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EGF and TGF-alpha are mitogenic

Transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) is required for the establishment of small colonies of human keratinocytes at clonal densities, but once small (10-15 cells) colonies have formed, the continued growth of these colonies can proceed in the absence of exogenous TGF-alpha or EGF. Equivalent receptor-binding concentrations of TGF-alpha and EGF are equipotent in stimulating colony formation. The growth of keratinocytes at high densities proceeds in the absence of exogenous peptide growth factors or hormones. The expression of TGF-alpha mRNA and protein is regulated by both cell density and the presence of exogenous growth factors. The addition of an antibody, which blocks the mitogenic effect of mature TGF-alpha, has no effect on the autocrine/paracrine growth of these cells at either density. However, monoclonal antibodies that antagonize ligand activation of the EGF receptor inhibit the autonomous proliferation of keratinocytes at high density and abrogate the exogenous TGF-alpha/EGF-independent expansion of colonies at clonal density. The results of these experiments are among the first to demonstrate that normal human epithelial cells in culture exhibit autocrine/paracrine-mediated proliferation. Exogenous growth factors initiate colonies of human keratinocytes that become self-perpetuating in culture. Keratinocytes regulate production of the mitogenic ligand, TGF-alpha, through a density-dependent mechanism, and cell density stringently controls proliferation (Pittelkow, 1993).

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) elicit quantitatively different cell proliferation responses even though they act via a common receptor, the epidermal growth factor receptor (EGFR). It is hypothesized that differential cellular trafficking of available ligand is responsible for the different mitogenic responses elicited by EGF and TGF alpha. Mitogenesis and ligand depletion were determined simultaneously in NR6 mouse fibroblasts expressing either wild-type (WT) or internalization-deficient cytoplasmic domain-truncated EGFR. The effects of both ligand-induced and low level constitutive ligand/receptor processing could be determined. For a given initial amount of growth factor, TGF alpha is a weaker stimulus than EGF in cells expressing either form of the EGFR. This difference in the mitogenic potencies correlates with increased depletion of TGF alpha observed during the growth assays. When this difference in ligand depletion is accounted for, or minimized, EGF and TGF alpha elicit quantitatively similar growth responses. Therefore, the relative mitogenic potencies of EGF and TGF alpha depend on ligand availability, as determined by the cellular trafficking of these ligands in conjunction with environmental circumstances. Interestingly, TGF alpha can be a less potent mitogenic stimulus than EGF under conditions where ligand availability is limited. Further, differences in ligand processing are sufficient to explain the different mitogenic potencies of these growth factors in either of the receptor trafficking scenarios. These results suggest a model of regulation of hormone responsiveness that favors dissociative ligands (such as TGF alpha) in receptor-limited situations and non-dissociative ligands (such as EGF) in the face of high receptor levels (Reddy, 1996).

Nerve growth factor (NGF) functions as a progression factor with both mitogenic and antimitogenic activities. When PC12 cells are treated with NGF, they advance to the G1 stage of the cell cycle before they differentiate. The correlation between cessation of proliferation and differentiation suggests that the antimitotic activity of NGF may be obligatory for differentiation. Although epidermal growth factor- (EGF) and NGF-treated PC12 cells share several common properties, including activation of the mitogen-activated protein (MAP) kinase pathway and induction of immediate early genes, EGF is mitogenic for PC12 cells and does not normally stimulate differentiation. However, combinations of EGF and low levels of cAMP stimulate differentiation even though neither agent alone does. Since EGF is mitogenic for PC12 cells and differentiation may not occur until proliferation is inhibited, differentiation caused by cAMP and EGF may be due to the antiproliferative activity of cAMP. To test this hypothesis, the effect of EGF or combinations of EGF and cAMP were examined on PC12 cell proliferation. EGF alone stimulates proliferation of PC12 cells and increases the levels of several cell cycle progression factors including cdk2, cdk4, and cyclin B1. Cyclic AMP inhibits the EGF-stimulated increases in cell cycle progression factors as well as proliferation. Other antiproliferative agents (including rapamycin, mimosine, and nitric oxide agonists) also synergize with EGF to stimulate differentiation. These data indicate that the coupling of antiproliferative signals with EGF modifies the biological properties of EGF and converts it to a differentiating growth factor (Mark, 1997).

Simultaneous addition of both TGF-alpha and TGF-beta induces the sustained, long-term outgrowth of chicken erythrocytic progenitor cells, referred to as T2ECs from both chick bone marrow and 2-day-old chicken embryos. By analysis for differentiation antigens and gene expression, these cells have been shown shown to represent very immature hematopoietic progenitors committed to the erythrocytic lineage. T2ECs differentiate into almost pure populations of fully mature erythrocytes within 6 days, when TGF-alpha and TGF-beta are withdrawn and the cells exposed to anemic chicken serum plus insulin. Outgrowth of these cells from various sources invariably requires both TGF-alpha and TGF-beta, as well as glucocorticoids. Proliferating, established T2ECs still require TGF-alpha, but are independent of exogenous TGF-beta. Using a TGF-beta-neutralizing antibody or expressing a dominant-negative TGF-beta receptor II, it has been demonstrated that T2ECs generate an autocrine loop involving TGF-beta during their establishment, which is required for sustained proliferation. Using specific inhibitors, it is also shown that signaling via Mek-1 is specifically required for induction and maintenance of cell proliferation driven by cooperation between the TGF-alpha and -beta receptors. These results establish a novel mechanism by which self-renewal of erythrocytic progenitors is induced and avian T2ECs are established as components of a new, quasi-optimal model system to study erythrocytic progenitors (Gandrillon, 1999).

The subventricular zone (SVZ) of the adult mammalian forebrain contains kinetically distinct precursor populations that contribute new neurons to the olfactory bulb. Because among forebrain precursors there are stem-like cells that can be cultured in the presence of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), it was asked whether distinct subsets of stem-like cells coexist within the SVZ or whether the proliferation of a single type of SVZ stem-like cell is controlled by several GFs. The latter is shown to be the case. Thus cells isolated from the SVZ coexpress the EGF and FGF receptors; by quantitative analysis, the number of stem-like cells isolated from the SVZ by either FGF2 or EGF is the same, whereas no additive effect occurs when these factors are used together. Furthermore, short-term administration of high-dose [3H]thymidine in vivo depletes both the EGF- and FGF2-responsive stem-like cell populations equally, showing they possess closely similar proliferation kinetics and likely belong to the constitutively proliferating SVZ compartment. By subcloning and population analysis, it has been demonstrated that responsiveness to more than one GF endows SVZ cells with an essential stem cell feature: the ability to vary self-renewal, which has been until now undocumented in CNS stem-like cells. The multipotent stem cell-like population that expands slowly in the presence of FGF2 in culture switches to a faster growth mode when exposed to EGF alone and expands even faster when exposed to both GFs together. Analogous responses are observed when the GFs are used in the reverse order, and furthermore, these growth rate modifications are fully reversible (Gritti, 1999).

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. Neural stem cells that have the capacity to self-renew and differentiate into neurons and glia can be cultured from the adult SVZ. These cells grow as spherical floating clusters (neurospheres) in the presence of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF). The SVZ germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, it has been shown that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. It is concluded that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain (Doetsch, 1999).

Many astrocytes express EGF and bFGF receptors. Interestingly, using these growth factors, neural stem cells can be isolated in vitro not only from the adult SVZ but also from other brain regions, including the spinal cord, diencephalon, and hippocampus. The results described here raise the intriguing possibility that neural stem cells that have been cultured from other brain regions may actually be derived from astrocyte-like cells in vivo. Astrocytes encompass a heterogeneous population of cells that are widely distributed in the adult brain and continue to divide in situ. After injury, astrocytes proliferate to form glial scars but not neurons. It is possible that a neurogenic potential is latent in many astrocytes throughout the CNS but that inhibitory signals may suppress these cells from producing neurons. Neurogenic factors may only be present close to the brain ventricles or within the adult SVZ. Alternatively, SVZ astrocytes that act as stem cells in vitro and generate neurons in vivo may correspond to a fundamentally different cell type that resembles astrocytes and expresses GFAP and other glial markers. If this is the case, then this work suggests that use of markers such as GFAP, a hallmark of glial cells, may not be a reliable indication of fully differentiated glia. Further work is required to characterize those SVZ astrocytes that can act as primary neuronal precursors and to determine how they differ from other astrocytes within the SVZ and the rest of the brain (Doetsch, 1999 and references).

During neural development, astrocytes are derived from radial glia. Adult cortical astrocytes assume the characteristics of radial glia upon exposure to embryonic brain extracts. Interestingly, radial glia divide and have been hypothesized to function as neuronal precursors. The identification of SVZ astrocytes as neuronal precursors in the adult brain further suggests that what has classically been considered an astrocytic lineage, including radial glia, may in fact correspond to embryonic and adult neuroepithelial cells that retain some of the properties of neural stem cells. The present results demonstrate that SVZ astrocytes are the in vivo primary precursors for new neurons during regeneration and under normal conditions. Furthermore, neurospheres in vitro can be derived from SVZ astrocytes labeled in vivo, indicating that these cells can act as neural stem cells. Exploitation of the regenerative capacity and neurogenic potential of SVZ astrocytes may have powerful implications for brain repair (Doetsch, 1999).

EGF and stem cells

Neural stem cells in the subventricular zone (SVZ) continue to generate new neurons in the adult brain. SVZ astrocytes (Type B cells) function as the primary precursors of rapidly dividing transit-amplifying Type C cells (secondary precursors), which generate neuroblasts (Type A cells) destined for the olfactory bulb. SVZ cells exposed to EGF in culture grow to form neurospheres that are multipotent and self-renewing. The majority of these EGF-responsive cells are not derived from relatively quiescent stem cells in vivo, but from the highly mitotic, Dlx2+, transit-amplifying C cells. When exposed to EGF, C cells downregulate Dlx2, arrest neuronal production, and become highly proliferative and invasive. Killing Dlx2+ cells dramatically reduces the in vivo response to EGF and neurosphere formation in vitro. Furthermore, purified C cells are 53-fold enriched for neurosphere generation. It is concluded that transit-amplifying cells retain stem cell competence under the influence of growth factors (Doetsch, 2002).

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

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