Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas

We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30–40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-α, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms. Received: 18 November 1997 / Revised, accepted: 10 March 1998     

The gene for heparin-binding epidermal growth factor-like growth factor is stress-inducible: its role in cerebral ischemia.

The functions of the epidermal growth factor (EGF) family members in the adult brain are not known. This study investigated the changes in the expression of members of the EGF family following global ischemia employing in situ hybridization and immunohistochemical techniques to elucidate their roles in pathological conditions. EGF mRNA was not detected in either the control or the postischemic rat brain. Although transforming growth factor-alpha (TGF-alpha) mRNA was widely expressed in the normal brain, its expression did not change appreciably following ischemia. By contrast, heparin-binding EGF-like growth factor (HB-EGF) mRNA expression was rapidly increased in the CA3 sector and the dentate gyrus of the hippocampus, cortex, thalamus, and cerebellar granule and Purkinje cell layers. EGF receptor mRNA, which was widely expressed, also showed an increase in the CA3 sector and dentate gyrus. Conversely, HB-EGF mRNA did not show any increase prior to ischemic neuronal injury in the CA1 sector, the region most vulnerable to ischemia. Immunohistochemical detection of HB-EGF in the postischemic brain suggested a slight increase of immunostaining in the dentate gyrus of the hippocampus and the cortex. These findings showed that the gene encoding HB-EGF is stress-inducible, indicating the likelihood that HB-EGF is a neuroprotective factor in cerebral ischemia.     

Multiple trophic actions of heparin-binding epidermal growth factor (HB-EGF) in the central nervous system

The epidermal growth factor (EGF) family of ligands interacts with the epidermal growth factor receptor (EGF-R) to produce numerous direct and indirect actions on central nervous system cells. They induce the proliferation of astrocytes and multipotent progenitors ('stem' cells) and promote the survival and differentiation of postmitotic neurons. Heparin-binding epidermal growth factor (HB-EGF) interacts with both EGF-R and a related receptor, ErbB4, whereas transforming growth factor alpha (TGFalpha) interacts only with EGF-R. Because of the unique characteristics of HB-EGF and the potential utility of EGF family members in brain repair, we examine the effects of HB-EGF on rat and mouse CNS cells in vitro and compare them to those of TGFalpha. We find that, like TGFalpha, HB-EGF stimulates the proliferation of CNS astrocytes and multipotent progenitors. These proliferative effects require the expression of EGF-R, as no such effects are observed in cells derived from EGF-R-/- mice. Both HB-EGF and TGFalpha enhanced the survival of neurons derived from the neocortex and the striatum. Within these neuron-enriched cultures, nestin-positive cells but not neurons express EGF-R mRNA, indicating that the neurotrophic actions of EGF-R ligands are a result of indirect stimulation mediated by non-neuronal cells. The neurotrophic actions of HB-EGF and TGFalpha are accompanied by an elevation in immunoreactive dual phosphorylated mitogen-activated protein kinase (MAP kinase) in neurons, providing evidence that the MAP kinase cascade mediates these actions. In situ hybridization studies demonstrate that HB-EGF mRNA is present within the brainstem as early as E14 and subsequently is found in the developing cortical plate, hippocampus, cerebellar Purkinje cells and ventrobasal thalamus, among other brain areas. These findings indicate that HB-EGF may be an important trophic factor in the developing CNS and is a useful candidate molecule for brain repair strategies.     

Comparison between epidermal growth factor, transforming growth factor-alpha and EGF receptor levels in regions of adult rat brain.

Examination of adult rat brain regions by specific radioimmunoassays revealed a widespread distribution of transforming growth factor-alpha (TGF-alpha), but not epidermal growth factor (EGF), the peptide that had previously been reported to be present in rodent brain. Polyadenylated RNA samples from the different regions of rat brain were analyzed by Northern blot to identify mRNA species encoding precursor proteins for EGF (preproEGF), TGF-alpha (preproTGF-alpha), and the EGF/TGF-alpha receptor. The results indicate that TGF-alpha is the most abundant ligand for the EGF/TGF-alpha receptor in most parts of the brain analyzed. Message for preproEGF was only detectable after prolonged autoradiographic exposure; levels of preproEGF mRNA were between two and three orders of magnitude lower in brain than those expressed in control tissue (kidney), and one to two orders of magnitude lower than preproTGF-alpha mRNA levels in all brain regions. These results were confirmed by analysis of mRNA by RT/PCR, and support the hypothesis that expression of preproEGF mRNA in the brain is limited to smaller discrete areas, whereas preproTGF-alpha gene expression is almost ubiquitous.     

Abnormal expression of epidermal growth factor and its receptor in the forebrain and serum of schizophrenic patients

Epidermal growth factor (EGF) comprises a structurally related family of proteins containing heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGFalpha) that regulates the development of dopaminergic neurons as well as monoamine metabolism. We assessed the contribution of EGF to schizophrenia by measuring EGF family protein levels in postmortem brains and in fresh serum of schizophrenic patients and control subjects. EGF protein levels were decreased in the prefrontal cortex and striatum of schizophrenic patients, whereas the levels of HB-EGF and TGFalpha were not significantly different in any of the regions examined. Conversely, EGF receptor expression was elevated in the prefrontal cortex. Serum EGF levels were markedly reduced in schizophrenic patients, even in young, drug-free patients. Chronic treatment of animals with the antipsychotic drug haloperidol had no influence on EGF levels in the brain or serum. These findings suggest that there is abnormal EGF production in various central and peripheral tissues of patients with both acute and chronic schizophrenia. EGF might thus provide a molecular substrate for the pathologic manifestation of the illness, although additional studies are required to determine a potential link between impaired EGF signaling and the pathology/etiology of schizophrenia.     

Epidermal growth factor and transforming growth factor alpha induce c-fos gene expression in retinal Muller cells in vivo.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are peptides that act at a common receptor and are mitogenic for immature astrocytes and trophic for developing brain neurons in vitro. However, a role for these growth factors in the mature nervous system has not been established. To investigate the actions of EGF and TGF alpha in the adult central nervous system (CNS) in vivo, the growth factors were injected into the vitreous cavity of adult male rabbits. After varying intervals, the retinas were examined for c-fos mRNA by Northern blot hybridization or Fos (and Fos-related antigen) protein by immunocytochemistry. EGF induction of c-fos mRNA occurs within 30 min and persists more than 4 hr. Fos nuclear immunostaining is induced selectively in nuclei of Muller cells by both EGF and TGF alpha. Fos-like immunoreactivity appears within 1 hr and persists more than 9 hr after EGF injection. These observations demonstrate that mature retinal Muller cells respond to exogenously applied EGF and TGF alpha in vivo, although the effect of the growth factors is not necessarily direct. The expression of c-fos and other immediate early genes provides a short-term marker that can be used to investigate the role of growth factors in normal retinal physiology and responses to injury.     

Heparin-binding epidermal growth factor-like growth factor stimulates cell proliferation in cerebral cortical cultures through phosphatidylinositol 3'-kinase and mitogen-activated protein kinase

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.     

Transforming growth factor alpha attenuates the functional expression of AMPA receptors in cortical GABAergic neurons

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor alpha (TGFalpha), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFalpha treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFalpha treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFalpha effect. Moreover, TGFalpha counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFalpha with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFalpha, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.     

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) regulates survival of midbrain dopaminergic neurons

Summary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF-family of ligands signaling via the EGF-receptor tyrosine kinase. In the present study we show that HB-EGF which is expressed in close proximity of developing mesencephalic dopaminergic neurons promotes the survival of TH-positive neurons in vitro. The survival promoting effect of HB-EGF is mediated via astroglial cells and utilizes the MAPK as well as the Akt-signaling pathway. Most notably endogenous HB-EGF significantly contributes to the survival of TH-+ neurons in control cultures, suggesting a relevant developmental role of HB-EGF for dopaminergic neurons. These findings indicate that HB-EGF may be an important molecule for developing dopaminergic neurons of the ventral midbrain. Received March 12, 2001; accepted April 27, 2001     

Prenatal ontogeny of the epidermal growth factor receptor and its ligand,transforming growth factor alpha,in the rat brain

Transforming growth factor alpha (TGFα) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGFα induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGFα and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGFα and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGFα in many brain structures preceded that of EGF-R mRNA. TGFα mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGFα mRNA. Northern blot analysis demonstrated that mRNA species for both TGFα and EGF-R were similar in embryos and adults. These data indicate that TGFα and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain. J. Comp. Neurol. 380:243–261, 1997. © 1997 Wiley-Liss, Inc.     

Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are potent mitogenic proteins capable of inducing cell division in a wide variety of cell types. In addition to their mitogenic properties, both proteins have recently been shown to enhance survival and process outgrowth from neurons of central nervous system origin. The full spectrum of neuronal subtypes responding to these factors has not been elucidated. In the present study, EGF was found to enhance survival and process outgrowth of primary cultures of cerebellar neurons of neonatal rat brain. This effect was dose-dependent and was observed with EGF concentrations as low as 100 pg/ml. In marked contrast, bFGF was ineffective in enhancing survival or neurite elongation from cerebellar neurons when tested in the range of 0.1 to 10.0 ng/ml. However, within this concentration range, bFGF did prove effective in stimulating an increase in [3H]thymidine incorporation into primary cultures of cerebellar astrocytes, demonstrating that bFGF was active and that cells in the cerebellum do respond to bFGF. These results suggest that EGF or an EGF-like peptide may act as a neurite elongation and maintenance factor for cerebellar neurons. EGF has now been shown to support striatal, cortical, and cerebellar neurons, suggesting that this factor may have trophic activity throughout the central nervous system. bFGF, in contrast, appears to exert its effects on limited populations of neurons.     

EGF and NGF injected into the brain of old mice enhance BDNF and ChAT in proliferating subventricular zone

The response of cells localized in the brain subventricular zone (SVZ) to growth factor stimulation has been largely described for development and adult life, whereas no information on their behavior during aging is available. To address the question of whether the cells in the SVZ of old mice respond to the intracerebroventricular administration of epidermal growth factor (EGF) and nerve growth factor (NGF), we studied the distribution of proliferating cells and the effects on ChAT and brain-derived neurotrophic factor (BDNF) synthesis in forebrain and SVZ. It was found that the conjoint administration of EGF + NGF produced a major increase in ChAT expression in both forebrain and SVZ. The ChAT mRNA levels and the number of ChAT positive cells localized in the ventricular border and in the parenchyma of SVZ area were also increased significantly in the mice receiving EGF + NGF. Enhanced numbers of SVZ cells expressing proliferative markers were also discovered in EGF + NGF treated mice and some of these cells expressed cholinergic markers, as demonstrated by double immunostaining. In addition, EGF and NGF treatments significantly upregulate BDNF protein and mRNA levels in this brain region. The present study demonstrates that cells localized in SVZ of aged mouse brain retain the capacity to respond to EGF and NGF and that after stimulation with these two growth factors, the synthesis of ChAT and BDNF also increases. The implication that cells of the SVZ remain a reservoir of cholinergic and BDNF-positive neurons in aged brain opens a new perspective for understanding the role of growth factors during neurodegenerative disorders associated with aging.     

Opposing actions of the EGF family and opioids: heparin binding-epidermal growth factor (HB-EGF) protects mouse cerebellar neuroblasts against the antiproliferative effect of morphine

Endogenous opioids and opiate drugs of abuse inhibit the proliferation of cerebellar external granular layer (EGL) neuroblasts by mechanisms that are incompletely understood. Opioids do not act alone, rather multiple extracellular factors regulate granule cell neurogenesis and these undoubtedly act in concert with opioids to shape developmental outcome. We examined whether, heparin binding-epidermal growth factor-like growth factor (HB-EGF), a recently described member of the epidermal growth factor (EGF) family, might compete with an inhibitory opioid signal. The results confirmed our ongoing studies that morphine inhibited neuroblast proliferation, while HB-EGF enhanced cell replication. HB-EGF not only counteracted the antiproliferative morphine signal, but invariably enhanced DNA synthesis irrespective of morphine treatment. Our findings suggest that regional and temporal differences in the availability of endogenous HB-EGF may serve to limit the response of EGL neuroblasts to opioids, and HB-EGF may be neuroprotective in opiate drug abuse. If similar responses occur in vivo, then the EGF family and the opioid system may represent distinct and contrasting components of an extracellular signaling system serving to coordinate EGL neurogenesis.     

人脑胶质瘤的表皮生长因子基因表达

采用Ｎｏｒｔｈｅｒｎ杂交和斑点杂交的方法检测了５０例胶质瘤、４个恶性胶质瘤体外细胞系和７例正常脑组织表皮生长因子（ＥＧＦ）ｍＲＮＡ表达水平。结果显示：上述组织或细胞都可表达５．０ｋｂ的ＥＧＦｍＲＮＡ片断，高恶度胶质瘤较低恶度胶质瘤和正常脑组织ＥＧＦｍＲＮＡ表达水平增高，低恶度胶质瘤与正常脑组织的表达无显著差异。在５０例胶质瘤中２９例（５８％）ＥＧＦｍＲＮＡ过表达，其中多见于恶性胶质瘤，ＥＧＦｍＲＮＡ表达与肿瘤的分级呈正相关。提示胶质瘤可以合成ＥＧＦ，在胶质瘤的发生发展过程中，ＥＧＦ参与或促进了其恶性进展。     

Localization of cells synthesizing transforming growth factor-alpha mRNA in the mouse brain

The distribution of transforming growth factor (TGF)-alpha and TGF-beta 1 mRNA containing cells in adult mouse brain was examined using in situ hybridization histochemistry. There were no detectable TGF-beta 1 mRNA-containing cells found in the brain. TGF-alpha mRNA was localized to cell bodies of the caudate nucleus, dentate gyrus, anterior olfactory nuclei, and a laminar distribution of mitral cells in the olfactory bulb. TGF-alpha-synthesizing cells were localized in brain regions that have been shown to synthesize nerve growth factor, epidermal growth factor (EGF) and enkephalins. The function of local synthesis of TGF-alpha in the brain is as yet unknown.     

Post-ischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hypoxia-inducible, neuroprotective protein that also stimulates proliferation of neuronal precursor cells. Accordingly, HB-EGF may contribute to recovery from cerebral injury through direct neuroprotective effects, by enhancing neurogenesis, or both. When administered by the intracerebroventricular route 1-3 days after focal cerebral ischemia in adult rats, HB-EGF decreased the volume of the resulting infarcts and reduced post-ischemic neurological deficits. HB-EGF also increased the incorporation of bromodeoxyuridine into cells expressing the immature neuronal marker protein TUC-4 in the dentate subgranular and rostral subventricular zones, consistent with increased proliferation of neuronal precursors. However, HB-EGF decreased the number of newborn neurons that migrated into the ischemic striatum, perhaps partly because reduction of infarct size by HB-EGF also reduced the stimulus to migration. To determine if HB-EGF might also directly inhibit migration of neuronal precursors, we co-cultured subventricular zone (SVZ) explants treated with HB-EGF or vehicle together with hypoxic cerebral cortical explants, and measured cell migration from the former toward the latter. HB-EGF reduced directed migration of SVZ cells toward the cortical explants, possibly due to a local chemoattractant effect on neuronal precursor cells, which may be mediated through the HB-EGF-specific receptor, N-arginine dibasic convertase. The delayed neuroprotective effect of HB-EGF may have implications for efforts to prolong the therapeutic window for intervention in stroke.     

Epidermal growth factor and its receptor in the developing human nervous system

Recent data suggest that epidermal growth factor and epidermal growth factor receptors (EGF/EGF-R) are present and functional in neurons within the central nervous system. Previously, EGF was detected in developing and mature rat brain and cerebrospinal fluid. Also, EGF-R was documented in discrete locations in normal adult human brain, as well as in senile plaques associated with Alzheimer's disease. Using two polyclonal sera, anti-EGF and anti-EGF-R, in conjunction with immunohistochemical staining, we examined formalin-fixed, paraffin-embedded neural tissues from 10 autopsied, human brains. These specimens were collected from patients who died during various stages of development ranging from 27 weeks of estimated gestational age to 63 years of age. Immunostaining for EGF and EGF-R was detected in hippocampal pyramidal cells. Purkinje cells, large multipolar neurons of the dentate nucleus, anterior horn cells, dorsal root ganglion cells, cells of the dorsal nucleus of Clark, intermediolateral column cells and ependymal cells. Positive binding studies with 125I-EGF confirmed that numerous EGF receptors are unoccupied, assessable, and available for interactions with potential ligands such as EGF and TGF alpha in developing rat brains. It appears that EGF and/or EGF-R may play a role during maturation and differentiation of the human central nervous system.     

Localization of immunoreactive epidermal growth factor receptor in neonatal and adult rat hippocampus

The regional and developmental expression of epidermal growth factor (EGF) receptor in rat hippocampus was investigated utilizing immunocytochemical techniques at the light and electron microscopic levels. EGF receptor immunoreactivity in adult hippocampus was compared to that found at postnatal day 7 (P7). While the receptor was observed in P7 hippocampus, immunostaining was more prominent in the adult hippocampus, especially in the pyramidal CA2 field. Ultrastructural analysis of this region revealed that the receptor was localized to the cell bodies of both P7 and adult neurons rather than the axons or dendrites. The expression of EGF receptor in selected regions of the adult brain was verified by Western blotting. These results demonstrate the presence of EGF receptor in rat hippocampus as early as P7, localize the receptor to the pyramidal cell body, and establish the hippocampal formation, particularly CA2, as a major site of EGF receptor expression in rat brain.