EGF receptor
EGFR and neural stem cells Temporal changes in progenitor cell responses to extrinsic
signals play an important role in development, but little is
known about the mechanisms that determine how these
changes occur. In the rodent CNS, expression of epidermal
growth factor receptors (EGFRs) increases during
embryonic development, conferring mitotic responsiveness
to EGF among multipotent stem cells. Cell-cell signaling controls this change. Whereas EGF-responsive
stem cells develop on schedule in explant and
aggregate cultures of embryonic cortex, co-culture with
younger cortical cells delays their development. Exogenous
BMP4 mimics the effect of younger cells, reversibly
inhibiting changes in EGFR expression and responsiveness.
Moreover, blocking endogenous BMP receptors in
progenitors with a virus transducing dnBMPR1B
accelerates changes in EGFR signaling. This involves a
non-cell-autonomous mechanism, suggesting that BMP
negatively regulates signal(s) that promotes the development
of EGF-responsive stem cells. FGF2 is a good candidate for
such a signal: it antagonizes the inhibitory
effects of younger cortical cells and exogenous BMP4.
These findings suggest that a balance between antagonistic
extrinsic signals regulates temporal changes in an intrinsic
property of neural progenitor cells (Lillien, 2000).
What then triggers the onset of the appearance of EGF-responsive
stem cells? It is appealing to consider a feedback
mechanism, whereby cells that produce BMPs or FGF2
achieve appropriate numbers or states of maturation at mid-embryonic
development, resulting in the generation of a net
positive signal. BMPs are produced by radial glial cells and by the choroid plexus. FGF2 is made by progenitor cells and
choroid plexus. The numbers of these
cells do not change in an appropriate manner at mid-embryonic
development to provide a trigger, suggesting that the cellular
event(s) that initiates the change in EGFR expression may be
more complex and involve a change in the level of expression
of FGF, BMP and/or their receptors. It has been reported that
the level of FGF2 increases during mid-late stages of
embryonic development. Thus, an increase in FGF2 expression could be
the trigger. The level of expression of BMPRs in the brain
appears to decline during embryonic development, suggesting that BMP signaling might decline. Given
the observation that BMPs in the limb negatively regulate the
expression of FGFs, together with the finding that dnBMPRs have a non-cell-autonomous positive effect, it is possible that a reduction
in BMP signaling triggers the increase in FGF2 expression in
the CNS. In explant cultures, it has been observed that a
lower concentration of FGF2 (1 ng/ml) has a greater effect on
E15 explants than on E12 explants. This observation is consistent with an increase in the level
of an endogenous positive signal, such as FGF2 with age, or
with a decline in the level or effectiveness of endogenous
BMPs (Lillien, 2000).
In addition to the change in EGFR expression and
responsiveness, cortical progenitor cells and stem cells change
in several other ways during mid-embryonic development.
Early progenitor cells and stem cells tend to generate more
neuronal progeny, while later progenitor cells and stem cells
tend to generate more glial progeny. Early
and late progenitor cells also differ in their competence to
generate deep layer neurons. The
developmental change in the latter property also appears to be
controlled by cell-cell signaling. Studies using viral transduction of EGFRs suggest
that mitotic stimulation of progenitor cells with EGF does not
control developmental changes in the ratio of neuronal and
glial progeny. It will be interesting to
determine whether the signals that have been identified as
regulators of EGFR expression and responsiveness also control
other properties of progenitor cells that change during
embryonic development, or whether these properties are
controlled by distinct mechanisms (Lillien, 2000).
It has been debated whether asymmetric distribution of cell surface receptors during mitosis could generate asymmetric cell divisions by yielding daughters with different environmental responsiveness and, thus, different fates. In mouse embryonic forebrain ventricular and subventricular zones, the EGFR can distribute asymmetrically during mitosis in vivo and in vitro. This occurs during divisions yielding two Nestin+ progenitor cells, via an actin-dependent mechanism. The resulting sibling progenitor cells respond differently to EGFR ligand in terms of migration and proliferation. Moreover, they express different phenotypic markers: the EGFRhigh daughter usually has radial glial/astrocytic markers, while its EGFRlow sister lacks them, indicating fate divergence. Lineage trees of cultured cortical glioblasts reveal repeated EGFR asymmetric distribution, and asymmetric divisions underlie formation of oligodendrocytes and astrocytes in clones. These data suggest that asymmetric EGFR distribution contributes to forebrain development by creating progenitors with different proliferative, migratory, and differentiation responses to ligand (Sun, 2005).
In the cortex, EGFR expression increases at E13, around peak neurogenesis and before gliogenesis (here, as in most CNS regions, neurons arise before glia). Around this time, EGF-reponsive stem cells arise. Ligand distribution also alters during development: from E12 in the rat, TGFα is produced largely from basal forebrain and the choroid plexu, while from E17, heparin binding EGF is expressed in the SVZ and by developing neurons in the cortical plate (Sun, 2005).
At E13-15, cortical divisions that are asymmetric for EGFR usually occur in the VZ, are mostly parallel to the ventricular surface, and result in Numb asymmetry, with Numb moving into the EGFRhigh daughter. While Numb can sustain the progenitor phenotype, it can also stimulate neuroblast differentiation in vertebrates, analogous to its role in Drosophila neural development, and it was found that in cultured cortical progenitors at midgestation, Numb preferentially moves into the differentiating neuronal daughter at P/N divisions. Overexpression of EGFR in cortical progenitor cells in vivo causes them to migrate to the SVZ, toward localized ligand, indicating that translocation of progenitors from the VZ to the SVZ compartments may be EGFR dependent. The SVZ is now appreciated as a major location for terminal mitoses of restricted neuroblasts. The EGFRhigh daughter exhibits further EGF-dependent migration than its EGFRlow sister. Hence, one possibility is that, at around E13, asymmetric segregation of EGFR into the VZ daughter that receives Numb will stimulate its translocation to the SVZ to undergo its final divisions and then differentiate. Because asymmetric EGFR divisions at terminal divisions produce a progenitor and a neuron, and EGFR is weak in the SVZ at E13-14, it is believed that asymmetric distribution of EGFR is infrequent in SVZ neuroblasts. If this scenario does occur in vivo, the EGFRhigh cell must rapidly lose EGFR expression once it reaches the SVZ (Sun, 2005).
The cell that remains in the VZ has FGFR, but lacks Numb, so it has active Notch and is therefore likely to continue as a progenitor; it might be either a multipotent neuroblast or a multipotent FGF-dependent stem cell. At all stages examined, a low frequency of EGFRhigh Numb- daughters are observed. These may retain progenitor cell status, becoming EGF-responsive stem cells (Sun, 2005).
At later stages, E16–17, most VZ cell divisions that are EGFR-asymmetric occur perpendicular to the cell surface and have symmetric Numb. Again, EGFR acquisition might help one daughter to translocate to the SVZ. High levels of EGFR stimulation, especially in late-stage cells, slow their proliferation, probably as part of the astrocyte differentiation step. Hence, asymmetric EGFR segregation at this stage, along with Numb acquisition, may help ensure that the EGFRhigh daughter begins to differentiate into an astrocyte and translocates toward the cortical plate and higher ligand concentration. At E16–17, asymmetric EGFR cell divisions also occur in the SVZ, usually parallel to the nearby ventricle surface. It is speculated that the EGFRlow cells generated in the VZ or SVZ may take on a nonastrocytic fate, either neuronal, or, possibly, oligodendroglial (Sun, 2005).
In cultured asymmetric EGFR pairs from E16-E17 cortex, the EGFRhigh daughter usually has progenitor/astrocyte lineage markers, while the EGFRlow daughter usually lacks them. It is possible that the EGFRlow daughter can later reexpress astrocyte markers. However, the simplest explanation is that the sibling cells have become different, and that after losing progenitor/astrocyte markers, the newly generated EGFRlow daughter go on to acquire a different fate. These studies suggest that in vitro it likely acquires oligodendrocyte characteristics, stimulated by FGF2 signaling. After longer periods of culture, cells derived from these progenitors are RC2-NG2+ and EGFR- (RC2 refers to staining with a monoclonal antibody and NG2 refers to Neurogenin2). The fact that EGFR overexpression pushes the cells into the astrocyte pathway at the expense of oligodendrocytes strengthens the idea that creating cells lacking EGFR is important for this fate choice in cultured cells (Sun, 2005).
While progenitors in vitro can robustly produce both oligodendrocytes and astrocytes, these bipotent cells are found rarely in vivo, comprising only 10%-17% of the progenitor population. It is, of course, possible that cells exhibiting glial bipotency are rare, because once the lineages diverge, they may be amplified by symmetric divisions of restricted progenitors. Moreover, EGFR asymmetry is a relatively rare event. It was found that NG2 overlaps rarely with EGFR in vitro and in embryonic forebrain sections, indicating that from early stages, oligodendrocyte lineage cells don't express this receptor, which is consistent with the recent finding that PDGFRα-expressing progenitors in basal forebrain lack EGFR. Overexpression of EGFR in vivo pushes cortical progenitor cells toward the astrocyte lineage, but whether this happens at the expense of oligodendrocyte generation has not been examined, and knockouts of EGFR die too early to determine an effect on oligodendrogenesis. Hence, it will be important in the future to determine whether glial divergence in the developing forebrain in vivo has a dependence on EGFR asymmetric distribution (Sun, 2005).
In conclusion, these data show that the EGFR can be asymmetrically segregated in dividing neural progenitor cells in vivo and in vitro and can generate functionally different sister cells in the presence of ligand. EGFR ligands are present in germinal zones and are secreted from the choroid plexus into the cerebrospinal fluid; hence, an effective way to reduce EGFR signaling in the presence of abundant ligand is simply to remove the surface receptor. Asymmetric distribution of surface receptors during mitosis is another means of generating receptor complexity on CNS progenitor cells, allowing divergent proliferative, migratory, and differentiation responses that contribute to the emerging cellular complexity (Sun, 2005).
Coordinated regulation of neuronal progenitor differentiation in the subventricular zone (SVZ) is a fundamental feature of adult neurogenesis. However, the molecular control of this process remains mostly undeciphered. This study investigated the role of neuregulins (NRGs) in this process and showed that a NRG receptor, ErbB4, is primarily expressed by polysialylated neural cell adhesion molecule immature neuroblasts but is also detected in a subset of GFAP(+) astroglial cells, ependymal cells, and Dlx2(+) precursors in the SVZ. Of the NRG ligands, both NRG-1 and -2 are expressed by immature polysialylated neural cell adhesion molecule neuroblasts in the SVZ. NRG2 is also expressed by some of the GFAP(+) putative stem cells lining the ventricles. Infusion of exogenous NRG1 leads to rapid aggregation of Dlx2(+) cells in the SVZ and affects the initiation and maintenance of organized neuroblast migration from the SVZ toward the olfactory bulb. In contrast, the infusion of NRG2 increased the number of Sox2 and GFAP(+) precursors in the SVZ. An outcome of this NRG2 effect is an increase in the number of newly generated migrating neuroblasts in the rostral migratory stream and GABAergic interneurons in the olfactory bulb. The analysis of conditional null mice that lack NRG receptor, ErbB4, in the nervous system revealed that the observed activities of NRG2 require ErbB4 activation. These results indicate that different NRG ligands affect distinct populations of differentiating neural precursors in the neurogenic regions of the mature forebrain. Furthermore, these studies identify NRG2 as a factor capable of promoting SVZ proliferation, leading to the formation of new neurons in vivo (Ghashghaei, 2006).
Many organs rely on undifferentiated stem and progenitor cells for tissue regeneration. Whether differentiated cells themselves can contribute to cell replacement and tissue regeneration is a controversial question. This study shows that differentiated heart muscle cells, cardiomyocytes, can be induced to proliferate and regenerate. An underlying molecular mechanism was identified for controlling this process that involves the growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor, ErbB4. NRG1 induces mononucleated, but not binucleated, cardiomyocytes to divide. In vivo, genetic inactivation of ErbB4 reduces cardiomyocyte proliferation, whereas increasing ErbB4 expression enhances it. Injecting NRG1 in adult mice induces cardiomyocyte cell-cycle activity and promotes myocardial regeneration, leading to improved function after myocardial infarction. Undifferentiated progenitor cells did not contribute to NRG1-induced cardiomyocyte proliferation. Thus, increasing the activity of the NRG1/ErbB4 signaling pathway may provide a molecular strategy to promote myocardial regeneration (Bersell, 2009).
EGFR, cell fate, differentiation and cell transformation Embryonic mouse skin undergoes a drastic morphological change from 13 to 16 gestational days, i.e., formation of rudiments of hair
follicles and stratification and cornification of interfollicular epidermis. To investigate underlying molecular mechanisms of the
morphogenesis, an organ culture system was established that allows skin tissues isolated from 12.5- or 13.5-days postcoitus embryos
to develop in a manner that is histologically and temporally similar to the process in vivo. Expression of differentiation markers of
epidermal keratinocytes including cholesterol sulfotransferase and cytokeratin K1, was induced in culture, since it also occurs in vivo. The
morphogenic process was observed by time-lapse videomicrography. In this culture system, epidermal growth factor (Egf) and
transforming growth factor alpha specifically and completely inhibit the hair follicle formation with marginal effects on interfollicular
epidermis. The inhibitory action by Egf is reversible and stage specific, i.e., at an early stage of the development of hair rudiments.
Among known ligands to the Egf receptor, Schwannoma-derived growth factor and heparin-binding Egf are expressed in in vivo
epidermis during the period of the initial formation of hair follicles. Egf receptor is expressed in epidermis throughout the developing
period examined (Kashiwagi, 1997).
Embryonic mouse skin undergoes a drastic morphological change from 13 to 16 gestational days: the
formation of rudiments of hair follicles and stratification and cornification of interfollicular epidermis.
To investigate the underlying molecular mechanisms of this morphogenesis, an organ culture
system was established to allow skin tissues isolated from 12.5- or 13.5-days postcoitus embryos to develop in a
manner that is histologically and temporally similar to the process in vivo. Expression of differentiation
markers for epidermal keratinocytes, including cholesterol sulfotransferase and cytokeratin K1, is
induced in culture, the same as it normally occurs in vivo. The morphogenic process was observed by time-lapse
videomicrography. In this culture system, epidermal growth factor (EGF) and transforming growth
factor alpha specifically and completely inhibit the hair follicle formation with marginal effects on
interfollicular epidermis. The inhibitory action by EGF is reversible and stage specific, that is, it occurs at an early
stage of the development of hair rudiments. Among known ligands to the EGF receptor,
Schwannoma-derived growth factor and heparin-binding EGF are expressed in in vivo epidermis
during the period of the initial formation of hair follicles. EGF receptor is expressed in epidermis
throughout the developing period examined (Kashiwagi, 1997).
Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate in vitro branching
morphogenesis in the fetal mouse submandibular gland (SMG) rudiments. The EGF system (EGF,
TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in
the extracellular matrix. Inhibition of EGFR tyrosine-kinase activity by a tyrphostin
retards the in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact
mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse
transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these
mRNAs were quantitatively determined by nuclease protection assays. These findings suggest that the
EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized
by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of the cultured
SMG rudiments is inhibited by anti-alpha6 antibodies. Synthesis of the alpha6-subunit in cultured SMGs is increased by EGF and drastically reduced
by tyrphostin. mRNAs for the alpha6- and beta1- and beta4-integrin subunits are
expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that (1)
the EGF system is a physiologic regulator of development of fetal mouse SMG, and (2) it may act by regulating expression of integrins, which in turn control interaction of
epithelial cells with the extracellular matrix (Kashimata, 1997).
Peptide growth factors likely play an important role in cardiac development, but growth factors which
inhibit or prevent differentiation in cardiac myocytes are largely unknown. Epidermal growth
factor (EGF) significantly inhibits differentiation and promotes proliferation in cultured human fetal
ventricular cardiac myocyte cell lines. In enriched cell lines and in a pure myocyte cell strain, EGF
inhibits increases in immunoreactive sarcomeric actin and sarcomeric myosin heavy chain (SMHC); these are responses
normally seen after serum withdrawal. In the pure myocyte strain, EGF induces a cardiomyoblastic
phenotype: it causes a complete loss of detectable sarcomeric proteins in the majority of cells and
is also mitogenic. EGF inhibits expression of cardiac alpha-actin and SMHC mRNAs, but inhibition
of SMHC expression is predominantly of the beta-MHC isoform. Removal of EGF is followed by
reexpression of sarcomeric proteins. Blocking the EGF receptor (EGFR) with monoclonal anti-receptor
antibody completely abolishes the dedifferentiating effects of EGF and also significantly reduces the
mitogenic effect of the peptide. The results indicate that activation of the EGFR both inhibits
differentiation and promotes proliferation of human fetal ventricular myocytes in vitro. These findings
suggest an important role for EGF in human cardiac differentiation and development (Goldman, 1996).
Upon binding of TGF
alpha to its receptor, the EGF receptor (EGFR),
TGF alpha (Drosophila homolog: Spitz) can exert diverse biological activities, such as induction of cell proliferation
or differentiation. To explore the possibility that TGF alpha might regulate cell fate
during murine eye development, transgenic mice were generated that express human
TGF alpha in the lens under the control of the mouse alpha A-crystallin promoter. The
transgenic mice displayed multiple eye defects, including corneal opacities, cataracts
and microphthalmia. At early embryonic stages TGF alpha induces the perioptic
mesenchymal cells to migrate abnormally into the eye and accumulate around the lens.
In situ hybridization reveals that the EGFR mRNA is highly expressed in the
perioptic mesenchyme, suggesting that the migratory response is mediated by receptor
activation. In order to test this model, the TGF alpha transgenic mice were bred to
EGFR mutant waved-2 mice. The eye defects of the TGF alpha
transgenic mice are significantly abated in the wa-2 homozygote background. Because
the EGFR mutation in the wa-2 mice is located in the receptor kinase domain, this
result indicates that the receptor tyrosine kinase activity is critical for signaling the
migratory response. Taken together, these studies demonstrate that TGF alpha is
capable of altering the migratory decisions and behavior of perioptic mesenchyme
during eye development (Reneker, 1995).
To address the influence of EGF receptor level on responses of retinal progenitor cells to
TGF-alpha, additional copies of EGF-Rs were introduced in vitro and in vivo with a
retrovirus. Low concentrations of TGF-alpha in vitro will normally stimulate proliferation,
whereas high concentrations bias choice of cell fate, inhibiting differentiation into
rod photoreceptors while promoting differentiation into Muller glial cells. Introduction of extra EGF-Rs into progenitor cells in vitro reduces the
concentration of TGF-alpha required for changes in rod and Muller cell differentiation
but does not enhance proliferation. Introduction of extra EGF-Rs in vivo increases the
proportion of clones that contained Muller glial cells, suggesting that receptor level is
normally limiting. These findings demonstrate that responsiveness to extracellular
signals during development can be modulated by the introduction of additional
receptors, and suggest that the level of expression of receptors for these signals
contributes to the regulation of cell fate (Lillien, 1995).
The level of epidermal growth factor receptors (EGF-Rs) expressed by progenitor cells in the newborn (P0) rat retina is
limiting for the generation of Muller glial cells but not for proliferation. To determine whether EGF-R signaling biases cells to generate a specific cell type or
regulates more general processes during progenitor cell development, extra copies of the EGF-R were introduced into progenitor cells at earlier stages
(E15 and E18), when different cell types are produced. Progenitor cells in early embryonic retina (E15) normally express lower levels of
EGF-Rs than progenitor cells in later retina (E18 and P0). Whereas lower levels of stimulation of endogenous and virally transduced EGF-Rs enhance
proliferation, higher levels reduce proliferation, resulting in premature differentiation. At E15, very few EGF-R-infected progenitor cells differentiate
prematurely into Muller glial cells, unlike E18 and P0 cells, even when they are exposed to an older retinal environment. Higher levels of EGF-R-mediated
signaling alone therefore do not specify a glial fate, indicating that competence to generate glia is temporally regulated by additional mechanisms. The
differences in EGF-R expression observed among retinal progenitor cells at distinct developmental stages may instead help to define signaling thresholds
that delay or accelerate their differentiation (Lillien, 1998).
The mammalian lung develops through branching morphogenesis, which is controlled by growth factors,
hormones, and extracellular matrix proteins. The role of EGF-receptor signaling has been evaluated in
lung morphogenesis by analyzing the developmental phenotype of lungs in mice using an inactivated
EGF-receptor gene, both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often
show evidence of lung immaturity that can result in visible respiratory distress. The lungs of these
mutant mice have impaired branching and deficient alveolization and septation, resulting in a 50%
reduction in alveolar volume and thus a markedly reduced surface for gas exchange. The
EGF-receptor inactivation also results in the immaturity of type II pneumocytes, which is apparent from
their increased glycogen content and a reduced number of lamellar bodies. The defective branching
is already evident at Day 12 of embryonic development. When explants of embryonic lungs from
Day 12 embryos are cultured under defined conditions, the branching defect in
EGF-receptor-deficient lungs is even more pronounced, with only half as many terminal buds as
normal lungs. EGF treatment stimulates the expression of surfactant protein C and thyroid transcription
factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor
signaling regulates the expression of these marker genes during type II pneumocyte maturation. The localization of EGF expression in pulmonary mesenchyme suggests that the mesenchyme might provide ligands that induce EGF-R signaling in the epithelial cells. These data indicate that signal transduction through the EGF receptor plays a major role in lung
development and that its inactivation leads to a respiratory distress-like syndrome (Miettinen, 1997).
Epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG): the EGF receptor
(EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG. The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among
them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. An investigation was carried out to see whether EGF utilizes
the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18, placed into wells of defined medium (BGJb), and exposed to EGF for 5 or 30 min. EGF induces the
appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots show the specific
appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increases at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for
MEK-1/2 (the MAPK kinase that activates ERK-1/2) increases near the cell membrane after EGF stimulation. Phospho-ERK-1/2 localizes in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF
for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibits branching morphogenesis and, furthermore, decreases the stimulatory effect of EGF on branching. Western blots confirm that this inhibitor blocks phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this
cascade perturbs branching morphogenesis. However, EGF does not cause phosphorylation of two other MAPKs (SAPK/JNK or p38MAPK) in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG (Kashimata, 2000).
Mesenchymal-epithelial interactions are crucial during development of the embryonic mammary bud, when mammary mesenchyme induces the overlying ectoderm to form epithelial buds at day 12 of gestation. Stromal-epithelial interactions are critical in determining patterns of growth, development and ductal morphogenesis
in the mammary gland, and their perturbations are significant components of tumorigenesis. Growth factors such
as epidermal growth factor (EGF) contribute to these reciprocal stromal-epithelial interactions. To determine the
role of signaling through the EGF receptor (Egfr) in mammary ductal growth and branching, mice with
a targeted null mutation in the Egfr were used. Because Egfr-/- mice die perinatally, transplantation methods were used to
study these processes. When neonatal mammary glands are transplanted under the renal capsule of
immuno-compromised female mice, Egfr is found to be essential for mammary ductal growth and branching
morphogenesis, but not for mammary lobulo-alveolar development. Ductal growth and development is normal in
transplants of mammary epithelium from Egfr-/- mice into wild-type (WT) gland-free fat pads and in tissue
recombinants prepared with WT stroma, irrespective of the source of epithelium. However, ductal growth and branching is impaired in tissue recombinants prepared with
Egfr-/- stroma. Thus, for ductal morphogenesis, signaling through the Egfr
is required only in the stromal component, the mammary fat pad. These data indicate that the Egfr pathway plays
a key role in the stromal-epithelial interactions required for mammary ductal growth and branching morphogenesis.
In contrast, signaling through the Egfr is not essential for lobulo-alveolar development. Stimulation of
lobulo-alveolar development in the mammary gland grafts by inclusion of a pituitary isograft under the renal
capsule as a source of prolactin results in normal alveolar development in both Egfr-/- and wild-type transplants.
Through the use of tissue recombinants and transplantation, new insights have been gained into the nature of
stromal-epithelial interactions in the mammary gland, and how they regulate ductal growth and branching
morphogenesis. Thus, signaling through the Egfr may promote fibroblast survival, which in turn induces ductal epithelial cell proliferation (Wiesen, 1999).
Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the
TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF
receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and
fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless,
pups born to triple null dams frequently die or are runted, suggesting a mammary gland defect.
Comparison of individual and combinatorial knockouts establishes that specific loss of AR severely stunts
ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal
developing ducts. Surprisingly, loss of all three ligands does not significantly affect cellular proliferation,
apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females,
but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in
mammopoiesis and lactogenesis. In triple null glands, alveoli are poorly organized and differentiated, and
milk protein gene expression is decreased. Additionally, Stat5a activation is frequently reduced in AR
single and combinatorial nulls in association with impaired lactation. Collectively, these results provide genetic
confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary
gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of
growth factors from pups nursed by triple null dams further worsen their survival and growth,
establishing functions for both maternal- and neonatal-derived growth factors (Luetteke, 1999).
An amino-truncated variant form of the epidermal growth factor receptor (EGFRvIII) has been
identified in human brain, breast, lung and ovarian tumors. Overexpression of
this mutant EGF receptor in NIH3T3 cells results in transformation as a result of the activation of
the receptor kinase via ligand-independent dimerization. Transformation is correlated with
tyrosine phosphorylation of only a subset of the proteins observed in cells overexpressing the
normal EGF receptor. This suggests that further studies on cells expressing the EGFRvIII might
provide insights into the pathways most relevant to transformation. In clones expressing high levels
of mutant EGF receptor, the levels of both Grb2 and SHC are decreased. Despite this decrease,
much of the endogenous Grb2 immunoprecipitates with EGFRvIII. Interestingly, no increase in
ras-GTP loading is found in clones expressing EGFRvIII. MAP kinase assays indicate
only a small increase in activity. These results indicate that high-level expression of EGFRvIII
induces down-regulation of the ras-MAP kinase pathway and that other components involved in
EGF receptor signal transduction may play a greater role in neoplastic transformation by
EGFRvIII (Moscatello, 1996).
An EGF-R-mutant lacking the autophosphorylation sites
phosphorylates Shc and retains mitogenic activity. In these cells,
in response to EGF, Ras (See Drosophila Ras) is fully activated with formation of the tyrosine-phosphorylated
Shc-Grb2-mSOS complex without the receptor. This pointed out the importance of Shc in
EGF-induced Ras activation. The EGF-R phosphorylates Shc, but not the Shc
SH2 mutant, lacking binding ability for phosphotyrosine. This suggests that intact Shc SH2 is
essential for the full-length Shc to become phosphorylated, probably by inducing a conformational
change in Shc. Thus a Shc SH2 peptide may inhibit competitively Shc phosphorylation. The Shc SH2 domain was microinjected into NIH3T3 cells overexpressing the EGF-R. Microinjected
Shc SH2 greatly suppresses EGF-induced DNA synthesis. But neither microinjection of the Shc
SH2 mutant nor the PLC-gamma 1 SH2 has any effect. This suppressing effect is rescued by
comicroinjection of the full-length Shc, suggesting Shc SH2 specifically suppressed the Shc
pathway. Thus it is concluded that, in EGF-induced mitogenesis, Shc phosphorylation is crucial, whereas receptor
autophosphorylation is dispensable (Gotoh, 1995).
The EGF receptor (EGFR) is required for skin development and is implicated in epithelial tumor formation. Transgenic mice expressing a dominant form of Son of Sevenless (SOS-F) in basal keratinocytes develop skin papillomas with 100% penetrance. However, tumor formation is inhibited in a hypomorphic (wa2) and null EGFR background. Similarly, EGFR-deficient fibroblasts are resistant to transformation by SOS-F and rasV12, however, tumorigenicity is restored by expression of the anti-apoptotic bcl-2 gene. The K5-SOS-F papillomas and primary keratinocytes from wa2 mice display increased apoptosis, reduced Akt phosphorylation, and grafting experiments imply a cell-autonomous requirement for EGFR in keratinocytes. Therefore, EGFR functions as a survival factor in oncogenic transformation and provides a valuable target for therapeutic intervention in a broader range of tumors than anticipated (Sibilia, 2000).
Feather buds form sequentially in a hexagonal array. Bone morphogenetic protein (BMP) signaling from the feather bud inhibits bud formation in the adjacent interbud tissue, but whether interbud fate and patterning is actively promoted by BMP or other factors has been unclear. Epidermal growth factor (EGF) signaling is shown to act positively to establish interbud identity. EGF and the active EGF receptor (EGFR) are expressed in the interbud regions. Exogenous EGF stimulates epidermal proliferation and expands interbud gene expression, with a concurrent loss of feather bud gene expression and morphology. Conversely, EGFR inhibitors result in the loss of interbud fate and increased acquisition of feather bud fate. EGF signaling acts directly on the epidermis and is independent of BMP signaling. The timing of competence to interpret interbud-promoting signals occurs at an earlier developmental stage than previously anticipated. These data demonstrate that EGFR signaling actively promotes interbud identity (Atit, 2003).
A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioning future hair follicle epithelium from interfollicular epidermis. Although many developmental signalling pathways are implicated in follicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7) receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, the underlying mechanisms have not been characterised. This study shows that receptors for EGF and KGF undergo marked downregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persist throughout hair follicle initiation. Using embryonic skin organ culture, it was shown that when skin from the sites of primary pelage and whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. Downstream molecular markers and microarray profiling were then used to provide evidence that, in response to KGF and EGF signalling, epidermal differentiation is promoted at the expense of hair follicle fate. It is proposed that hair follicle initiation in placodes requires downregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicular epidermis. A previously unrecognised role has also been uncovered for KGF signalling in the formation of hair follicles in the mouse (Richardson, 2009).
Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. It is hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, it was demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. It was determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention (Inda, 2010).
Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, this study identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. This study shows that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced Kras(G12D)-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR (see Drosophila Egfr) and blocks EGFR-induced RAS activation. Moreover, inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. These results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer (Humbert, 2022).
EGF receptor
:
Biological Overview
| Regulation
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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