Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRß1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRß1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRß1 population were significantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRß1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.

V-erbA generates ribosomes devoid of RPL11 and regulates translational activity in avian erythroid progenitors.

The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.

Transcription factor NF-Y is a functional regulator of the transcription of core clock gene Bmal1.

The circadian clock enables organisms to adjust to daily environmental changes and synchronize multiple molecular, biochemical, physiological, and behavioral processes accordingly. In mammalian clock work, Bmal1 is the most important core clock gene, which works with another core clock gene Clock to drive the expression of other clock genes and clock-controlled genes. However, the regulation of Bmal1 has not been fully understood. This work was aimed at identifying the positive regulator(s) of Bmal1 transcription. A series of 5' deletion reporter constructs was generated, and binding site mutations of mouse Bmal1 promoter fragments were cloned into pGL3-basic and pGL3(R2.1)-basic plasmids and transfected into NIH 3T3 cells. Luciferase activity was either measured 48 h after transfection or recorded for 4 days after serum shock. DNA affinity precipitation assay was used to detect the transcription factors binding to Bmal1 promoter. Small interfering RNA against nuclear factor Y, subunit A (NF-YA) and dominant negative NF-YA were employed to study the role of NF-Y in Bmal1 transcription regulation. Deletion and mutation analyses identified two clusters of CCAAT/GC-boxes at the proximal region of Bmal1 promoter as the activating cis-elements. Bmal1 promoter activity was up-regulated by NF-Y and/or Sp1 and repressed by dominant negative NF-YA or siRNA against NF-YA. The activation of Bmal1 promoter activity by NF-Y and Sp1 was inhibited by Rev-Erbα. DNA affinity precipitation assay showed that NF-Y and Sp1 bound to the two CCAAT/GC clusters of Bmal1 promoter. These results indicate that NF-Y is a functional activator of Bmal1 transcription and it cooperates with Sp1 and Rev-Erbα to generate the daily cycle of Bmal1 expression.

Multiple novel signals mediate thyroid hormone receptor nuclear import and export.

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor ß1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor ß1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.

Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

Recruitment of the oncoprotein v-ErbA to aggresomes.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.

The effect of oncoprotein v-erbA on thyroid hormone-regulated genes in hepatocytes and their potential role in hepatocellular carcinoma.

Mutant forms of thyroid hormone receptor (TR) with dominant negative activity are frequently found in human hepatocellular carcinoma (HCC). Interestingly, the v-erbA oncogene, known to exert a dominant-negative effect on the expression of thyroid hormone (T3)-responsive genes, led to the development of HCC in a transgenic mouse model. Thus it is possible that the oncogenic activity of v-erbA in hepatocytes may be mediated by its dominant negative activity on T3-responsive genes. Microarray analysis was used to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to T3. The Affymetrix GeneChip Mouse Genome 430 2.0 array consisted of over 39,000 transcripts representing well-known genes. We have identified twenty T3-responsive genes that are negatively regulated by v-erbA at 3 h, and eighteen genes at 24 h, such as follistatin, activin ßC, thrombomodulin, Six1, Rasgrp3 and Ndrg2, as well as genes that are regulated by v-erbA only, such as angiopoietin 1 and Igfr2. We have identified T3 responsive genes that are dysregulated by v-erbA. These genes are known to be involved in carcinogenesis. Our studies may provide insight into the potential role of mutant forms of TR in the pathogenesis of HCC.

Association of v-ErbA with Smad4 disrupts TGF-beta signaling.

Disruption of the transforming growth factor-beta (TGF-beta) pathway is observed in the majority of cancers. To further understand TGF-beta pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-beta responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-beta-induced cell growth inhibition and that dysregulation of TGF-beta signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-beta-induced growth inhibition and that TGF-beta sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-beta signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.

Modulation of expression of RA-regulated genes by the oncoprotein v-erbA.

Retinoic acid (RA) modulates the expression of genes involved in embryogenesis, development and differentiation processes in vertebrates. The v-erbA oncogene is known to exert a dominant-negative effect on the expression of RA-responsive genes. v-erbA belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid. While RA inhibits cell proliferation and promotes cell differentiation and apoptosis in a variety of tissues, v-erbA seems to play a role in oncogenesis, namely in the development of hepatocellular carcinoma (HCC) in a transgenic mouse model. In order to study the effect of v-erbA on RA-responsive genes, we used microarray analysis to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA for 3 h or 24 h. We have identified RA-responsive genes that are affected by v-erbA, as well as genes that are regulated by v-erbA alone. We have found that v-erbA can affect gene expression in the presence of RA and at the level of basal transcription. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC.

Involvement of the TGF-beta and mTOR/p70S6Kinase pathways in the transformation process induced by v-ErbA.

v-ErbA is the oncogenic form of TRalpha/c-ErbA which transforms chicken erythrocytic progenitors by blocking differentiation. The oncogenic property of v-ErbA has been correlated with its ability to antagonize ligand-dependent gene activation by TRalpha/c-ErbA and retinoic acid receptors. Nevertheless, its cytoplasmic retention suggests that v-ErbA could interfere with intracellular signaling pathways. We demonstrate that only the transforming form of v-ErbA confers to chicken erythroid progenitors a TGF-beta independency and induces an activation of the mTOR/p70S6K pathway. In these cells, TGF-beta and mTOR/p70S6K pathways regulate the expression of a known target gene of v-ErbA, band3. This is the first demonstration that v-ErbA is able to modulate specifically some signaling pathways leading to changes in the expression level of a gene involved in transformation.

Multiple mutations contribute to repression by the v-Erb A oncoprotein.

The v-Erb A oncoprotein of avian erythroblastosis virus is derived from c-Erb A, a hormone-activated transcription factor. Notably, v-Erb A has sustained multiple mutations relative to c-Erb A and functions as a constitutive transcriptional repressor. We report here an analysis of the contributions of these different mutations to v-Erb A function. Our experiments demonstrate that two amino-acid differences between v-Erb A and c-Erb A, located in the 'I-box,' alter the dimerization properties of the viral protein, resulting in more stable homodimer formation, increased corepressor binding, and increased target gene repression. An additional amino-acid difference between v- and c-Erb A, located in helix 3 of the hormone binding domain, renders corepressor binding by the viral protein more resistant to release by thyroid hormone. Finally, we report that a C-terminal truncation in v-Erb A not only inhibits exchange of corepressor and coactivator, as previously noted, but also permits v-Erb A to recruit both SMRT and N-CoR corepressors, whereas c-Erb A is selective for N-CoR. The latter two mutations in v-Erb A also impair its ability to suppress c-Jun function in response to T3 hormone. We propose that the acquisition of oncogenic potential by the v-Erb A protein was a multistep process involving a series of mutations that alter the transcriptional repressive properties of the viral protein through multiple mechanisms.

Cancer promoted by the oncoprotein v-ErbA may be due to subcellular mislocalization of nuclear receptors.

The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.

The orphan receptor Rev-erbalpha gene is a target of the circadian clock pacemaker.

Rev-erbalpha is a ubiquitously expressed orphan nuclear receptor which functions as a constitutive transcriptional repressor and is expressed in vertebrates according to a robust circadian rhythm. We report here that two Rev-erbalpha mRNA isoforms, namely Rev-erbalpha1 and Rev-erbalpha 2, are generated through alternative promoter usage and that both show a circadian expression pattern in an in vitro system using serum-shocked fibroblasts. Both promoter regions P1 (Rev-erbalpha1) and P2 (Rev-erbalpha2) contain several E-box DNA sequences which function as response elements for the core circadian-clock components: CLOCK and BMAL1. The CLOCK-BMAL1 heterodimer stimulates the activity of both P1 and P2 promoters in transient transfection assay by 3-6-fold. This activation was inhibited by the overexpression of CRY1, a component of the negative limb of the circadian transcriptional loop. Critical E-box elements were mapped within both promoters. This regulation is conserved in vertebrates since we found that the CLOCK-BMAL1 heterodimer also regulates the zebrafish Rev-erbalpha gene. In line with these data Rev-erbalpha circadian expression was strongly impaired in the livers of Clock mutant mice and in the pineal glands of zebrafish embryos treated with Clock and Bmal1 antisense oligonucleotides. Together these data demonstrate that CLOCK is a critical regulator of Rev-erbalpha circadian gene expression in evolutionarily distant vertebrates and suggest a role for Rev-erbalpha in the circadian clock output.

V-erba homodimers mediate the potent dominant negative activity of v-erba on everted repeats.

The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.

Nuclear export of the oncoprotein v-ErbA is mediated by acquisition of a viral nuclear export sequence.

v-ErbA, an oncogenic derivative of the thyroid hormone receptor alpha (TRalpha) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRalpha and another isoform, TRbeta, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRalpha has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA.

Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

Sequences required for the transition from monomeric to homodimeric forms of thyroid hormone receptor alpha and v-erbA.

Thyroid hormone receptor alpha (TRalpha) and the oncoprotein v-erbA (a mutated form of TRalpha incapable of binding T3) bind as heterodimers with retinoid X receptor (RXR) to DNA sequences with different orientations of AGGTCA half sites. v-erbA can also form homodimers, whereas, TRalpha1 homodimerizes poorly. Therefore, in order to obtain a better understanding for the distinct homodimerization properties between TRalpha1 and v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on direct and everted repeats (DRs, ERs). We found that the enhanced homodimerization properties of v-erbA compared to TRalpha1 map to isoleucine at position 339 in conjunction with serine at position 351 and alanine at position 358. Our data indicate that the methyl group in isoleucine at position 339 plays an important role in v-erbA homodimerization, particularly on ER 6. Functional studies with I339V+S351P+A358T, a v-erbA mutant unable to homodimerize but still able to heterodimerize with RXR on ERs and DRs, indicate that v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on DRs. However, the repressor activity of this mutant is weaker than that of the wild type v-erbA on ERs, suggesting that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the potent dominant negative activity of v-erbA on ERs.

Dimerization of v-erbA on inverted repeats.

Thyroid hormone receptors (TRs) and the oncoprotein v-erbA can heterodimerize with retinoid X receptor (RXR) on core motifs arranged as inverted repeats (IR0) which contain the consensus sequence AGGTCA. On this core motif, v-erbA can also form homodimers whereas TRs homodimerize very poorly. Therefore to obtain a better understanding of distinct homodimerization properties of TR alpha 1 as compared to those of v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on IR0. We found that the enhanced homodimerization properties of v-erbA compared to those of TR alpha 1 on IR0 map to amino acids 107-156 in v-erbA/121-170 in TR alpha 1 (VT-2 chimera). Furthermore, functional studies on transient transfections showed that v-erbA-RXR heterodimers do not mediate the dominant negative activity of v-erbA on an inverted repeat response element. These data, in conjunction with our previous studies, indicate that v-erbA homodimers mediate the repressor activity of v-erbA on IR0.

Sarcoma and thyroid disorders: a common etiology?

We have recently observed that many of our sarcoma patients presented also with thyroid disorders. Literature data are almost unavailable on this topic. The relationship between the sarcoma and thyroid disorders is examined. Retrospective analysis of files of patients with sarcoma and clinically overt thyroid disorders was carried out. Of the 375 patients with soft tissue sarcomas (STS) and 235 with bone sarcoma (BS) including small blue round cell tumors (SBRC), 28 patients (4.6%) had an associated significant thyroid disorder. The types of sarcoma were mainly liposarcoma followed by malignant fibrous histiocytoma, leiomyosarcoma and bone sarcoma. The primary sites were mainly limb and trunk. The interval between the diagnosis of the thyroid disorder and the sarcoma varied between -14 years (thyroid first) and +16.5 years (thyroid later) with a median of -0.2 years. Thyroid disorders included goiter, thyroiditis and carcinoma. There are both basic-science and clinical evidence to a possible common pathway that leads to the association between overt thyroid disorders and sarcomas of bone or soft tissues. Oncogene erbA activity is related to thyroid receptors to T3 and to development of sarcoma. Cross talk of the sarcoma oncogene and the erbA might contribute to the development of sarcoma. The thyroid hormone receptor and the highly related viral oncoprotein v-erbA are found exclusively in the nucleus as stable constituents of chromatin. It has been shown that v-erbA can block the spontaneous differentiation in erythroid cells transformed by various retroviral oncogenes. V-erbA can alter the spectrum of neoplasia induced by the v-src oncogene, which causes predominantly sarcomas and erythroblastosis in chicks. The erbA can cooperate with other oncogenes such as v-erbB or with v-fms, v-ras, and c-kit. Cooperation with v-myc may play a role in the development of rhabdomyosarcoma especially in thyroid hormone deficiency state. The possible clinical implications are the need to screen patients with sarcoma to thyroid disorders, and patients with thyroid disorders for malignant diseases.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.