endothelin-1 and Cell-Transformation--Viral

Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.

Topics: Adaptor Proteins, Signal Transducing; Adenoviridae; Cell Line, Transformed; Cell Transformation, Viral; Endothelin-1; Gene Transfer Techniques; Glomerular Mesangium; Guanine Nucleotide Exchange Factors; Humans; Mesangial Cells; Proto-Oncogene Proteins c-crk

Beta1Pix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor (GEF) for the Rho family small G protein Cdc42/Rac. On stimulation with extracellular signals, GEFs induce the exchange of guanosine diphosphate to guanosine triphosphate, resulting in the activation of the small guanosine 5C-triphosphatases. This activation enables the signal to propagate to downstream effectors. Herein, we show that G(salpha) stimulation by cholera toxin increased Cdc42 activation by endothelin-1 (ET-1), whereas pertussis toxin had no effect. H-89, a protein kinase A (PKA) inhibitor, strongly inhibited Cdc42 activation by ET-1. Moreover, the overexpression of beta1Pix enhanced ET-1-induced Cdc42 activation. The essential role of beta1Pix in ET-1-induced Cdc42 activation was evidenced by the blocking of Cdc42 activation in cells expressing beta1Pix mutant lacking the ability to bind PAK (beta1Pix SH3m[W43K]) or mutant lacking GEF activity (beta1PixdeltaDH). The overexpression of mutant lacking the pleckstrin homology domain beta1PixdeltaPH, which is unable to bind phospholipids, had no effect on Cdc42 activation. These results demonstrate that beta1Pix, along with PKA, plays a crucial role in the regulation of Cdc42 activation by ET-1.

Topics: cdc42 GTP-Binding Protein; Cell Cycle Proteins; Cell Line, Transformed; Cell Transformation, Viral; Cholera Toxin; Endothelin-1; GTP-Binding Protein alpha Subunits, Gs; Guanine Nucleotide Exchange Factors; Humans; Mesangial Cells; Rho Guanine Nucleotide Exchange Factors

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), alpha-melanocyte-stimulating hormone (MSH), and the endogenous opioid, beta-endorphin (EP). ACTH-dependent Cushing's syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing's patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and beta-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing's syndrome and cardiovascular diseases.

Topics: Adenoviridae; Adenovirus E1A Proteins; Adrenocorticotropic Hormone; beta-Endorphin; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Endothelial Cells; Endothelin-1; Gene Transfer Techniques; Genetic Vectors; Green Fluorescent Proteins; Humans; Neovascularization, Physiologic; Pro-Opiomelanocortin; Recombinant Proteins; Serotyping

Endothelin (ET)-1 is an angiogenic factor that, among others, is secreted by endothelial cells during development of several neoplasias. In particular, Kaposi sarcoma (KS) skin lesions show overexpression of the ET-1 system. Spindle cells, which characterize tumor lesions, are of endothelial origin and during disease are infected by human herpesvirus 8 (HHV-8). The majority of these cells are latently infected, suggesting that latent genes are sufficient for maintenance of viral infection and development of KS. The establishment of a reliable infection system is required to better understand the role of viral and cellular angiogenic factors involved in KS progression. For this purpose, we used human microvascular endothelial cells (HMEC-1) to establish an ET-1-producing model of infection with HHV-8. Viral particles purified from BCBL-1 cells were used to infect HMEC-1 monolayer, and infection was assessed by polymerase chain reaction, reverse transcription polymerase chain reaction, and confocal microscopy. Mitochondrial activity and cell viability, measured at 24, 48, and 72 hours after infection by 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was reduced in HHV-8-infected cells compared with control. In contrast, 1 week after infection, HHV-8-positive cells showed higher mitochondrial functionality. Endothelin production was measured in culture media collected at 24, 48, and 72 hours after infection. The levels of endothelin precursor big endothelin-1 was increased 3 days after infection, although big ET-1 and ET-1 production did not differ significantly between infected and uninfected cells. These results indicate this model as a useful tool to further characterize the effects of HHV-8 in the early and late phases of infection, and to determine its ability to interfere with the endothelin system.

Topics: Cell Line, Transformed; Cell Transformation, Viral; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Herpesviridae Infections; Herpesvirus 8, Human; Humans; Interleukin-6

Simian virus 40 (SV40) is an oncogenic DNA virus that induces malignant transformation. Endothelin (ET), a 21 amino acid peptide with mitogenic and anti-apoptotic effects, binds to G-protein coupled ETA and ETB receptors. This report examines the effect of SV40 transformation on the expression of ET receptors. Results from receptor binding and reverse transcription (RT)-polymerase chain reaction (PCR) studies show that human lung fibroblasts IMR90 and WI38 express both ETA and ETB receptors, and that the expression of both receptors is significantly down-regulated in IMR90-SV40 and WI38-SV40, cell lines derived from IMR90 and WI38 with SV40 virus transformation. Receptor binding and RT-PCR analysis of 3A(tPA-30-1), a cell line derived from human placenta that expresses a higher level of SV40 large T-antigen at the permissive temperature (33 degrees C) than at the restrictive temperature (40 degrees C), further demonstrates that there is an inverse correlation between the expression of SV40 T-antigen and the expression of ET receptor. ET-1 and fetal bovine serum stimulate DNA synthesis in non-transformed cells; however, proliferation of transformed cells is independent of either fetal bovine serum or ET-1. We conclude that SV40 transformation down-regulates the expression of ET receptors, and that expression of ET receptors is inversely correlated with expression of SV40 large T-antigen.

Topics: Antigens, Viral, Tumor; Binding Sites; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Endothelin-1; Gene Expression Regulation; Humans; Receptors, Endothelin; RNA, Messenger; Simian virus 40

For many applications, specificity of gene expression by recombinant retroviral vectors is necessary. We wished to obtain transcriptional targeting in endothelial cells as part of an antivasculature approach to cancer treatment and have achieved specificity by using the promoter for human prepro-endothelin-1. In particular, we have inserted this heterologous promoter within the 3' long terminal repeat (LTR), replacing all viral upstream transcriptional regulatory sequences, to generate a hybrid LTR with precise fusion at the TATA box for initiation of transcription at the viral start site. Reverse transcription and integration resulted in duplication of this hybrid promoter in the 5' LTR of the provirus for transcription of the internal transgene. An important feature of our vectors is the absence of a selectable marker gene or additional promoters to avoid potential complications of silencing or interference and because selection will be inappropriate for clinical application. This vector design showed endothelial cell specificity of beta-galactosidase expression when tested on a panel of human cell lines and primary breast microvascular endothelial cells, matching the specificity of expression of the endogenous promoter. Such simplified vectors exhibiting transcriptional specificity are likely to be useful for the development of a gene therapy approach to targeting tumor vasculature.

Topics: 3T3 Cells; Animals; Cattle; Cell Line; Cell Transformation, Viral; Cloning, Molecular; Endothelin-1; Endothelins; Endothelium, Vascular; Gene Expression; Gene Targeting; Genetic Vectors; Humans; Leukemia Virus, Murine; Mice; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Precursors; Terminal Repeat Sequences; Transcription, Genetic; Tumor Cells, Cultured

Some human papillomaviruses (HPVs) such as HPV type 16 (HPV16) and HPV18 are involved in cervical carcinoma, and they can immortalize and transform keratinocytes. Endothelin-1 (ET-1) is produced in keratinocytes and has been shown to act through ETA receptors as an autocrine growth factor for keratinocytes. This study examines whether HPV16 alters the ET-1-mediated autocrine loop in human keratinocytes, providing a selective growth advantage for transformed cells. ET-1 is released in similar amounts from normal and HPV-transfected keratinocytes. All HPV-transfected cell lines express high-affinity ETA receptors. A two-fold increase in ET-1 binding sites is present in HPV16-immortalized keratinocytes, and this effect seems to be linked to the overexpression of mRNA for this receptor rather than to differences in the surface/internalized ratio of the receptors. ET-1 induces significant increases in [3H]thymidine incorporation and cell proliferation. Furthermore, HPV-transfected keratinocytes can proliferate in the absence of any growth factor added to the growth medium, and the ETA receptor antagonist BQ123 prevents this proliferation. These data suggest a new mechanism in the growth control of HPV-transformed cells mediated by the upregulation of ET-1 autocrine loop.

Topics: Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Child; Endothelin-1; Humans; Keratinocytes; Mitogens; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Receptors, Endothelin; Repressor Proteins; RNA, Messenger