligands for natural killer (NK) cells concomitant with a drastic decrease in hepatic NK cell frequency. In conclusion, our study provides a comprehensive characterization of sequential molecular changes during a stepwise progression of preneoplastic lesions toward HCC and highlights a critical role of metabolic disorders and innate immunity at the early stages of liver cancer.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.

The mechanisms of progression of chronic kidney disease (CKD) are poorly understood. Epidemiologic studies suggest a strong genetic component, but the genes that contribute to the onset and progression of CKD are largely unknown. Here, we applied an experimental model of CKD (75% excision of total renal mass) to six different strains of mice and found that only the FVB/N strain developed renal lesions. We performed a genome-scan analysis in mice generated by back-crossing resistant and sensitive strains; we identified a major susceptibility locus (Ckdp1) on chromosome 6, which corresponds to regions on human chromosome 2 and 3 that link with CKD progression. In silico analysis revealed that the locus includes the gene encoding the EGF receptor (EGFR) ligand TGF-α. TGF-α protein levels markedly increased after nephron reduction exclusively in FVB/N mice, and this increase preceded the development of renal lesions. Furthermore, pharmacologic inhibition of EGFR prevented the development of renal lesions in the sensitive FVB/N strain. These data suggest that variable TGF-α expression may explain, in part, the genetic susceptibility to CKD progression. EGFR inhibition may be a therapeutic strategy to counteract the genetic predisposition to CKD.

Background: Adrenocorticotrophin (ACTH)-secreting pituitary adenomas account for approximately 7% - 14% of all pituitary adenomas, but its pathogenesis is still enigmatic. This study aimed to explore mechanisms underlying the pathogenesis of ACTH-secreting pituitary adenomas., Methods: We used fiber-optic beadarray to examine gene expression in three ACTH-secreting adenomas compared with three normal pituitaries. Four differentially expressed genes from the three ACTH-secreting adenomas and three normal pituitaries were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway., Results: Fiber-optic beadarray analysis showed that the expression of 28 genes and 8 expressed sequence tags (ESTs) were significantly increased and the expression of 412 genes and 31 ESTs were significantly decreased. Bioinformatic and pathway analysis showed that the genes HIGD1B, EPS8, HPGD, DAPK2, and IGFBP3 and the transforming growth factor (TGF)-β signaling pathway and extracellular matrix (ECM)-receptor interaction pathway may play important roles in tumorigenesis and progression of ACTH-secreting pituitary adenomas., Conclusions: Our data suggest that numerous aberrantly expressed genes and several pathways are involved in the pathogenesis of ACTH-secreting pituitary adenomas. Fiber-optic beadarray combined with pathway analysis of differential gene expression appears to be a valid method of investigating tumour pathogenesis.

Hepatocellular carcinoma (HCC) results from the cumulative effects of deregulated tumor suppressor genes and oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and their downstream signaling pathway components. The overexpression of transforming growth factor alpha (TGF-alpha) and the inhibition of TGF-beta signaling are especially common in human liver cancer. Thus, we assessed whether TGF-alpha overexpression and TGF-beta signaling inactivation cooperate in hepatocarcinogenesis using an in vivo mouse model, MT1/TGFa;AlbCre/Tgfbr2(flx/flx) mice ("TGFa;Tgfbr2(hepko)"), which overexpresses TGF-alpha and lacks a TGF-beta receptor in the liver. TGF-beta signaling inactivation did not alter the frequency or number of cancers in mice with overexpression of TGF-alpha. However, the tumors in the TGFa;Tgfbr2(hepko) mice displayed increased proliferation and increased cdk2, cyclin E and cyclin A expression as well as decreased Cdkn1a/p21 expression compared to normal liver and compared to the cancers arising in the TGF-alpha overexpressing mice with intact TGF-beta receptors. Increased phosphorylated ERK1/2 expression was also present in the tumors from the TGFa;Tgfbr2(hepko) mice and correlated with downregulated Raf kinase inhibitor protein expression, which is a common molecular event in human HCC. Thus, TGF-beta signaling inactivation appears to cooperate with TGF-alpha in vivo to promote the formation of liver cancer that recapitulates molecular features of human HCC.

Although J2N-k strain of cardiomyopathic hamsters is an excellent model of dilated cardiomyopathy, the presence and mechanisms of apoptosis in the hearts of these genetically modified animals have not been investigated. This study examined the hypothesis that cardiac dysfunction and apoptosis in the cardiomyopathic hamsters were associated with tumour necrosis factor-alpha (TNF-alpha)-mediated signalling pathway involving the activation of some pro-apoptotic proteins and/or deactivation of some antiapoptotic proteins. Echocardiographic assessment of 31-week-old hamsters indicated an increase in the internal dimension of the left ventricle as well as decreases in the ejection fraction, fractional shortening and cardiac output without any evidence of cardiac hypertrophy. Increased level of TNF-alpha and apoptosis in cardiomyopathic hearts were accompanied by increased protein content for protein kinase C (PKC) -alpha and -epsilon isozymes as well as caspases 3 and 9. Phosphorylated protein content for p38 MAPK and NF kappaB was increased whereas that for Erk1/2, BAD and Bcl-2 was decreased in cardiomyopathic hearts. These results support the view that TNF-alpha and PKC isozymes may promote apoptosis due to the activation of p38 MAPK and deactivation of Erk1/2 pathways, and these changes may contribute toward the development of cardiac dysfunction in dilated cardiomyopathy.

Stem cell-based therapies offer promising potential for myocardial infarction (MI), but endogenous molecules released in response to injury likely impair posttransplantation stem cell function. Stem cell-mediated cardioprotection occurs in part via paracrine effects, and transforming growth factor-alpha (TGF-alpha) has been shown to enhance paracrine function. However, it is unknown whether pretreating stem cells with TGF-alpha increases stem cell-mediated cardioprotection after acute MI. Mesenchymal stem cells (MSCs) were treated with TGF-alpha (250 ng/ml) for 24 h. Adult male Sprague-Dawley rat hearts were isolated and perfused using the Langendorff method. MI was induced by ligating the left anterior descending coronary artery. Postligation (30 min), vehicle or 1 x 10(6) MSCs with or without pretreatment were injected in the infarct border zones, and the hearts were perfused for an additional 60 min. Left ventricular function was continuously measured, and infarct size was assessed with Evans blue dye and 2,3,5-triphenyltetrazolium chloride staining. Myocardial production of interleukin (IL)-1beta and IL-6 and caspase 3 activation was also measured. Left ventricular function decreased significantly following coronary artery ligation but improved following injection of untreated MSCs and to a greater extent after injection of pretreated MSCs. In addition, the infarct area, myocardial caspase 3 activation, and IL-6 production were lowest in hearts injected with pretreated cells. Intramyocardial injection of TGF-alpha-pretreated MSCs after acute MI is associated with increased myocardial function and decreased myocardial injury. This strategy may be useful for optimizing the therapeutic efficacy of stem cells for the treatment of acute MI.

During Drosophila oogenesis, nurse cells undergo changes in chromosomal morphology, first from the polytenic form to a transient condensed phase known as the five-blob configuration, then into a diffuse polytenic-polyploid state for the remainder of oogenesis. The mechanism by which nurse-cell chromosome dispersal is regulated remains elusive. Mutations in several genes, including the heterogeneous ribonucleoprotein genes squid (sqd) and hrb27C, the alternative splicing factor gene poly U binding factor 68 kDa (pUf68, also known as half-pint), and the germ-line-specific gene ovarian tumor (otu), that produce defects in nurse-cell chromosome dispersal also produce defects in oocyte polarity, suggesting a link between these two processes. Here, we characterize a novel gene named poly, which, when mutated in the germ line, disrupts nurse-cell chromosome dispersal, as well as localization of anteroposterior and dorsoventral determinants in the oocyte. We also show that poly interacts genetically with hrb27C and otu. We conclude that poly is required for nurse-cell chromosome dispersal and oocyte polarization in the Drosophila germ-line. In addition, our interaction data suggest that poly is probably a member of the characterized mRNP complex that mediates both processes.

The development of the gastric mucosa is controlled by hormones, growth factors and feeding behavior. Early weaning (EW), which means the abrupt interruption of suckling, increases proliferation and differentiation in the rat gastric epithelium. Transforming growth factor alpha (TGFalpha) is secreted in the stomach, binds to the epidermal growth factor receptor (EGFR) and may control cell proliferation, differentiation and migration. Here, we investigated the influence of suckling-weaning transition on the differentiation of mucous neck cells in the stomach and its association to the expression of TGFalpha and EGFR. Fifteen-day-old Wistar rats were divided into two groups: suckling (control), in which pups were kept with the dam, and early weaning (EW), in which rats were separated from their mother and fed with hydrated powdered chow. TGFalpha and EGFR levels were increased at 18 days in EW animals compared to control ones (p<0.05). Histochemical reactions with Periodic Acid-Schiff reagent+Alcian Blue or Bandeiraea simplicifolia II lectin were used to stain the mucous neck cells and showed an increase in this cell population throughout EW, which was more pronounced at 17 days when compared to suckling pups (p<0.05). These morphological results were confirmed by RT-PCR for mucin 6. The levels of mucin 6 mRNA were higher in EW animals from the 16th to the 18th day (1-3 days post-weaning) when compared to the respective control group. Inhibition of EGFR through AG1478 administration to EW animals prevented the expansion of mucous neck cell population induced by EW (p<0.05). Therefore, early weaning up regulated TGFalpha/EGFR expression and induced differentiation of mucous neck cells. Moreover, we showed that EGFR takes part in the maturation of this cell population. We conclude that regular suckling-weaning transition is crucial to guarantee the development of the gastric mucosa.

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle alpha-actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 +/- 42 and 575 +/- 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist N6-cyclohexyladenosine. Maximum TGF responses (0-30 nl/min flow step) were increased from 8.4 +/- 0.9 mmHg in WT (n = 21) to 14.2 +/- 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 +/- 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5-10 and 10-15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 +/- 1.5 nl/min compared with 2.6 +/- 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses.

Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.

Estrogen signaling in auditory and vestibular sensory epithelia is a newly emerging focus propelled by the role of estrogen signaling in many other proliferative systems. Understanding the pathways with which estrogen interacts can provide a means to identify how estrogen may modulate proliferative signaling in inner ear sensory epithelia. Reviewed herein are two signaling families, EGF and TGFbeta. Both pathways are involved in regulating proliferation of supporting cells in mature vestibular sensory epithelia and have well characterized interactions with estrogen signaling in other systems. It is becoming increasingly clear that elucidating the complexity of signaling in regeneration will be necessary for development of therapeutics that can initiate regeneration and prevent progression to a pathogenic state.

Transforming growth factor-alpha (TGFalpha) is a powerful endogenous mitogen and neurotrophic factor, which has previously been shown to induce a massive proliferative response in the brains of Parkinson's disease model rats injured by an acute neurotoxic lesion. We now show that TGFalpha can also produce a massive proliferative response in rat brains subjected to stroke caused by a middle cerebral artery occlusion (MCAO), even when the growth factor is administered as late as 4 weeks after injury. This combination of stimuli provokes DNA synthesis, shown by 5'-bromo-2-deoxyuridine incorporation, throughout the ependymal layer and subventricular zone (SVZ) of the forebrain during the 4 weeks of growth factor administration. The newly generated cells migrate preferentially along and ventral to the corpus callosum (CC) and external capsule to the site of the injury where many of them differentiate into several site-appropriate neuronal phenotypes in association with near complete (99%) behavioral recovery. We conclude that the injury response of endogenous neural stem cells as well as behavioral recovery can be significantly enhanced by application of TGFalpha, and that this approach represents a potential therapeutic strategy for chronic stroke and other neurological damage in human patients.

Epithelial and endothelial tight junctions act as a rate-limiting barrier between an organism and its environment. Continuing studies have highlighted the regulation of the tight junction barrier by cytokines. Elucidation of this interplay is vital for both the understanding of physiological tight junction regulation and the etiology of pathological conditions. This review will focus on recent advances in our understanding of the molecular mechanisms of tight junctions modulation by cytokines.

To elucidate the mechanism of action of prostaglandin I(2) (PGI(2)) and carbaprostacyclin, we studied their effect on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Hepatocyte parenchymal cells, maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of PGI(2) or carbaprostacyclin in a time- and dose-dependent manner. PGI(2) was less potent than carbaprostacyclin in stimulating hepatocyte mitogenesis. These effects of PGI(2) and carbaprostacyclin were abolished by treatment with a specific IP-receptor antagonist, CAY10441 (10(-9) - 10(-7) M). Hepatocyte mitogenesis induced by the IP-receptor agonists was almost completely blocked by specific inhibitors of growth-related signal transducers such as AG1478 (5 x 10(-7) M), LY294002 (10(-7) M), PD98059 (10(-6) M), and rapamycin (10 ng/ml). In addition, PGI(2) or carbaprostacyclin significantly increased the kinase activity of a (p175 kDa) receptor tyrosine kinase and the phosphorylation of extracellular signal-regulated kinase (ERK) 2. Addition of a monoclonal antibody against transforming growth factor (TGF)-alpha, but not insulin-like growth factor-I, to the culture dose-dependently inhibited the PGI(2)- or carbaprostacyclin-induced hepatocyte mitogenesis. Furthermore, treatment with the IP-receptor agonists significantly increased the secretion of TGF-alpha to the culture medium. These results indicate that the IP receptor agonist-induced hepatocyte mitogenesis is mediated by autocrine secretion of TGF-alpha followed by activation of a receptor tyrosine kinase / ERK pathway.

The growth factor, transforming growth factor-alpha (TGF-alpha) is strongly expressed in the hypothalamic circadian pacemaker, the suprachiasmatic nucleus (SCN). TGF-alpha is one of several SCN peptides recently suggested to function as a circadian output signal for the regulation of locomotor activity rhythms in nocturnal rodents. When infused in the brain, TGF-alpha suppresses activity. TGF-alpha suppresses other behaviors as well including feeding, resulting in weight loss. Elevated TGF-alpha is correlated with some cancers, and it is possible the TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), mediate fatigue and weight loss associated with cancer. If true for cancers outside of the brain, then systemic TGF-alpha should also affect behavior. We tested this hypothesis in hamsters with intraperitoneal injections or week-long subcutaneous infusions of TGF-alpha. Both treatments suppressed activity and infusions caused reduced food consumption and weight loss. To identify areas of the brain that might mediate these effects of systemic TGF-alpha, we used immunohistochemistry to localize cells with an activated MAP kinase signaling pathway (phosphorylated ERK1). Cells were activated in two hypothalamic areas, the paraventricular nucleus and a narrow region surrounding the third ventricle. These sites could not only be targets of TGF-alpha produced in the SCN but could also mediate effects of elevated TGF-alpha from tumors both within and outside the central nervous system.

The growth and maturation of the ovarian follicle requires the coordinate function of somatic cells and the oocyte. Over the past three decades, numerous growth factors involved in the bidirectional signals between the somatic and germ cells have been identified. A possible function of epidermal growth factor (EGF) signaling at selected stages of follicle maturation had been proposed early on and is supported by many observations of in vitro effects of this growth factor on steroidogenesis, oocyte maturation, and cumulus expansion. However, attempts to link EGF levels in the follicular fluid with the state of follicle and oocyte maturation have been inconclusive. More recently, data generated using mouse genetic models perturbing ovulation and fertility indicate that EGF-like growth factors, rather than EGF itself, accumulate in the follicle at the time of ovulation. EGF-like growth factor mRNA is regulated by the luteinizing hormone surge, and corresponding proteins are detected in the follicle. The EGF-like growth factors amphiregulin, epiregulin, and betacellulin are potent stimulators of oocyte maturation and cumulus expansion, and perturbation of this EGF network in vivo impairs ovulation. Similar findings in species other than the mouse confirm an important physiological role for this network at the time of ovulation. Whether this network also plays a critical role in humans and whether it can be used as a biological marker of follicle development or for the improvement of fertility remains to be determined. This review summarizes the most recent findings on the EGF network during ovulation and the potential clinical applications of manipulating this intercellular communication pathway in the control of fertility.

Viral infection is a major trigger for exacerbation of asthma and induces overproduction of mucins. We investigated whether dsRNA could amplify the induction of mucin by TGF-alpha in human bronchial epithelial cells, as well as the molecular mechanisms regulating MUC5AC expression. Human pulmonary mucoepidermoid carcinoma (NCI-H292) cells and normal human bronchial epithelial cells were exposed to polyinosinic-cytidyric acid (poly(I:C)) and TGF-alpha. Then, MUC5AC protein production, mRNA expression, and promoter activity were evaluated. Cells were pretreated with a selective inhibitor of ERK, and phosphorylation of ERK was examined by Western blotting. Furthermore, the expression of MAPK phosphatase 3 (MKP3) mRNA was evaluated and the effect of MKP3 overexpression was assessed. Poly(I:C) synergistically increased MUC5AC induction by TGF-alpha in both NCI-H292 and normal human bronchial epithelial cells. This increase was dependent on MUC5AC gene transcription. A MEK1/2 inhibitor (U0126) significantly inhibited MUC5AC production. Phosphorylation of ERK was enhanced by poly(I:C). TGF-alpha stimulation up-regulated MKP3 mRNA expression, while costimulation with poly(I:C) inhibited this up-regulation dose-dependently. Enhanced expression of MUC5AC mRNA by poly(I:C) in wild-type cells was completely suppressed in cells transfected with the MKP3 expression vector. dsRNA can synergistically amplify the induction of MUC5AC mucin by TGF-alpha. This synergistic effect on MUC5AC production may be due to enhanced activation of ERK through inhibition of MKP3 by poly(I:C).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL.

In secondary hyperparathyroidism, enhanced expression of TGF-alpha in the parathyroid leads to its own upregulation, generating a feed-forward loop for TGF-alpha activation of its receptor, EGFR receptor (EGFR), which promotes parathyroid hyperplasia. These studies examined the role of activator protein 2alpha (AP2), an inducer of TGF-alpha gene transcription, in the upregulation of parathyroid TGF-alpha in secondary hyperparathyroidism. In rat and human secondary hyperparathyroidism, parathyroid AP2 expression strongly correlated with TGF-alpha levels and with the rate of parathyroid growth, as expected. Furthermore, the increases in rat parathyroid content of AP2 and its binding to a consensus AP2 DNA sequence preceded the increase in TGF-alpha induced by high dietary phosphate. More significant, in A431 cells, which provide a model of enhanced TGF-alpha and TGF-alpha self-induction, mutating the core AP2 site of the human TGF-alpha promoter markedly impaired promoter activity induced by endogenous or exogenous TGF-alpha. Important for therapy, in five-sixths nephrectomized rats fed high-phosphate diets, inhibition of parathyroid TGF-alpha self-induction using erlotinib, a highly specific inhibitor of TGF-alpha/EGFR-driven signals, reduced AP2 expression dosage dependently. This suggests that the increases in parathyroid AP2 occur downstream of EGFR activation by TGF-alpha and are required for TGF-alpha self-induction. Indeed, in A431 cells, erlotinib inhibition of TGF-alpha self-induction caused parallel reductions in AP2 expression and nuclear localization, as well as TGF-alpha mRNA and protein levels. In summary, increased AP2 expression and transcriptional activity at the TGF-alpha promoter determine the severity of the hyperplasia driven by parathyroid TGF-alpha self-upregulation in secondary hyperparathyroidism.

Using a mouse predisposed to neoplasia by a germ line mutation in Apc (Apc(Min)), we tested whether induced hyperplasia is sufficient to increase intestinal tumor multiplicity or size in the intestine. We found that hyperplasia in the jejunum correlated with a significant increase in tumor multiplicity. However, tumor multiplicity was unchanged in the hyperplastic colon. This result indicates that even an intestine predisposed to neoplasia can, in certain regions including the colon, accommodate net increased cell growth without developing more neoplasms. Where hyperplasia correlated with increased tumor multiplicity, it did not increase the size or net growth of established tumors. This result suggests that the event linking hyperplasia and neoplasia in the jejunum is tumor establishment. Two novel observations arose in our study: the multiple intestinal neoplasia (Min) mutation partially suppressed both mitosis and transforming growth factor alpha-induced hyperplasia throughout the intestine; and zinc treatment alone increased tumor multiplicity in the duodenum of Min mice.

Regulation of human keratinocyte (HK) migration is critical for skin wound healing. Profiling HK migration-specific genes could help us gain a comprehensive understanding of the process. The main challenge is to separate genes that are unrelated to migration, but simultaneously induced by the same growth factor. In this study, we took advantage of a unique response of HKs to transforming growth factor-beta (TGF-beta), which inhibits proliferation but not migration of HKs, to suppress selectively the proliferation-related genes. Furthermore we stimulated HKs independently with TGF-alpha or insulin and identified the common genes and eliminated TGF-alpha- or insulin-specific genes. Under these conditions, we obtained profiles of the immediate-early genes (IEGs, at 30 minutes), early genes (EGs, at 60 minutes), and delayed-early genes (DEGs, at 120 minutes) by microarray analyses, followed by quantitative real-time reverse transcription-PCR (QRT-PCR) validation and functional characterization by RNA interference (RNAi). Our results revealed the following: (1) 25 upregulated and 1 downregulated IEGs; (2) 58 upregulated and 15 downregulated EGs, and (3) 13 upregulated and 3 downregulated DEGs in both TGF-alpha- and insulin-stimulated HKs. Three genes, all encoding secreted molecules, were investigated in HK migration. These cell motility-specific gene profiles may prove useful to skin wound healing.

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.

The maturation of many neural functions occurs during puberty. An abnormal development of these processes, in the context of genetic vulnerability, may result in sex- and age-dependent penetrance of neuropsychiatric disorders. Reduced transforming growth factors-alpha (TGF-alpha) expression in Waved-1 (Wa-1) mice impairs the stress response and fear memory in adult males, but are absent or far less prominent in adult females and in pubertal males. Gonadectomy around the onset of puberty, when the mutant anatomical and behavioral phenotypes are undetectable, results in significant gene x environment effects. Adult control males show reduced physiological stress response as a result of gonadectomy, but not adult Wa-1 males. In females, pubertal gonadectomy elevates specific anxiety parameters only in adult control mice. There also are general sex-specific effects of pubertal gonadectomy on adult stress and fear memory. Surgical stress alone also induces sex- and genotype-dependent effects, albeit in different behavioral parameters than those affected by gonadectomy. We conclude that normal development of stress and memory processes is reliant on the levels of stress and gonadal factors during puberty, the effects of which are modulated by genetic factors and sex.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are members of the polypeptide growth factor family. The epidermal growth factor-receptor (EGF-R) is a receptor tyrosine kinase of the ErbB family. Many types of cancer, including ovarian cancer, display enhanced EGF-R immunoreactivity on their cell surface membranes. Also, an increase in TGF-alpha synthesis and secretion usually occurs in human carcinoma cell lines. In this study, we compared the immunoreactivities of TGF-alpha and EGF-R in ovarian tumors and related immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumor and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin-eosin and labeled for binding of primary antibodies against TGF-alpha and EGF-R using an avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare immunohistochemical labeling intensities. Increased immunoreactivity of EGF-R and moderate immunoreactivity of TGF-alpha was detected in adenocarcinomas. There was no significant difference in the immunoreactivity of TGF-alpha among the histologic types of ovarian tumors. The results of this study support the hypothesis that EGF-R may be a more useful marker than TGF-alpha in epithelial ovarian tumors.