Wilson O. Endege

Downregulation of vascular endothelial growth factor receptors by tumor necrosis factor-alpha in cultured human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) potently stimulates angiogenesis, whereas TNF-alpha has both pro- and anti-angiogenic activity. By measuring thymidine uptake, we found that TNF-alpha blocked a 2.3-fold increase in DNA synthesis induced by VEGF in human endothelial cells. To explore the possibility that the two interact to regulate endothelial cell proliferation, we examined the effect of TNF-alpha on VEGF receptor expression. In venous and arterial endothelial cells, TNF-alpha potently reduced mRNA transcripts of the two VEGF receptors (KDR/flk-1 and flt-1) in a dose- and time-dependent fashion. TNF-alpha at 1 ng/ml induced maximal inhibition of mRNA expression, which fell by approximately 70% after 24 h. TNF-alpha treatment did not significantly affect the KDR/flk-1 half-life but did decrease its rate of transcription to 40% of control. The decrease in KDR/flk-1 mRNA depended partially on new protein synthesis and was abolished by phorbol ester pretreatment. TNF-alpha decreased the amount of 35S-labeled KDR/flk-1 immunoprecipitated by an antibody specific for KDR/flk-1 to 18% of control. We conclude that TNF-alpha downregulates expression of both VEGF receptors in human endothelial cells and that this effect is transcriptional (at least for KDR/flk-1). These data support the hypothesis that TNF-alpha exerts its antiangiogenic effect in part by modulating the VEGF-specific angiogenic pathway.

Disappearance of cyclin A correlates with permanent withdrawal of cardiomyocytes from the cell cycle in human and rat hearts.

The regulated expression of cyclins controls the cell cycle. Because cardiomyocytes in adult mammals withdraw permanently from the cell cycle and thus cannot regenerate after injury, we examined cyclin expression during development by comparing cyclin A-E mRNA levels in fetal and adult human hearts. Cyclin B mRNA was detectable in adult hearts, although at a level markedly lower than that in fetal hearts. Levels of cyclin C, D1, D2, D3, and E mRNA were essentially identical in the two groups. In contrast, cyclin A mRNA was undetectable in adult hearts whereas cyclin A mRNA and protein were readily detectable in fetal hearts and cardiomyocytes, respectively. We then measured cyclin A mRNA and protein levels in rat hearts at four stages of development (fetal and 2, 14, and 28 d). Cyclin A mRNA and protein levels decreased quickly after birth (to 37% at day 2) and became undetectable within 14 d, an observation consistent with reports that cardiomyocytes stop replicating in rats by the second to third postnatal week. This disappearance of cyclin A gene expression in human and rat hearts at the time cardiomyocytes become terminally differentiated suggests that cyclin A downregulation is important in the permanent withdrawal of cardiomyocytes from the cell cycle.

Collagen VIII is expressed by vascular smooth muscle cells in response to vascular injury

To identify genes involved in vascular remodeling, we applied differential mRNA display analysis to the rat carotid artery balloon injury model. One polymerase chain reaction product showing increased expression at days 2 to 14 after vascular injury was nearly identical to the mouse alpha 1 chain of type VIII collagen, a heterotrimeric short-chain collagen of uncertain function expressed by a limited number of cell types. By Northern analysis, expression of both chains of the type VIII collagen heterotrimer increased: collagen alpha 1 (VIII) mRNA expression was almost 4-fold higher than control by 7 days after vascular injury, and collagen alpha 2 (VIII) mRNA expression reached a maximum of almost 6-fold above baseline by 3 days after injury. By immunohistochemical analysis, type VIII collagen expression increased in the media and neointima in a localized pattern consistent with the distribution of activated dedifferentiated vascular smooth muscle cells (VSMCs). Cultured VSMCs expressed higher levels of type VIII collagen in response to serum and growth factors, notably platelet-derived growth factor (PDGF)-BB. VSMCs adhered significantly less to type VIII collagen than to type I collagen substrata and showed greater PDGF-BB-stimulated migration (by 2.2-fold) on type VIII collagen than on type I collagen. We hypothesize that increased expression of type VIII collagen by VSMCs after arterial injury may contribute to vascular remodeling through the promotion of VSMC migration.

Degradation of E2A Proteins through a Ubiquitin-conjugating Enzyme, UbcE2A

The helix-loop-helix E2A proteins (E12 and E47) govern cellular growth and differentiation. To identify binding partners that regulate the function of these ubiquitous transcription factors, we screened for proteins that interacted with the C terminus of E12 by the yeast interaction trap. UbcE2A, a rat enzyme that is highly homologous to and functionally complements the yeast ubiquitin-conjugating enzyme UBC9, was identified and cloned. UbcE2A appears to be an E2A-selective ubiquitin-conjugating enzyme because it interacts specifically with a 54-amino acid region in E47-(477-530) distinct from the helix-loop-helix domain. In contrast, most of the UbcE2A protein is required for interaction with an E2A protein. The E2A proteins appear to be degraded by the ubiquitin-proteasome pathway because the E12 half-life of 60 min is extended by the proteasome inhibitor MG132, and E12 is multi-ubiquitinated in vivo Finally, antisense UbcE2A reduces E12 degradation. By participating in the degradation of the E2A proteins, UbcE2A may regulate cell growth and differentiation.

APEG-1, a Novel Gene Preferentially Expressed in Aortic Smooth Muscle Cells, Is Down-regulated by Vascular Injury

Despite the importance of phenotypic alterations in arterial smooth muscle cells (ASMC) during the pathogenesis of arteriosclerosis, little is known about genes that define differentiated ASMC. Using differential mRNA display, we isolated a novel gene preferentially expressed in the rat aorta and termed this gene APEG-1. The cDNA of rat APEG-1 contained an open reading frame encoding 113 amino acids, which would predict a basic protein of 12.7 kDa. The amino acid sequence of rat APEG-1 was highly conserved among human and mouse homologues (97 and 98%, respectively). Using an APEG-1 fusion protein containing an N-terminal c-Myc tag, we identified APEG-1 as a nuclear protein. By in situ hybridization, APEG-1 mRNA was expressed in rat ASMC. Although APEG-1 was expressed highly in differentiated ASMC in vivo, its expression was quickly down-regulated and disappeared in dedifferentiated ASMC in culture. In vivo, APEG-1 mRNA levels decreased by more than 80% in response to vascular injury as ASMC changed from a quiescent to a proliferative phenotype. Taken together, these data indicate that APEG-1 is a novel marker for differentiated ASMC and may have a role in regulating growth and differentiation of this cell type.

Interferon-γ-mediated Inhibition of Cyclin A Gene Transcription Is Independent of Individual cis-Acting Elements in the Cyclin A Promoter

Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-γ on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-γ-treated VSMCs. IFN-γ treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-γ treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-γ treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-β1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-γ treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-γ inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.

The Polymyositis-Scleroderma Autoantigen Interacts with the Helix-Loop-Helix Proteins E12 and E47

The basic helix-loop-helix (bHLH) transcription factors E12 and E47 regulate cellular differentiation and proliferation in diverse cell types. While looking for proteins that bind to E12 and E47 by the yeast interaction trap, we isolated the rat (r) homologue of the human (h) polymyositis-scleroderma autoantigen (PM-Scl), which has been localized to the granular layer of the nucleolus and to distinct nucleocytoplasmic foci. The rPM-Scl and hPM-Scl homologues are 96% similar and 91% identical. We found that rPM-Scl mRNA expression was regulated by growth factor stimulation in cultured rat aortic smooth muscle cells. rPM-Scl bound to E12 and E47 but not to Id3, Gax, Myb, OCT-1, or Max. The C terminus of rPM-Scl (amino acids 283–353) interacted specifically with a 54-amino acid domain in E12 that is distinct from the bHLH domain. Finally, cotransfection of rPM-Scl and E47 specifically increased the promoter activity of a luciferase reporter construct containing an E box and did not affect the basal activity of the reporter construct. rPM-Scl appears to be a novel non-HLH-interacting partner of E12/E47 that regulates E2A protein transcription.

Evaluation of Tyro3 expression, Gas6-mediated Akt phosphorylation, and the impact of anti-Tyro3 antibodies in melanoma cell lines.

Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.