Thyroid hormone receptor/c-erbA: control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12

The c-erbA proto-oncogenes encode nuclear receptors for thyroid hormone (T3), a hormone intimately involved in mammalian brain maturation. To study thyroid hormone receptor (TR) action on neuronal cells in vitro, we expressed the chicken c-erbA/TR alpha-1 as well as its oncogenic variant v-erbA in the adrenal medulla progenitor cell line PC12. In the absence of T3, exogenous TR alpha-1 inhibits NGF-induced neuronal differentiation and represses neuron-specific gene expression. In contrast, TR alpha-1 allows normal differentiation and neuronal gene expression to occur in the presence of T3. Finally, TR alpha-1- expressing cells become NGF-responsive for proliferation when T3 is absent, but NGF-dependent for survival in presence of T3. A similar differentiation induction by NGF plus T3 was observed in a central nervous system-derived neuronal cell line (E 18) expressing exogenous TR alpha-1. Together with the finding that TR alpha-1 constitutively blocked dexamethasone-induced differentiation of PC12 cells into the chromaffin pathway, these results suggest that TR alpha-1 plays an important role in regulating commitment and maturation of neuronal progenitors. In contrast, the v-erbA oncogene, a mutated, oncogenic version of TR alpha-1, partially but constitutively inhibited NGF- induced neuronal differentiation of PC12 cells and potentiated dexamethasone-induced chromaffin differentiation, giving rise to an aberrant "interlineage" cell phenotype.     

Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR.

V-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways.     

A single point mutation in erbA restores the erythroid transforming potential of a mutant avian erythroblastosis virus (AEV) defective in both erbA and erbB oncogenes.

We have characterized the v-erbA and v-erbB oncogenes of td359, a transformation-defective mutant of avian erythroblastosis virus (AEV) unable to transform erythroblasts, and the revertant r12, obtained after in vivo passage of the mutant. Molecular cloning, sequencing, construction of chimeric viruses and testing of their oncogenic capacities revealed that both oncogenes of td359 are mutated and biologically defective. The r12 virus, although still containing a mutant v-erbB gene, recovered its erythroid transforming potential by acquiring a highly active gag-erbA gene. These results demonstrate that two co-operating oncogenes, an active v-erbA and a defective v-erbB, can transform a cell type not transformed by either oncogene alone. Furthermore, a single amino acid substitution inactivated the td359 v-erbA protein and we show that its reversion led to the reactivation of the protein. This lesion is located in the same region as several previously described inactivating mutations of glucocorticoid receptors, suggesting that the structure/function relationship of the virally transduced form of the c-erbA/thyroid hormone receptor is closely similar to that of steroid hormone receptors.     

Genetic dissection of functional domains within the avian erythroblastosis virus v-erbA oncogene.

The avian erythroblastosis virus v-erbA locus potentiates the oncogenic transformation of erythroid and fibroblast cells and is derived from a host cell gene encoding a thyroid hormone receptor. We report here the use of site-directed mutagenesis to identify and characterize functional domains within the v-erbA protein. Genetic lesions introduced into a putative hinge region or at the extreme C-terminus of the v-erbA coding domain had no significant effect on the biological activity of this polypeptide. In contrast, mutations introduced within the cysteine-lysine-arginine-rich center of the v-erbA coding region, a DNA-binding domain in the thyroid and steroid hormone receptors, abolished or severely compromised the ability of the viral protein to function. Our results suggest that the mechanism of action of the v-erbA protein in establishing the neoplastic phenotype is closely related to its ability to interact with DNA, presumably thereby altering expression of host target genes by either mimicking or interfering with the action of the normal c-erbA gene product.     

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells

To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon nuclear expression of the cTR-alpha protein the cells become responsive to thyroid hormone, as detected by expression of a number of genes (malic enzyme, phosphoenolpyruvate carboxykinase, and Na+/K(+)-ATPase) reported to be indirectly induced by the hormone in vivo. In addition, our data show that the c-erbA product directly activates the Moloney murine leukemia virus promoter in a ligand-dependent manner. The data show that the chicken c-erbA-alpha protein can modulate the expression of rat genes under either direct or indirect control by thyroid hormone.     

EGF-R as a hemopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.     

Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo

In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.     

The estrogen receptor cooperates with the TGF alpha receptor (c-erbB) in regulation of chicken erythroid progenitor self-renewal.

A unique combination of growth promoting factors is described that allows growth of large amounts (10(10)-10(11)) of normal erythroid progenitors from chick bone marrow. These erythroid progenitors express the estrogen receptor (ER) as well as the receptor tyrosine kinase TGF alpha R/c-erbB. They require both TGF alpha and estradiol for sustained self-renewal in vitro, but terminally differentiate upon withdrawal of TGF alpha and inactivation of the ER by an antagonist (ICI 164.384). Overexpression of the human ER in erythroblasts devoid of endogenous ER revealed that the hormone-activated ER alone arrested erythroid differentiation and repressed a large group of erythrocyte genes. When similarly overexpressed, TGF alpha R/c-erbB inhibited the expression of a distinct, but overlapping, set of genes. The endogenous ER and TGF alpha R/c-erbB affect erythrocyte gene expression in a similar, but less pronounced fashion. Surprisingly, suppression of ER function by antagonist efficiently inhibited erythroblast transformation by tyrosine kinase oncogenes, suggesting a role of the endogenous ER in leukemogenesis. We speculate that the oncogenes v-erbB and v-erbA cooperate in erythroleukemia induction by a mechanism that is employed by TGF alpha R/c-erbB and ER to regulate normal progenitor self-renewal in response to external signals.     

v-erbA oncogene function in neoplasia correlates with its ability to repress retinoic acid receptor action.

The v-erbA oncoprotein of avian erythroblastosis virus is an aberrant version of a thyroid hormone receptor and functions in neoplasia by blocking erythroid differentiation and by modifying the growth properties of fibroblasts. v-erbA has been proposed to represent a novel dominant negative oncogene, acting in the cancer cell by interfering with the actions of its normal cell homologs, the thyroid hormone receptors. We report here that v-erbA can actually interfere with the actions of a variety of members of the steroid/retinoid receptor family and that the ability of v-erbA to act in neoplasia best correlates not with suppression of c-erbA action, but with interference with the retinoic acid receptor response. We suggest that v-erbA may act in neoplasia by promiscuously interfering with a retinoid-mediated differentiation process.