Interactive Fly, Drosophila

Signaling downstream of EGF

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. These two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these dimodified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression (Cheung, 2000).

A clue to the functional link between H3 phosphorylation and acetylation seen in vivo is provided by the in vitro finding that yeast Gcn5 displays a stronger (up to 10-fold) preference for Ser-10 phosphorylated H3 peptide as substrate when compared to an unmodified control peptide. This effect is likely due to phosphate-dependent stabilization of the enzyme-substrate complex, since a single amino acid substitution (R164K) of Gcn5 at the predicted site of phospho-Ser-10 interaction completely abolishes Gcn5s higher affinity for phosphorylated H3. These data show that the observed differences in enzymatic rates are greatest when substrate concentrations are at or below the Km value. Since most enzymes operate in this concentration range, it is suspected that these inherent differences in substrate affinity have significant physiological consequences. At present, the roles of H3 phosphorylation in gene expression in yeast are not well defined. Analyses of the phenotype of yeast strains bearing mutations in Gcn5 at R164 are currently underway to further identify links between H3 phosphorylation and acetylation in yeast (Cheung, 2000).

The preference of wild-type Gcn5 for phosphorylated H3 peptide, which is referred to as phosphate-directed substrate recognition, provides evidence that one covalent modification can alter the efficiency of a subsequent enzymatic reaction on the same histone molecule. Several human HATs such as PCAF and p300 also prefer phosphorylated H3 as substrate; however, whether other families of HATs such as CBP or TAFII250 also display such a preference remains to be determined. Transcription-associated H3 phosphorylation occurs only in discrete regions of the genome, and therefore acetylation of bulk chromatin at lysine 14 is clearly not dependent on phosphorylation. However, for signaling pathway-activated genes such as c-fos, the coupling of histone phosphorylation and acetylation at specific loci may contribute to mechanisms that allow them to be rapidly activated in response to external stimuli. The concept of coupled modifications need not be confined to relationships between histone kinases and HATs. The recent discovery of a nuclear receptor coactivator-interacting protein, CARM1, which possesses arginine-specific H3 methyltransferase activity (HMT), provides evidence that histone methylation also contributes to transcriptional activation. Interestingly, methylated H3 has been reported to occur in a subset of hyperacetylated H3 molecules in several systems; therefore, these two modifications may also be coupled in some functional way. Different combinations of modifications may allow the limited set of histone modifications that are targeted to histone N-terminal tails to regulate a multitude of cellular processes. Moreover, this general concept can easily be extended to nonhistone substrates such as p53, which is also regulated by phosphorylation and acetylation (Cheung, 2000).

Although the phosphorylated H3 peptide is acetylated more efficiently by Gcn5 in vitro, only a subset of phosphorylated H3 is acetylated in EGF-stimulated cells. The acetylation of phosphorylated H3 in vivo is undoubtedly further determined by the abundance and targeting of the relevant HATs. In response to EGF stimulation, only a small set of genes are activated. Consistent with this, immunofluorescence studies using the Phos H3 and Phos/Ac H3 antibodies show punctate nuclear staining in the EGF-stimulated cells, in contrast to the more diffuse nuclear staining observed with antibodies directed against acetylated H3. Therefore, targeting of histone kinases and HATs is likely to play an important role in regulating the mitogen response. It has been shown that pp90Rsk (see Drosophila S6kII) is associated with CBP in mitogen-stimulated cells, and the Rsk-2 isoform is required for EGF-stimulated phosphorylation of H3 at Ser-10 in vivo. These findings suggest the possibility that kinase and HAT activities in a single complex can be targeted together to selected promoters to activate expression (Cheung, 2000).

Traditionally, nuclear responses to signal transduction pathways are thought to be mediated by activation of transcription factors via a number of well-documented phosphorylation cascades. The discovery that H3 is a physiologically relevant substrate of some of these activated kinases suggests that chromatin structure also plays a role in regulating the expression of immediate-early genes. The synergism between H3 phosphorylation and acetylation reported here further suggests that H3 phosphorylation may itself serve as an important integrator for signaling-mediated chromatin remodeling. It is postulated that phosphorylation cascades induced upon EGF stimulation lead to targeting of histone kinases (such as Rsk-2) to selected gene loci and rapid phosphorylation of H3 in specific nucleosomes. With the enhanced efficiency of acetylation of phosphorylated H3, and the possible recruitment of a single complex containing both a histone kinase and a HAT, these effects could expedite acetylation of H3 at targeted promoters. Such localized changes in chromatin structure may then facilitate binding of EGF-activated transcription factors to activate expression of the appropriate downstream genes. By linking histone acetylation to the EGF-stimulated phosphorylation cascade, this model provides an attractive explanation of how the chromatin structure of genes, regulated by different factors, can be altered in a coordinate fashion in response to external stimuli (Cheung, 2000).

TGF-alpha, EGF and Differentiation

Reciprocal signaling between distinct tissues is a general feature of organogenesis. Despite the identification of developmental processes in which coordination requires reciprocal signaling, little is known regarding the underlying molecular details. The development of the uterine-vulval connection in the nematode C. elegans is here used as a model system to study reciprocal signaling. In C. elegans, development of the uterine-vulval connection requires the specification of uterine uv1 cells and morphogenesis of primary-derived vulval cells. LIN-3, an epidermal growth factor (EGF) family protein, is first produced by the gonadal anchor cell to induce vulval precursor cells to generate vulval tissue. In a second step, lin-3 is expressed in the primary vulval lineage after vulval induction. The primary vulva, the recipient of the initial EGF signal, is necessary to signal back to induce the uv1 uterine cell fate. Using genetic and cell biological analyses, it has been found that the specification of uterine uv1 cells is dependent on EGF signaling from cells of the primary vulval lineages to a subset of ventral uterine cells of the gonad. RAS and RAF are necessary for this signaling. EGL-38, a member of the PAX family of proteins, is necessary for transcription of lin-3 in the vulva but not in the anchor cell. A let-23 mutation that confers ligand-independent activity bypasses the requirement for EGL-38 in specification of the uv1 cell fate. It has been shown how relatively simple EGF signals can be used reciprocally to specify the uterine-vulval connection during C. elegans development (Chang, 1999).

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).

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).

Upon binding of TGF alpha to its receptor, the EGF receptor (EGFR), TGF alpha 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).

It is believed that signaling through the epidermal growth factor (EGF) receptor plays a critical role in the development of the Drosophila eye. The role of EGF-mediated signaling in vertebrate retinal development was analyzed. During late retinal neurogenesis, EGF delays rod photoreceptor differentiation; this effect of EGF involves the modulation of expression of a homolog of the Drosophila proneural gene, Mash1. EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retina in vitro. The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. This suggests that Mash1 positive cells are rod precursors. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. This study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. The sensitivity of precursors to signaling through Notch and EGF receptors and the similarity of their functions suggest that during retinal development these two distinctive signaling mechanisms complement one another in keeping cells uncommitted. While Notch signaling can keep a cell uncommitted by repressing proneural homologs, signaling through the EGF receptor is likely to occur by regulating the decision of a cell to quit mitosis or to reenter the cell cycle. In Drosophila, cells that quit mitosis participate in the formation of clusters (developing ommatidia) at the morphogenetic furrow, whereas those that reenter the cell cycle join clusters at a later stage as late-born neurons. Therefore, in gain of function Epidermal growth factor receptor Drosophila mutants, more cells in the eye imaginal disc remain in the cell cycle and fewer ommatidia are formed. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors, it is proposed that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina (Ahmad, 1998).

Sp1 (Drosophila homolog: Buttonhead) nuclear levels have been shown to directly correlate with the proliferative state of the cell. Changes in the abundance of Sp1 were studied in a rat pituitary cell line (GH4) whose growth rate is regulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nM EGF for at least 16 h show a 50% decrease in Sp1 binding to a GC-rich DNA sequence element present in the gastrin promoter. The decrease in binding correlates with a decrease in cell proliferation, a loss of nuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancer element in transfection assays. Okadaic acid, a phosphatase inhibitor, is synergistic with the effect of EGF on Sp1 protein levels, suggesting that the loss of Sp1 is mediated by phosphorylation events. A 2-fold increase in orthophosphate-labeled Sp1 occurs with EGF treatment an okadaic acid. Cycloheximide prevents the expected loss of Sp1 mediated by EGF and okadaic acid. This suggests that the synthesis of a protease may mediate these events. This hypothesis was tested directly by showing that the cysteine protease inhibitor leupeptin prevents Sp1 degradation. Sp1 has a domain with a high concentration of proline, glutamic acid, serine, and threonine residues, as reported for a number of proteins with inducible rates of degradation. Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade of phosphorylation events that promotes Sp1 proteolysis, decreases Sp1 nuclear levels and decreases cellular proliferation (Mortensen, 1997).

Table of contents

spitz: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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