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[Display omitted] • Mucor circinelloides transformed lathyranes 4 and 5 into nine derivatives, including jatrophane derivatives. • Compounds 12 induces NRG1 release and differentiation of NSC into neurons like other novel PKC activating compounds. • Compound 12 binds to the PKC enzyme pocket via hydrogen bonds with Gly-253, Leu-251, and Thr242. • Oxygenated groups at C-5 and C-8 are essential for H-bond interactions with PKC. • Compound 12 shows stronger interaction with PKC δ-C1B compared to PKC α-C1B. Promoting endogenous neurogenesis for brain repair is emerging as a promising strategy to mitigate the functional impairments associated with various neurological disorders characterized by neuronal death. Diterpenes featuring tigliane, ingenane, jatrophane and lathyrane skeletons, frequently found in Euphorbia plant species, are known protein kinase C (PKC) activators and exhibit a wide variety of pharmacological properties, including the stimulation of neurogenesis. Microbial transformation of these diterpenes represents a green and sustainable methodology that offers a hitherto little explored approach to obtaining novel derivatives and exploring structure–activity relationships. In the present study, we report the biotransformation of euphoboetirane A (4) and epoxyboetirane A (5), two lathyrane diterpenoids isolated from Euphorbia boetica , by Mucor circinelloides MC NRRL3631. Our findings revealed the production of nine biotransformation products (6 – 14), including jatrophane derivatives originated through an unprecedented rearrangement from the parent lathyranes. The chemical structures and absolute configurations of the new compounds were elucidated through comprehensive analysis using NMR and ECD spectroscopy, as well as MS. The study evaluated how principal metabolites and their derivatives affect TGF α and NRG1 release, as well as their potential to promote proliferation or differentiation in cultures of NSC isolated from the SVZ of adult mice. In order to shed some light on the mechanisms underlying the ability of 12 as a neurogenic compound, the interactions of selected compounds with PKC δ -C1B were analyzed through molecular docking and molecular dynamics. Based on these, it clearly appears that the ability of compound 12 to form both acceptor and donor hydrogen bonds with certain amino acid residues in the enzyme pocket leads to a higher affinity compound-PKC complex, which correlates with the observed biological activity. [ABSTRACT FROM AUTHOR]

Objective To explore the effect of LncRNA NUTM2A-AS I on interleukin-I 13 (IL-I 13) -induced chondrocyte damage and its molecular mechanism. Methods Chondrocytes were randomly divided into control group, model group (5 μg/ L IL-I 13 ), miR-183-Sp+model group, miR-NC+model group, si-LncRNA NUTM2A-ASl+model group, si-TGFa+model group, si-NC+ model group, pcDNA-TGFa+si-LncRNA NUTM2A-ASI +model group, pcDNA+si-LncRNA NUTM2A-ASI +model group; realtime fluorescent quantitative PCR (RT-qPCR ) was used to detect the expression levels of LncRNA NUTM2A-AS1, miR-183-Sp and TGFa mRNA; cell counting kit 8 ( CCK-8 ) was used to detect cell viability; flow cytometry was used to detect chondrocyte apoptosis; enzyme-linked immunosorbent assay ( ELISA) was used to detect the levels of TNF-a and IL-6; dual luciferase reporter experiment was used to detect the targeting relationship between NUTM2A-AS1, miR-183-Sp, and TGFa. Results In IL-113- induced chondrocytes, the expressions of LncRNA NUTM2A-AS I and TGFa were increased, the expression of miR-183-Sp was decreased, the activity of chondrocytes was decreased, the apoptosis rate was increased, and the levels of TNF-a and IL-6 were increased. Low expression of LncRNA NUTM2A-AS1, low expression of TGFa or overexpression of miR-183-Sp could promote cell proliferation and inh伽t apoptosis and inflammatory responses. Conclusion The low expression of LncRNA NUTM2A-AS I can inhibit IL-1[3-induced chondrocyte apoptosis and inflammation by regulating miR-183-Sp/TGFa, and promote cell survival. [ABSTRACT FROM AUTHOR]

Background: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer. Methods: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα). Results: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed. Conclusions: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein. [ABSTRACT FROM AUTHOR]

Abstract Colon cancer is one of the most common cancers in the developed countries. The association between transforming growth factor TGFα and human cancer incidence has been suggested, yet, the regulatory roles of TGFα and the molecular mechanisms remain unknown, especially in colon cancer. We aim to investigate the functional regulations of TGFα in colon cancer progression. Two colon cancer cell lines were applied, and plasmid overexpression and siRNA-mediated depletion techniques were used to verify the role of TGFα in colon cancer. Cell proliferation was analyzed by MTS assay and colony formation assay, and western blot assay was used to examine protein expression. Migration, invasion, and reporter assays were also carried out to study the regulations of TGFα in colon cancer. Our results evidenced that expression of TGFα facilitates short-term and long-term proliferations of colon cancer cells. Moreover, TGFα was suggested as a migration-and-invasion promoting factor of colon cancer. Finally, our data indicated that TGFα modulates epithelial-mesenchymal transition (EMT) markers and NFκB signaling pathway in colon cancer cells. We provide the first time evidence of the promoting role TGFα plays in colon cancer tumorigenesis with proposed regulatory mechanisms involving EMT alteration and NFκB signaling pathway. Graphical abstract Image Highlights • TGFα facilitates short-term and long-term proliferations of colon cancer cells. • TGFα was suggested as a migration-and-invasion promoting factor of colon cancer. • TGFα modulates epithelial-mesenchymal transition markers and NFκB signaling pathway in colon cancer cells. [ABSTRACT FROM AUTHOR]

Background: Nonsyndromic cleft lip with/without cleft palate (nsCL ± P) is one of the most common birth defects of complex etiology, occurring in ∼1/700 live births worldwide. A series of epidemiological studies were conducted to investigate the association between a transforming growth factor alpha (TGFα) polymorphism and nsCL ± P risk. The aim of this study was to investigate the association between the TGFα/HinfI polymorphisms and nsCL ± P in Turkish patients. Methods: One hundred fifty-five Turkish subjects were enrolled: 70 nsCL ± P patients and 85 unrelated control individuals. Genomic DNA was isolated from peripheral blood leukocytes, and molecular analysis of gene polymorphisms was carried out using polymerase chain reaction and restriction enzyme digestions. Results: We found significant difference between the TGFα gene HinfI allele frequencies of the controls and: 1) the occurrence of nonsyndromic cleft lip (p = 0.029,); 2) the occurrence of nonsyndromic cleft lip and palate (nsCL + P) cases (p = 0.024; and 3) the occurrence of both nsCL ± nsCLP cases (p = 0.0365). The association between age of parents, gender, maternal exposures, socioeconomic status and clefts was assessed in each group separately. Conclusion: Our study indicates that the TGFαHinfI gene polymorphism might be associated with nsCL ± P susceptibility, thus contributing to the occurrence of nsCL ± P in Turkish patients. The relatively small sample size of our study is one limitation of our study, and future research with larger specimen sets from different ethnicities will be required to validate our findings. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Colorectal cancer (CRC) is the 3 rd most common cancer worldwide. Recently, long non-coding RNAs (lncRNAs) were found to be critical modulators in the CRC progression. The aim of this study is to investigate the potential roles of lncRNA P73 antisense RNA 1T (TP73-AS1) in CRC development and progression. METHODS: Quantitative real-time PCR (qRT-PCR) was performed to determine relevant gene expression levels; western blot was performed to determine protein expression levels; CCK-8, colony formation, wound healing and Transwell invasion assays were used to determined CRC cell proliferation, migration and invasion; in vivo tumor growth was assessed in xenograft mice model. RESULTS: TP73-AS1 was up-regulated in both CRC tissues and CRC cell lines. Overexpression of TP73-AS1 was associated with metastasis and advanced clinical stages in CRC patients. Overexpression of TP73-AS1 promoted CRC cell growth, proliferation, migration and invasion in vitro; and knockdown of TP73-AS1 significantly inhibited CRC cell growth, proliferation, migration and invasion in vitro as well as tumor growth in vivo. Bioinformatics analysis and luciferase reporter assay indicated that TP73-AS1 could bind directly with miR-194, and TP73-AS1 negatively regulated the expression of miR-194 in CRC cells. Further study indicated that miR-194 negatively regulated the downstream target of transforming growth factor alpha (TGF α) via targeting its 3’ untranslated region, and TP73-AS1 positively regulated the expression of TGF α in CRC cells. Moreover, overexpression of miR-194 suppressed CRC cell proliferation and invasion, and attenuated the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. Silence of TGF α inhibited CRC cell proliferation and invasion, and also reversed the effects of TP73-AS1 overexpression on CRC cell proliferation and invasion. CONCLUSIONS: this study demonstrated that TP73-AS1 regulated CRC progression by acting as a competitive endogenous RNA to sponge miR-194 to modulate the expression of TGF α. [ABSTRACT FROM AUTHOR]

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes. These results suggest that proper formation of the tendon and tarsal plate in the mouse eyelid is required for normal morphogenesis of the meibomian gland. [ABSTRACT FROM AUTHOR]

Background In secondary hyperparathyroidism (SHPT), enhanced parathyroid levels of transforming growth factor-α (TGFα) increase EGF receptor (EGFR) activation causing parathyroid hyperplasia, high parathyroid hormone (PTH) and also reductions in vitamin D receptor (VDR) that limit vitamin D suppression of SHPT. Since anti-EGFR therapy is not an option in human SHPT, we evaluated ADAM17 as a therapeutic target to suppress parathyroid hyperplasia because ADAM17 is required to release mature TGFα, the most potent EGFR-activating ligand. Methods Computer analysis of the ADAM17 promoter identified TGFα and C/EBPβ as potential regulators of the ADAM17 gene. Their regulation of ADAM17 expression, TGFα/EGFR-driven growth and parathyroid gland (PTG) enlargement were assessed in promoter–reporter assays in A431 cells and corroborated in rat and human SHPT, using erlotinib as anti-EGFR therapy to suppress TGFα signals, active vitamin D to induce C/EBPβ or the combination. Results While TGFα induced ADAM17-promoter activity by 2.2-fold exacerbating TGFα/EGFR-driven growth, ectopic C/EBPβ expression completely prevented this vicious synergy. Accordingly, in advanced human SHPT, parathyroid ADAM17 levels correlated directly with TGFα and inversely with C/EBPβ. Furthermore, combined erlotinib + calcitriol treatment suppressed TGFα/EGFR-cell growth and PTG enlargement more potently than erlotinib in part through calcitriol induction of C/EBPβ to inhibit ADAM17-promoter activity, mRNA and protein. Importantly, in rat SHPT, the correction of vitamin D deficiency effectively reversed the resistance to paricalcitol induction of C/EBPβ to suppress ADAM17 expression and PTG enlargement, reducing PTH by 50%. Conclusion In SHPT, correction of vitamin D and calcitriol deficiency induces parathyroid C/EBPβ to efficaciously attenuate the severe ADAM17/TGFα synergy, which drives PTG enlargement and high PTH. [ABSTRACT FROM AUTHOR]

Background: Secondary hyperparathyroidism (SH) is major problem in chronic renal failure. There are studies to examine proliferation and apoptosis associated biomarkers expressions in parathyroid lesions to reveal specific features. In this study, we evaluated the expression of some growth factors and their receptors in parathyroid gland of patients with SH or primary hyperparathyroidism (PH). Materials and Methods: A total of 49 patients had been operated for PH and 26 for SH. Parathyroid tissue samples were evaluated histopathologically and immunohistochemically using antibodies to human p53, Kİ-67, anti-human p21, antitransforming growth factor (TGF) α, CPP32 (caspase 3), and epidermal growth factor receptor (EGFR). Results: Adenoma was higher in PH compared with SH as 48/49 and 3/26, respectively (P = 0.000). Parathyroid hyperplasia was found in 23/26 patients with SH and 1/49 patient with PH. In parathyroid tissue there were no difference between PH and SH for p53, Ki-67, caspase, EGFR expressions; while there were significantly difference for TGFα (P = 0.047) and borderline significant difference for p21 (P = 0.06) expressions. Conclusion: Adenoma was priority present in PH patients, hyperplasia was present in SH. There were no differences between primary and SH or adenoma and hyperplasia for expressions of cycline-dependent kinase inhibitor p21, p53, EGFR, Ki67, caspase; while TGFα expression was found to be different. [ABSTRACT FROM AUTHOR]

EGF receptor (EGFR) and its signaling have been investigated for many years, but how its different ligands regulate signaling has not been thoroughly explored. When investigating EGFR activation and downstream signaling in HeLa cells using a panel of ligands, we found a ligand-dependent differential activation of EGFR and the signaling pathways Akt, PLCγ and STAT with HB-EGF and BTC being the most potent ligands. All the tested ligands induced full activation of Erk signaling at 1 nM, whereas only HB-EGF and partly BTC and EGF induced strong activation of Akt, STAT3 and PLCγ at this concentration. Interestingly, we also found that the high activation potencies of HB-EGF and BTC could only partially be explained by their binding affinities, and are therefore likely to be regulated by other mechanisms. We thus suggest that the signaling pathways initiated from the EGFR vary depending on the ligands bound in a cell specific manner. [ABSTRACT FROM AUTHOR]

Neuroendocrine neoplasms (NENs) are a heterogeneous family of malignancies whose proliferation is partially dependent on growth factors secreted by the microenvironment and the tumor itself. Growth factors which were demonstrated to be important in experimental models of NENs include EGF (epidermal growth factor), TGF (transforming growth factor) α, TGFβ and CTGF (connective tissue growth factor). EGF and TGFα bind to the EGF receptor to stimulate an intact RAS/RAF/MAPK pathway, leading to the transcription of genes associated with cell proliferation, invasion and metastasis. Theoretically, TGFα stimulation can be inhibited at several points of the MAPK pathway, but success is limited to NEN models and is not evident in the clinical setting. TGFβ(1) stimulates TGFβ receptors (TGFβRI and TGFβRII) resulting in inhibition of neuroendocrine cell growth through SMAD-mediated activation of the growth inhibitor P21(WAF1/CIP1). Although some NENs are inhibited by TGFβ(1), paradoxical growth is seen in experimental models of gastric and small intestinal (SI) NENs. Therapeutic targeting of TGFβ(1) in NENs is therefore complicated by uncertainty of the effect of TGFβ(1) secretion on the direction of proliferative regulation. CTGF expression is associated with more malignant clinical phenotypes in a variety of cancers, including NENs. CTGF promotes growth in gastric and SI-NEN models, and is implicated as a mediator of local and distant fibrosis caused by NENs of enterochromaffin cell origin. CTGF inhibitors are available, but their anti-proliferative effect has not been tested in NENs. In summary, growth factors are essential for NEN proliferation, and although interventions targeting these proteins are effective in experimental models, only limited clinical efficacy has been identified. [ABSTRACT FROM AUTHOR]

Abstract: ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR–ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR–ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR–ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR–ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR–ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway. [Copyright &y& Elsevier]

Background: Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15– 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis. Methods: Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability. Results: Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells. Conclusions: Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells. [ABSTRACT FROM AUTHOR]

Background: Microglia are the resident immune cells of the central nervous system and are accepted to be involved in a variety of neurodegenerative diseases. Several studies have demonstrated that microglia, like peripheral macrophages, exhibit two entirely different functional activation states, referred to as classical (M1) and alternative (M2) activation. TGFβ is one of the most important anti-inflammatory cytokines and its effect on inhibiting microglia or macrophage classical activation has been extensively studied. However, the role of TGFβ during alternative activation of microglia has not been described yet. Methods: To investigate the role of TGFβ in IL4-induced microglia alternative activation, both, BV2 as well as primary microglia from new born C57BL/6 mice were used. Quantitative RT-PCR and western blots were performed to detect mRNA and protein levels of the alternative activation markers Arginase1 (Arg1) and Chitinase 3-like 3 (Ym1) after treatment with IL4, TGFβ or both. Endogenous TGFβ release after IL4 treatment was evaluated using the mink lung epithelial cell (MLEC) assay and a direct TGFβ2 ELISA. TGFβ receptor type I inhibitor and MAPK inhibitor were applied to address the involvement of TGFβ signalling and MAPK signalling in IL4-induced alternative activation of microglia. Results: TGFβ enhances IL4-induced microglia alternative activation by strongly increasing the expression of Arg1 and Ym1. This synergistic effect on Arg1 induction is almost completely blocked by the application of the MAPK inhibitor, PD98059. Further, treatment of primary microglia with IL4 increased the expression and secretion of TGFβ2, suggesting an involvement of endogenous TGFβ in IL4-mediated microglia activation process. Moreover, IL4-mediated induction of Arg1 and Ym1 is impaired after blocking the TGFβ receptor I indicating that IL4-induced microglia alternative activation is dependent on active TGFβ signalling. Interestingly, treatment of primary microglia with TGFβ alone results in up regulation of the IL4 receptor alpha, indicating that TGFβ increases the sensitivity of microglia for IL4 signals. Conclusions: Taken together, our data reveal a new role for TGFβ during IL4-induced alternative activation of microglia and consolidate the essential functions of TGFβ as an anti-inflammatory molecule and immunoregulatory factor for microglia. [ABSTRACT FROM AUTHOR]

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1+ progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1+ nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS. [ABSTRACT FROM AUTHOR]

Background: Liver carcinogenesis is associated with multiple genetic changes in affected cells, including alterations in the Hras signalling pathway. Aim: To define the biological contributions of Hras to mouse hepatocarcinogenesis, we quantified in vivo interactions between mutant Hras and other genetic alterations frequently associated with liver cancer, including overexpression of the transcription factor c-myc and the epidermal growth factor receptor ligand transforming growth factor alpha ( TGFα). Methods: To accomplish this aim, we initiated expression of an activated Hras in hepatocytes of adult mice with or without simultaneous overexpression of either c-myc or TGFα. Potential interactions also were assessed through the use of the comparative hepatocyte growth assay, a hepatocyte transplantation assay that measures effects of altered gene expression on hepatocyte growth in vivo. Results: Hras expression caused diffuse liver enlargement (hepatomegaly), and this phenotype was not changed by coexpression of c-myc or TGFα. Using the transplant system, we found that expression of mutant Hras alone was sufficient to induce hepatocyte focus growth in a quiescent liver. Paradoxically, adding expression of TGFα or c-myc reversed this Hras effect. Finally, the frequencies of transplant foci with the preneoplastic feature of extreme growth potential and of liver neoplasms were increased for Hras and both combinations when compared with control hepatocytes, but did not differ among oncogene-expressing groups. Conclusions: Hras-associated hepatocyte growth deregulation is not complemented by activation of c-myc or TGFα growth signalling pathways in mouse liver. This finding emphasizes the tissue-specific character of molecular growth regulation. [ABSTRACT FROM AUTHOR]

Abstract: High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression. [Copyright &y& Elsevier]

Abstract: Multiple neurotrophic factors play a role in proliferation, differentiation and survival in the olfactory epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic factors, fibroblast growth factor 2 (FGF2) and transforming growth factor alpha (TGFα), and that these neurotrophic factors have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased 20 h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse olfactory epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult olfactory epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal growth factor receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse olfactory epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development. [Copyright &y& Elsevier]

Abstract: Glial-derived transforming growth factor alpha (TGFα) activates the erbB1/erbB2 receptor complex on adjacent glial cells in the medial basal hypothalamus (MBH). This receptor activation stimulates the synthesis and release of prostaglandin-E2 (PGE2) from the glial cells, which then induces the release of prepubertal luteinizing hormone-releasing hormone (LHRH) secretion from nearby nerve terminals; thus, showing the importance of glial–neuronal communications at the time of puberty. Ethanol (EtOH) is known to cause depressed prepubertal LHRH secretion and delayed pubertal development. In this study, we assessed whether short-term EtOH exposure could alter the hypothalamic glial to glial signaling components involved in prepubertal PGE2 secretion. Immature female rats began receiving control or EtOH diets beginning when 27 days old. The animals were killed by decapitation after 4 and 6 days of treatment and confirmed to be in the late juvenile stage of development. Blood and brain tissues were collected for gene, protein, and hormonal assessments. Real-time polymerase chain reaction (PCR) analysis demonstrated that EtOH did not affect basal levels of erbB1 gene expression in the MBH. Expression of total erbB1 protein was also unaffected; however, the EtOH caused suppressed phosphorylation of erbB1 protein in the MBH at both 4 and 6 days (P <.01) as revealed by Western blotting. Phosphorylation and total protein levels of erbB2 receptor were not affected by EtOH exposure. Because this receptor is critical for PGE2 synthesis/release, which mediates the secretion of LHRH, we assessed whether in vivo EtOH exposure could affect the release of PGE2. EtOH exposure for 6 days suppressed (P <.01) basal levels of PGE2 released into the medium. The effects of 4- and 6-day EtOH exposure on gene and protein expressions of TGFα, an upstream component in the activation of erbB1/erbB2, were also studied. The levels of TGFα mRNA were increased markedly at 4 days (P <.001), but declined to near basal levels by 6 days in the EtOH-treated animals. The EtOH caused increases in TGFα protein expression at both 4 (P <.001) and 6 (P <.01) days; hence, suggesting that the EtOH inhibited release of the peptide. We confirmed this inhibition by showing decreased (P <.01) TGFα released from MBHs incubated in vitro following 6 days of EtOH exposure in vivo. Thus, these results demonstrate that EtOH is capable of interfering with hypothalamic glial to glial signaling processes involved in prepubertal PGE2 secretion. [Copyright &y& Elsevier]

Abstract: Objective/background: Signals for the expression of EGF, TGFα and related proteins are largely unknown. Having shown earlier, that activation of the afferent sensory nervous system by capsaicin induced epithelial cell proliferation and TGFα expression in vivo the aim of this study was to demonstrate, that neurotransmitters of the afferent sensory nervous system induce TGFα expression via mast cells and fibroblasts. Results: A significant increase in TGFα-mRNA and protein expression was detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells and SP-stimulated fibroblasts. Epithelial cell proliferation was only detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells but not in epithelial cells exposed to supernatants of SP-stimulated fibroblasts. TGFβ mRNA expression was increased in epithelial cells exposed to fibroblasts but not to mast cells. No increase in EGF-receptor expression was detected in epithelial cells exposed to either fibroblast or mast cell supernatants. Conclusion: Neurotransmitters of the afferent sensory nervous system induce TGFα expression in epithelial cells mediated by mast cells and fibroblasts. Release of CGRP and SP from neuroeffector junctions is an important signal for the TGFα expression after mucosal injury. Mucosal expression of TGFα may contribute to the protective effects of the sensory nervous system after mucosal injury. [Copyright &y& Elsevier]

Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-α (TGFα) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFα expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFα expression and epithelial cell proliferation in vivo. Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 μg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 μg/kg carbachol. TGFα expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFα expression. Sensory desensitisation did neither abolish the increased TGFα expression nor the increase in epithelial cell proliferation. Parasympathetic pathways are involved in the control of TGFα expression in gastrointestinal mucosa as well as in epithelial cell proliferation. [ABSTRACT FROM AUTHOR]

Abstract: 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 TGFß. 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. [Copyright &y& Elsevier]

Abstract: Bisphosphonates, and more specially nitrogen-containing bisphosphonates, which are in current use for the treatment of bone diseases, demonstrate proapoptotic, antiproliferative, antiangiogenic and anti-invasive properties on tumor cells. The amino-bisphosphonate alendronate is considered as a potential anticancer drug. In the case of A431 cells, which express high levels of VEGF, it had a two-step effect. At 24h, the antitumor properties of alendronate were counterbalanced by a survival process, which consisted of an enhancement of VEGF expression (mRNA and protein secretion) and TGFα secretion. It was only at 48h that alendronate displayed the expected antiproliferative and antiangiogenic properties. The first step, in which the PI3K pathway was engaged, could be prevented by the use of a VEGF-antisense oligonucleotide. The combination of such an antisense with small concentrations of alendronate (∼2μM), which is of the order of clinically used concentrations, was shown to have an antiangiogenic effect as soon as 12h. [Copyright &y& Elsevier]

Abstract: Ligands for the epidermal growth factor receptor ErbB1, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα), negatively regulate synaptic maturation of GABAergic neurons in the developing neocortex. Here, we evaluated the effects of these ligands in vivo on developing inhibitory neurons in the dentate gyrus. Hippocampal slices were prepared from postnatal mice repeatedly challenged with EGF or from transgenic mice overexpressing TGFα. We monitored paired pulse depression of field population spikes evoked by perforant path stimulation to estimate the strength of local inhibition. Administration of EGF increased the paired pulse ratio, suggesting a reduction of inhibitory strength. A similar reduction was observed in TGFα transgenic mice. Monitoring miniature and evoked synaptic currents, we estimated EGF effects on synaptic input and output of GABAergic neurons. EGF treatment diminished the amplitude of excitatory postsynaptic currents (EPSCs) in the GABAergic neurons without affecting their miniature EPSCs. EGF also affected output strength of the GABAergic neurons: The frequency of miniature inhibitory postsynaptic currents (IPSCs) and the evoked IPSC/evoked EPSC ratio were decreased in granule cells. In parallel, EGF down-regulated the protein level of vesicular GABA transporter. Thus, ErbB1 ligands influence GABAergic inhibitory synaptic transmission in the developing dentate gyrus. [Copyright &y& Elsevier]

Abstract: The epidermal growth factor (EGF) family plays an important role in reproductive function regulation. The aim of this work was to investigate the localization of EGF, transforming growth factor α (TGFα) and epidermal growth factor receptor (EGFr) in boar epididymis and accessory genital glands, as well as study the presence of EGFr and the effects of EGF on boar spermatozoa. In the epididymis, prostate and vesicular glands EGF, TGFα and EGFr were detected in the pseudostratified epithelium. None of them were observed in the bulbourethral glands. Epidermal growth factor receptor was detected by immunofluorescence in non-capacitated, capacitated and acrosome reacted spermatozoa. Confocal microscopy revealed different staining patterns over the head, midpiece and/or flagellum whereas, flow cytometry analysis showed that the population of positive spermatozoa did not exceed 58% and did not depend on the functional state. To study the effects of EGF, spermatozoa were capacitated in Tyrodés medium containing 0, 10 and 100ng/ml EGF. Acrosome status, membrane integrity and motility patterns were evaluated after capacitation and after the acrosome reaction (AR). Capacitation in the presence of 100ng/ml EGF significantly improved the quality of movement (P <0.01) after the AR. These findings suggest that EGF and TGFα are produced in the reproductive tract of the boar where they may act locally and/or on a population of spermatozoa, improving the quality of movement after the AR. [Copyright &y& Elsevier]

Abstract: The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFα) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0–3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFα/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFα to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFα was a similar to EGFR. However, the staining for TGFα slightly increased in the fertilized oocytes (1–2+) as compared to immature and mature oocytes (1+). TGFα was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5ng/ml TGFα was increased as compared to other treatments (P <0.05). There was no significant difference in the rate of development of blastocysts cultured with 5ng/ml TGFα, 20ng/ml EGF, 20ng/ml EGF+5ng/ml TGFα or the control treatment (P >0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P >0.05). However, TGFα alone enhanced cell survival rated (P <0.01) and reduced apoptosis. We concluded that TGFα can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro. [Copyright &y& Elsevier]

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFα) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFα mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/−) p53 mice did not acquire mammary lesions, p53+/− mice carrying the NRL-TGFα transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFα expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR−, and when combined with a heterozygous p53 genotype, ER−/PR−.Oncogene (2007) 26, 5238–5246; doi:10.1038/sj.onc.1210340; published online 5 March 2007 [ABSTRACT FROM AUTHOR]

Transforming growth factor-alpha (TGFα) is a member of the epidermal growth factor (EGF) family. Expression of TGFα is highly regulated in response to exogenous cellular signals including cytokines and other growth factors. The growth factor has been found to be indispensable for proper development of many tissues and organs. TGFα has also been implicated in numerous disease states including forms of breast cancer. This minireview summarizes the basic biology of TGFα and its actions during normal and pathogenic development of the mammary epithelium. [ABSTRACT FROM AUTHOR]

Abstract: Transforming growth factor-α (TGFα) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPKERK1,2); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPKERK1,2 for 1 h, induced by TGFα, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPKERK1,2, induced by FGF7, led to growth without branching. Unlike TGFα, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGFα indicated that the FGF7-induced MAPKERK1,2 signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGFα-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPKERK1,2 activation. [Copyright &y& Elsevier]

Abstract: Objective. : Progestin is an effective endocrine treatment for patients with atypical hyperplasia or with endometrial carcinoma that is estrogen receptor (ER) positive and progesterone receptor (PR) positive. However, long-term progestin treatment may lead to resistance. We have studied the progestin resistance phenotype that frequently develops in endometrial carcinoma. Methods. : Ishikawa endometrial carcinoma cells were cultured for a long period (10 months) in the presence of the synthetic progestin medroxyprogesterone acetate (MPA), thereby generating a subline refractory to the growth-suppressive effects of MPA. Results. : The MPA-resistant subline showed growth stimulation rather than inhibition after MPA treatment. Immunocytochemical analysis showed reduced ERα and PR-B expression and increased ERβ expression in this subline compared with parental Ishikawa cells. Progestin-resistant Ishikawa cells also showed increased expression of transforming growth factor α (TGFα), the epidermal growth factor receptor (EGFR), and EGFR tyrosine kinase (EGFR-TK); MPA treatment further stimulated the expression of TGFα in these cells. Additionally, progestin-resistant Ishikawa cells were highly sensitive to growth stimulation by TGFα and to growth inhibition by the EGFR-TK-specific inhibitor AG1478, and they showed increased dependence on TGFα-EGFR signaling. Conclusions. : Our results suggest that prolonged treatment of endometrial carcinoma cells with MPA induces resistance to the growth-suppressive effects of MPA and enhances cancer cell proliferation. The downregulation of ERα and PR-B, the upregulation of ERβ, and highly activated TGF-EGFR signaling are thus likely to contribute to progestin resistance in endometrial carcinoma. Therefore, an EGFR-TK-specific inhibitor might be useful in the treatment of progestin-resistant endometrial carcinoma. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-α (TGFα) is a well-known regulator of many developmental processes. However, its role in adult nervous system is yet unclear. Studies have shown that TGFα can regulate stress and memory behavior in adult mice. When TGFα is reduced in Waved-1 (Wa-1) mutant mice, the stress response and memory are impaired predominantly in males and only after puberty. To determine the neurochemical changes resulting from the reduced TGFα levels that could explain the reported behavioral outcomes, biogenic amine and amino acid levels were determined in the brain regions associated with stress and memory. Interestingly, sex-specific alterations in neurochemical levels were detected, including elevated noradrenaline and reduced glutamate levels in striatum of Wa-1 males, increased noradrenaline and reduced serotonin metabolite levels in hippocampus of Wa-1 females, reduced serotonin metabolite levels in cortex and amygdala of Wa-1 females, and reduced noradrenaline, dopamine, serotonin, glutamate and glycine levels in hypothalamus of Wa-1 females compared to their respective controls. Increased dopamine turnover in cortex and reduced dopamine and serotonin turnover in amygdala were observed in both male and female Wa-1 mice. The data indicate sex-specific alterations of specific neurochemicals as a result of reduced TGFα expression, which may underlie sex-dependent stress response and memory impairment in Wa-1 mice. [Copyright &y& Elsevier]

Abstract: Tumor cells traverse the basement membrane zone and gain access to the underlying mesenchyme to eventually form metastases. Laminin 5 is a major component of the basement membrane and connects keratinocytes at the level of hemidesmosomes to the mesenchyme. Underneath invading tumor cells anti-laminin 5 staining is diminished, and laminin 5 degradation products can stimulate cell migration and epidermal growth factor (EGF) receptor signaling. To investigate laminin 5 expression in parental HaCaT and tumorigenic c-Ha-ras-transformed HaCaT II-4rt keratinocytes, the cells were cultivated under monolayer and organotypic culture conditions. In monolayer cultures, HaCaT and c-Ha-ras-transformed HaCaT II-4rt keratinocytes secreted comparable amounts of laminin 5. After 7 days of organotypic cultures, collagen IV, β4-integrin, nidogen and laminin 5 were detected along the epithelial–mesenchymal interface of parental HaCaT keratinocytes, while staining for these proteins was patchy or absent in the organotypic cultures with c-Ha-ras-transformed HaCaT II-4rt cells. Immunoblotting analysis confirmed absence of laminin 5 deposition in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt while the protein was detected in organotypic cultures of HaCaT keratinocytes. Surprisingly, however, the α3 and γ2 laminin chain transcripts were strongly induced in c-Ha-ras-transformed HaCaT II-4rt cells by organotypic culture conditions, indicating that invasive epidermal tumor cells retain high mRNA levels for laminin 5 chains and suggesting an autocrine/paracrine induction of the laminin chain mRNAs. Moreover, as laminin 5 was absent in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt cells, it suggests immediate degradation of the protein. Degradation products may further contribute to the malignant phenotype by enhancing cellular migration and EGF-receptor activation. [Copyright &y& Elsevier]

Glial erbB1 receptors play a significant role in the hypothalamic control of female puberty. Activation of these receptors by transforming growth factor α (TGFα) results in production of prostaglandin E2, which then stimulates luteinizing hormone releasing hormone (LHRH) neurons to secrete LHRH, the neuropeptide controlling sexual development. Glutamatergic neurons set in motion this glia-to-neuron signaling pathway by transactivating erbB1 receptors via coactivation of AMPA receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Because the metalloproteinase tumor necrosis factor α converting enzyme (TACE) releases TGFα from its transmembrane precursor before TGFα can bind to erbB1 receptors, we sought to determine whether TACE is required for excitatory amino acids to activate the TGFα-erbB1 signaling module in hypothalamic astrocytes, and thus facilitate the advent of puberty. Coactivation of astrocytic AMPARs and mGluRs caused extracellular Ca2+ influx, a Ca2+/protein kinase C-dependent increase in TACE-like activity, and enhanced release of TGFα. Within the hypothalamus, TACE is most abundantly expressed in astrocytes of the median eminence (ME), and its enzymatic activity increases selectively in this region at the time of the first preovulatory surge of gonadotropins. ME explants respond to stimulation of AMPARs and mGluRs with LHRH release, and this response is prevented by blocking TACE activity. In vivo inhibition of TACE activity targeted to the ME delayed the age at first ovulation, indicating that ME-specific changes in TACE activity are required for the normal timing of puberty. These results suggest that TACE is a component of the neuron-to-glia signaling process used by glutamatergic neurons to control female sexual development. [ABSTRACT FROM AUTHOR]

Abstract: Using two-site enzyme immunoassays, we measured protein levels of epidermal growth factor (EGF), transforming growth factor alpha (TGFα), and heparin-binding epidermal growth factor (HB-EGF) in adult rat brain, and compared them with the phosphorylation levels of their receptor (ErbB1). There were significant variations in the brain distributions of each ErbB1 ligand. Among these ErbB1 ligands, HB-EGF protein levels were higher than those of TGFα and those of EGF were the lowest. TGFα protein was relatively enriched in the midbrain regions, while HB-EGF levels were most abundant in the cerebellum. Protein distributions of the EGF family members were discordant with previously reported mRNA distributions. In addition, there was significant basal ErbB1 phosphorylation detected with the largest amount of activation in the midbrain. These observations suggest that the activation of brain ErbB1 involves post-translational regulation of multiple EGF family members in a region-specific manner. [Copyright &y& Elsevier]

Abstract: Ras and Notch signaling have recently been shown to cooperate in the maintenance of neoplastic transformation. Here, we show that TGFα, a known activator of Ras signaling, can drive cell proliferation and at the same time induce the expression of the Notch target Hes-1 in the neuroblastoma cell line SK-N-BE(2)c. The up-regulation of Hes-1 occurred both at the transcriptional and protein levels and by use of EGFR and MEK inhibitors we could show that the Hes-1 response was dependent on activation of the MAP kinase ERK. Blocking Notch activation by γ-secretase inhibition did not profoundly affect the Hes-1 levels, neither in untreated nor in TGFα treated cells. The up-regulation of Hes-1 was associated with down-regulation of its pro-neuronal target gene Hash-1. Taken together, these results show that TGFα is a potent mitogen of neuroblastoma cells and suggest a connection between activation of ERK and Hes-1, thus providing a link between the Ras and Notch signaling pathways. [Copyright &y& Elsevier]

Epithelial metaplasia occurs when one predominant cell type in a tissue is replaced by another, and is frequently associated with an increased risk of subsequent neoplasia. In both mouse and human pancreas, acinar-to-ductal metaplasia has been implicated in the generation of cancer precursors. We show that pancreatic epithelial explants undergo spontaneous acinar-to-ductal metaplasia in response to EGFR signaling, and that this change in epithelial character is associated with the appearance of nestin-positive transitional cells. Lineage tracing involving Cre/lox-mediated genetic cell labeling reveals that acinar-to-ductal metaplasia represents a true transdifferentiation event, mediated by initial dedifferentiation of mature exocrine cells to generate a population of nestin-positive precursors, similar to those observed during early pancreatic development. These results demonstrate that a latent precursor potential resides within mature exocrine cells, and that this potential is regulated by EGF receptor signaling. In addition, these observations provide a novel example of rigorously documented transdifferentiation within mature mammalian epithelium, and suggest that plasticity of mature cell types may play a role in the generation of neoplastic precursors. [ABSTRACT FROM AUTHOR]

The HER2 gene encodes a tyrosine kinase receptor overexpressed in 25-30%of human breast cancers. Clinical trials have shown the efficacy of the anti-HER2 monoclonal antibody Trastuzumab in metastatic breast cancer patients. Nevertheless, 70%of patients are unresponsive from start of treatment and nearly all become unresponsive during treatment. Possible mechanisms for these failures could depend on impairment of the machinery responsible for receptor downregulation. To test this hypothesis, we analysed the genomic sequences encoding regions known to be critical for HER2 downregulation, of both HER2 and of the ubiquitin ligase Cbl. We investigated 63 breast cancers, and found no mutations in these regions. We thus considered alternative mechanisms-such as TGFaproduction-possibly interfering with HER2 downregulation. In selected cases, by comparing breast cancer neoplastic tissue before and after Trastuzumab treatment, we found induction of TGFaexpression. Moreover, by in vitro expression of exogenous TGFain breast cancer cells, we observed a dramatic reduction in Trastuzumab-induced HER2 endocytosis, downregulation and cell growth inhibition. Our results suggest that unresponsiveness to Trastuzumab may not be due to intrinsic defects in the machinery responsible for HER2 downregulation, but can be associated with a TGFa-related mechanism of escape to HER2 downregulation.Oncogene (2005) 24, 3002-3010. doi:10.1038/sj.onc.1208478 Published online 14 February 2005 [ABSTRACT FROM AUTHOR]

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor a (TGFa), and transforming growth factor ß1 (TGFß1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal dosesin vitroat the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGFa improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGFa. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGFß1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stagesin utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGFß1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25-27 pairs of somites and were 2.0-2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGFa, and TGFß1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos. [ABSTRACT FROM AUTHOR]

Abstract: The Epidermal Growth Factor Receptor (EGFR) and its structural relative erbB2 are frequently over-expressed in ovarian cancers and both are strongly associated with poor patient survival. To investigate the relative roles of these receptors in the regulation of cell growth and migration, a panel of ovarian carcinoma cell lines were stimulated with TGFα and NRG1β. TGFα had a much greater influence on cell migration than NRG1β where growth effects were equivalent. The extent of TGFα-stimulated migration on collagen in these assays could be associated with erbB2 expression levels. In addition, TGFα was found to stimulate activation of the ERK, PI3 kinase and PLCγ pathways. Direct blockade of the TGFα-interacting receptor EGFR inhibited both cell growth and migration, as well as downstream signaling induced by the growth factor. Specific blockade of the downstream proteins MEK and PI3 kinase significantly affected TGFα-induced mitogenesis in the cell lines tested but had less impact upon migration. Conversely, inhibition of the PLCγ pathway had little effect on cell growth but significantly decreased TGFα-driven migration. These results corroborate the likely importance of migration as well as growth in erbB receptor over-expressing ovarian cancers and directly implicate the roles of ERK and PI3 kinase in growth control, and PLCγ in the regulation of migration in this disease. [Copyright &y& Elsevier]

Abstract: The cytoplasmic domain of the transforming growth factor-α precursor (proTGFα) contains a C-terminal PSD-95/SAP90, Discs Large, and Zona Occludens-1 (PDZ) recognition motif (TVV). By yeast two-hybrid screening of a mouse embryo library, we have found that a third member of a family of PDZ-containing proteins, membrane associated guanylate kinase inverted-3 (MAGI-3), binds to TGFα''s TVV. MAGI-3 is widely expressed in multiple mouse tissues, including brain. Immunolocalization showed that MAGI-3 and TGFα were colocalized in neurons in the cortex and dentate gyrus, as well as in ependymal cells and some astrocytes. In vitro, proTGFα bound the PDZ-1 domain of MAGI-3 and MAGI-2, but not MAGI-1. MAGI-3 and the 17-kDa cell surface form of proTGFα interact transiently in MDCK cells stably transfected with both MAGI-3 and human proTGFα cDNAs. MAGI-3 and wild-type proTGFα colocalize at the cell surface. In contrast, MAGI-3 forms a stable complex with membrane-fixed TGFα early in the secretory pathway and interacts with immature and cell surface forms of membrane-fixed TGFα. Overexpression of MAGI-3 resulted in increased levels of TGFα in the basolateral medium of polarized MDCK cells, suggesting that MAGI-3 has a role in efficient trafficking of TGFα to the cell surface in polarized epithelial cells. [Copyright &y& Elsevier]

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix in response to signaling molecules. MEK kinase 1 (MEKK1) is a mitogen-activated protein kinase (MAPK) kinase kinase originally identified as an upstream activator for several MAPK pathways. During mouse embryogenesis, MEKK1 controls cell shape changes and formation of actin stress fibers that are required for sealing epidermis in the embryos in a process known as eyelid closure. MEKK1-null mice display eye-open at birth (EOB), a phenotype found also in mice impaired in activin, a subgroup of the transforming growth factor β (TGFβ) family, or in epidermal growth factor receptor (EGFR) or its ligand TGFα, or in transcription factor c-Jun. Molecular analyses have revealed at least two signaling mechanisms in the control of eyelid closure. One is originated from the activins and is transduced through MEKK1, leading to transcription-independent actin stress fiber formation and transcription-dependent keratinocyte migration. Another is the TGFα/EGFR signal that is transduced through a MEKK1-independent pathway to the activation of the ERK MAPK, which also leads to keratinocyte migration. c-Jun might serve as a connection between the two pathways. As embryonic eyelid closure is a specific morphogenetic process that is easily detectable, genetic mutant mice with EOB will be ideal models to understand the signaling mechanisms in the control of epithelial cell migration and the morphogenetic process of epithelial sheet movement. [Copyright &y& Elsevier]

We determined whether alterations in the expression of p53, p16INK4 and p21WAF1/CIP1 influence the invasiveness of a subset of gastric adenocarcinomas co-expressing TGFα and EGFR. Immunopositivity for TGFα-EGFR (26%) was observed in both early and advanced adenocarcinomas, and 88% of these showed immunoreactivity for p53. SSCP analysis revealed that in 81% of these tumors the p53 gene was mutated in exons 5–8. The intensity of p53 immunoreactivity was significantly higher (P<0.013) in deeply invasive tumors. p16INK4 and p21WAF1/CIP1 immunoreactivity was detected in 93 and 76% of the samples co-expressing TGFα-EGFR but the levels were not correlated with those of p53 and other clinico-pathological parameters. We conclude that gastric adenocarcinomas potentially dependent upon the TGFα-EGFR autocrine loop for growing exhibit increased aggressiveness in the presence of aberrant p53. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Background and aims: Transforming growth alpha (TGFα) and sensory neurons have been shown to promote gastric mucosal protection and healing. Aims were to examine in vitro interactions between gastric sensory neurons, the sensory neuropeptide calcitonin gene-related peptide (CGRP), and TGFα. Methods: Gastric mucosal/submucosal tissue fragments from Sprague–Dawley (SD) rats were incubated in short-term (30 min) culture. Peptide release into media and TGFα tissue content were measured by radioimmunoassay. Results: TGFα (1×10−8 to 1×10−6 M) caused dose-dependent stimulation of CGRP release. Maximal CGRP release (+87%) was observed with 1×10−6 M TGFα: 28.6±3.8 vs. control of 15.5±2.7 pg/g tissue; P<0.05. Both CGRP (1×10−7 to 1×10−5 M) and capsaicin (1×10−8 to 1×10−6M) significantly inhibited basal TGFα release in a dose-dependent fashion that ranged from −20% to −39%. In contrast, capsaicin-induced sensory denervation caused significant increases in both basal TGFα release and TGFα tissue content. Conclusion: Function interactions between TGFα and gastric sensory neurons are suggested by the observations that (1) TGFα stimulated CGRP release from gastric sensory neurons; (2) CGRP and acute capsaicin treatment inhibited TGFα release and; (3) capsaicin-induced sensory denervation caused significant increases in both gastric TGFα basal release and tissue content. [Copyright &y& Elsevier]

Elevated serum levels of parathyroid hormone (PTH) contribute to the increased morbidity and mortality in renal failure patients. Parathyroid gland hyperplasia is a major cause of high serum PTH. The present studies used the rat model of renal failure to address the mechanisms underlying uremia-induced parathyroid hyperplasia and the antiproliferative properties of vitamin D therapy (1,25-dihydroxyvitamin D (1,25(OH)2D3) or its less calcemic analogs). Enhanced TGFα/EGFR co-expression is the major mitogenic signal in uremic parathyroid glands. At early stages of renal failure, vitamin D therapy efficiently counteracts uremia- and high phosphorus-induced hyperplasia by inhibiting the increases in parathyroid-TGFα/EGFR co-expression. In established hyperparathyroidism, characterized by highly enhanced-TGFα/EGFR co-expression, vitamin D therapy arrests growth by suppressing EGFR-growth signals from the plasma membrane and nuclear EGFR actions as a transactivator of the cyclin D1 gene, an important contributor to parathyroid hyperplasia in humans. In advanced renal failure, reduced-parathyroid vitamin D receptor levels limits the antiproliferative efficacy of vitamin D therapy. However, non-antiproliferative doses of 1,25-dihydroxyvitamin D enhance the anti-EGFR actions of EGFR–tyrosine kinase inhibitors (TKI). In fact, combined 1,25-dihydroxyvitamin D/TKI therapy inhibits parathyroid hyperplasia more efficiently than phosphorus restriction, the most powerful promoter of parathyroid growth arrest available at present. [Copyright &y& Elsevier]

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity. [Copyright &y& Elsevier]

Transforming growth factor-α (TGFα), a member of the epidermal growth factor family, has neurotrophic actions on postmitotic neurons. We examined the chronic effects of TGFα on the electrophysiological properties of one type of GABAergic neuron, identified by its bipolar morphology, in neocortical primary culture. Approximately 85% of the bipolar neurons were GABA-immunoreactive. In response to depolarizing current injection, the bipolar neurons usually showed tonic firing of action potential under control conditions. After treatment with TGFα (20 ng/ml) for 2 days, these neurons failed to generate trains of action potentials. Furthermore, the treatment altered the action potential waveforms of the bipolar neurons, including the duration and amplitude of the fast after-hyperpolarization, which implies a reduction in voltage-gated potassium currents. In contrast, TGFα did not affect the firing properties of pyramidal-shaped non-GABAergic neurons. Voltage-clamp recordings from the bipolar neurons indicated that chronic treatment with TGFα markedly decreased the current densities of slow delayed rectifier (IK) and transient voltage-gated potassium currents, whereas the treatment had no effect on voltage-gated sodium current and fast delayed rectifier potassium current densities. Reverse transcription-polymerase chain reaction analysis of potassium channel mRNA in the bipolar neurons revealed that the reduction in the IK current density was caused by Kv2.2 mRNA down-regulation. Thus, chronic treatment with TGFα down-regulated slow delayed rectifier and transient voltage-gated potassium currents, and in parallel, suppressed repetitive generation of action potentials in the cortical GABAergic neurons. [Copyright &y& Elsevier]

The activation of transforming growth factor α (TGFcα)- erbB-1 and neuregulin- erbB-4 signaling pathways in hypothalamic astrocytes has been shown to play a key role in the process by which the neuroendocrine brain controls luteinizing hormone-releasing hormone (LHRH) secretion. Earlier studies suggested that tanycytes, an ependymoglial cell type of the median eminence, regulate LHRH release during the estrous cycle by undergoing plastic changes that alternatively allow or prevent direct access of the LHRH nerve terminals to the portal vasculature. Neither the molecules responsible for these plastic changes nor the underlying controlling mechanisms have been identified. Here we show that cultured tanycytes express erbB-1 and erbB-2, two of the four members of the erbB receptor family, and respond to TGFα with receptor phosphorylation, release of prostaglandin E[sub2] (PGE[sub2]), and a PGE[sub2]-dependent increase in the release of TGFβ[sub1], a growth factor previously implicated in the glial control of LHRH secretion. Blockade of either erbB-1 receptor signal transduction or prostaglandin synthesis prevented the stimulatory effect of TGFα on both PGE[sub2] and TGFβ[sub1] release. Time-lapse studies revealed that TGFα and TGFβ[sub1] have dramatically opposite effects on tanycyte plasticity. Whereas TGFα promotes tanycytic outgrowth, TGFβ[sub1] elicits retraction of tanycytic processes. Blockade of metalloproteinase activity abolished the effect of TGFβ[sub1], suggesting that TGFα induces tanycytic retraction by facilitating dissolution of the extracellular matrix. Prolonged (>12 hr) exposure of tanycytes to TGFα resulted in focal tanycytic retraction, an effect that was abolished by immunoneutralization of TGFβ[sub1] action, indicating that the retraction was attributable to TGFα-induced TGFβ[sub1] formation. These in vitro results identify tanycytes as targets. [ABSTRACT FROM AUTHOR]

Previous data disagree as to the role of extracellular signal-regulated kinase (ERK) in the EGF receptor-mediated stimulation of proliferation in hepatocytes. Using cultured rat hepatocytes, we here show that EGF receptor stimulation in mid/late G1 phase caused a sustained ERK activation that lasted for at least 48 h. Inhibition of the early part of this activity by a single addition of the MEK inhibitor PD98059 did not affect the DNA synthesis. However, inhibition of both the early and the sustained phase of ERK activation by washout and repeated administrations of PD98059 abolished the DNA synthesis induced by EGF and TGFα. EGF receptor stimulation also transiently activated p38, and inhibition of p38 by SB203580 markedly decreased the DNA synthesis. Furthermore, EGF and TGFα stimulated phosphorylation of Akt, a downstream target of the PI3-kinase pathway, and the PI3-kinase inhibitors wortmannin and LY294002 blocked the EGF-induced DNA synthesis. These results support a mechanism for EGF receptormediated mitogenic signalling in hepatocytes where ERK has an obligatory role, acting in concert with PI3- kinase, and augmented by p38. Furthermore, the data suggest that to perform this role ERK has to be activated for a prolonged period. [ABSTRACT FROM AUTHOR]

In polarized Madin–Darby canine kidney (MDCK) cells, newly synthesized transforming growth factor-α precursor (proTGFα) is directly sorted to the basolateral cell surface where it is sequentially cleaved and released into the basolateral conditioned medium (Dempsey, P.J., Coffey, R.J., J. Biol. Chem. 269 (1994) 16878–16889). In the present study, the role of the proTGFα cytoplasmic domain in basolateral sorting has been investigated using deletional and site-directed mutagenesis, as well as chimeric analyses of different TGFα constructs stably expressed in MDCK cells. The loss of polarized secretion of a proTGFα secretory mutant (TGFsec88) indicated that the proTGFα transmembrane and/or cytoplasmic domains contain essential basolateral sorting information. Using reporter chimeras with two apically sorted membrane proteins, p75 neurotrophin growth factor receptor and placental alkaline phosphatase, we show that the proTGFα cytoplasmic domain contains dominant basolateral sorting information. Analysis of proTGFα cytoplasmic domain truncation and internal deletion mutants, together with site-directed mutagenesis studies within the full-length proTGFα cytoplasmic domain, revealed redundant basolateral sorting motifs. Importantly, the C-terminal type I PDZ-binding motif was not required for basolateral sorting as determined by the integrity of basolateral sorting in deletion mutants lacking this motif. ProTGFα basolateral sorting may have important consequences for ligand presentation and spatial compartmentalization of epidermal growth factor receptor signaling networks in polarized epithelial cells. [Copyright &y& Elsevier]