Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Associated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells
© Punj et al; licensee BioMed Central Ltd. 2009
Received: 30 November 2008
Accepted: 6 August 2009
Published: 6 August 2009
Kaposi's sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS, a neoplasm characterized by proliferating spindle cells, extensive neoangiogenesis and a prominent inflammatory infiltrate. Infection of blood vascular endothelial cells with KSHV in vitro results in their spindle cell transformation, which is accompanied by increased expression of inflammatory chemokines and cytokines, and acquisition of lymphatic endothelial markers. Mimicking the effect of viral infection, ectopic expression of KSHV-encoded latent protein vFLIP K13 is sufficient to induce spindle transformation of vascular endothelial cells. However, the effect of K13 expression on global gene expression and induction of lymphatic endothelial markers in vascular endothelial cells has not been studied.
We used gene array analysis to determine change in global gene expression induced by K13 in human vascular endothelial cells (HUVECs). Results of microarray analysis were validated by quantitative RT-PCR, immunoblotting and a multiplex cytokine array.
K13 affected the expression of several genes whose expression is known to be modulated by KSHV infection, including genes involved in immune and inflammatory responses, anti-apoptosis, stress response, and angiogenesis. The NF-κB pathway was the major signaling pathway affected by K13 expression, and genetic and pharmacological inhibitors of this pathway effectively blocked K13-induced transcriptional activation of the promoter of CXCL10, one of the chemokines whose expression was highly upregulated by K13. However, K13, failed to induce expression of lymphatic markers in blood vascular endothelial cells.
While K13 may account for change in the expression of a majority of genes observed following KSHV infection, it is not sufficient for inducing lymphatic reprogramming of blood vascular endothelial cells.
Infection with Kaposi's Sarcoma (KS)-associated herpesvirus (KSHV), also known as the Human herpesvirus 8 (HHV8), has been linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease  KS is a highly vascular tumor that is induced by the infection of vascular or lymphatic endothelial cells with KSHV and is characterized by the presence of distinctive proliferating spindle-like cells, prominent neoangiogenesis and infiltration by inflammatory cells [2, 3]. The spindle cells not only represent the tumor cells in the KS lesion, but also produce a number of proinflammatory and angiogenic factors that drive the growth of the lesion . Latent infection of both micro- and macro-vascular endothelial cells with KSHV in vitro makes them acquire a spindle cell phenotype, which is accompanied by increased expression of a number of genes involved in the regulation of immune and inflammatory responses, cellular stress, apoptosis and angiogenesis [4–6]. Interestingly, KSHV infection of blood vascular endothelial cells also upregulates the expression of several of lymphatic markers, such as PROX-1, VEGFR-1, Podoplanin and XLKD1/LYVE1, which has led to the suggestion that KSHV infection results in lymphatic reprogramming of vascular endothelial cells [7–9].
The KSHV-encoded K13 protein is one of the few proteins to be expressed in latently-infected spindle cells. Although originally classified as a viral FLICE inhibitory protein (vFLIP), K13 was subsequently shown to be a potent activator of the NF-κB pathway [10–12], and to use this pathway to promote cellular survival, proliferation, transformation, cytokine secretion and KSHV latency [13–20]. Ectopic expression of K13 in human vascular endothelial cells is sufficient to transform them into spindle cells, which is accompanied by the upregulated expression of several proinflammatory cytokines and adhesion molecules known to be induced in KSHV-infected vascular endothelial cells [21, 22]. However, the effect of K13 on global gene expression in vascular endothelial cells has not been studied. It is also not clear whether ectopic expression of K13 in vascular endothelial cells, in the absence of other KSHV latent genes, is sufficient for inducing the changes in gene expression observed following infection with KSHV. To address these questions, we have examined the effect of ectopic K13 expression on global gene expression in human vascular endothelial cells (HUVECs). Our results indicate that K13 may account for change in the expression of a significant proportion of genes observed following KSHV infection. However, in contrast to KSHV infection, ectopic expression of K13 is incapable of inducing the expression of lymphatic endothelial markers.
Cells used in this study
Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Cambrex (East Rutherford, NJ) and were grown in EMB medium containing 10% FBS (fetal bovine serum) and supplemented with the bullet kit. Cells were used for experiments at passages 2 to 6. HUVECs stably transduced with an MSCVneo vector expressing a 4-Hydroxytamoxifen (4OHT)-inducible K13-ERTAM construct were selected in G418 and have been described previously . These cells were maintained under G418 selection for several passages prior to being used in the experiments to ensure that the experiments were conducted with stably transduced cells. An independent population of HUVECs stably transduced with a MSCV-hygro vector encoding the K13-ERTAM fusion construct were also generated and used to confirm the results of the microarray analysis.
Gene chip human array
We used the human genome HGU-133 plus 2.0 arrays (Affymatrix, Santa Clara, CA), an oligonucleotide-probe based gene array chip containing ~50,000 transcripts, which provides a comprehensive coverage of the whole human genome.
RNA isolation and hybridization to oligonucleotide arrays
HUVECs stably expressing empty vectors (MSCVneo and MSCV-hygro) or K13-ERTAM-encoding constructs were treated with 4OHT (50 nM) or solvent for 48 h. Total RNA was isolated using Qiagen RNeasy kit (Qiagen, Valencia, CA). Ten micrograms of total RNA was used to synthesize cDNA. T7 promoter introduced during the first strand synthesis was then used to direct cRNA synthesis, which was labeled with biotinylated deoxynucleotide triphosphate, following the manufacturer's protocol (Affymatrix, San Diego, California). After fragmentation, the biotinylated cRNA was hybridized to the gene chip array at 45°C for 16 h. The chip was washed, stained with phycoerytherin-streptavidin, and scanned with the Gene Chip Scanner 3000. After background correction, preliminary data analysis was done in the Microarray Suite 5.0 software (MAS 5.0, Stratagene, La Jolla, CA). For primary analysis we used PLIER as recommended in the work flow of software Gene Spring GX10.0 (Agilent Technologies, Santa. Clara).
Gene array data analysis
Fluorescence intensities were uploaded to the Array Assist 6.5 and Gene Spring GX10.0 (Agilent Technologies, Santa Clara) software. Data was normalized by quantitative normalization, and then transferred logarithmically for further analysis to determine changes in a particular gene induced by K13. In order to compare the changes in gene expression, the data was further normalized by using the 50 RFU fluorescence value as threshold, and statistical analysis showing fold changes was determined (p ≤ 0.05). The microarray experiment design, setup, and data have been deposited in National Center for Biotechnology Information's Gene Expression Omnibus and are accessible through GEO series accession number GSE16051.
Luciferase reporter assay
A luciferase reporter plasmid containing the CXCL10/IP-10 promoter (pGL3IP-10) was kindly provided by Dr. Dan Muruve (University of Calgary). Expression constructs for K13, K13-58AAA and MC159 have been described previously . 293T cells were transfected in a 24-well plate with various test plasmids along with the CXCL10 luciferase reporter constructs (50 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), as described previously . Cells were lysed 24-36 h later, and cell extracts were used to measure firefly luciferase and β-galactosidase activities, respectively. Luciferase activity was normalized by the β-galactosidase activity to control for differences in transfection efficiency.
Western blot analysis was performed as described previously . Primary antibody dilutions used in these experiments were Flag (1:5000; Sigma, St. Louis), COX2 (1:1000; Cayman Chemicals, Michigan), β-actin (1:5000; Sigma, St. Louis), IκB-α (1:2000: Santa Cruz, Santa Cruz, CA) and Tubulin ((1:1000; Sigma, St. Louis).
Network, gene ontology and canonical pathways analysis
Genes, which qualified in the stringent statistical tests, were used for gene ontology and pathway analysis. Expression data sets containing gene identifier and their corresponding expression values, as fold-changes, were uploaded as a tab-delimited text file to the Ingenuity pathway Analysis (IPA) software (Ingenuity systems, Mountain view, CA). Genes, which mapped to the ingenuity pathway database, were categorized based on molecular functions, gene ontology and biological processes. Each class was grouped based on their p-value. The identified genes named as focused genes were also mapped to genetic networks in the IPA database and ranked by score. The calculated probability score represented whether a collection of genes in a network could be found by chance alone.
mRNA expression assay by quantitative reverse transcript-polymerase chain reaction (qRT-PCR)
cDNA was synthesized from RNA samples by PCR RNA core kit (Applied Biosystems, Bedford, MA). Real time quantitative reverse transcript-polymerase chain reaction (qRT-PCR) with SYBER Green, using gene-specific PCR primers, was performed to verify the microarray data. Eleven genes were selected randomly and the primers used to amplify each gene are listed in the Additional File-1. Samples were run in triplicate, and PCR was performed by an ABI 7700 thermocycler (Applied Biosystems, Bedford, MA). Expression of multiple house keeping genes (GNB, β-actin, GAPDH and tubulin) were simultaneously determined for normalization, following the geNorm method . A linear regression analysis was performed and the coefficient of variation was calculated to assess a correlation between the RT-PCR and gene array results of these 11 randomly selected genes.
Multiplex analysis for cytokines and chemokines affected by K13
The LabMAP technology (Luminex) combines the principle of a sandwich immunoassay with the fluorescent-bead-based technology. A Luminex-based Multiplexed assay was purchased from Biosource International (Camarillo, CA) and used to measure the presence of cytokines and chemokines in culture supernatants of HUVEC-K13-ERTAM that had been mock-treated or treated with 4OHT for 48 h. For comparison, supernatant from HUVECs infected with KSHV for 48 h was included.
Induction of host gene expression by K13
In our previous work, we used retroviral-mediated gene transfer to generate human umbilical endothelial cells (HUVECs) with stable expression of a K13-ERTAM fusion construct in which the K13 cDNA is fused in-frame to the ligand-binding domain of a mutated estrogen receptor . The mutated estrogen receptor does not bind to its physiological ligand estrogen, but binds with very high affinity to the synthetic ligand 4OHT (4-hydroxytamoxifen) and regulates the activity of K13 in a 4OHT-dependent manner . In the absence of 4OHT treatment, the K13-ERTAM-HUVECs maintain their cobblestone appearance and are indistinguishable from the empty vector-expressing cells in their growth characteristics. However, these cells acquire spindle morphology within 24 h of induction by 4OHT treatment, thus mimicking the effect of KSHV infection .
To comprehensively identify the spectrum of genes induced by K13 expression, HUVECs with long-term stable expression of empty vector (MSCV) and K13-ERTAM were plated in parallel cultures. Each group of cells was then mock treated or treated with 4OHT for 48 h and RNA was harvested from all plates simultaneously. The RNA was then quantified and subjected to high density oligonucleotide microarray analysis using the Affymetrix HG-U133 plus 2 gene array representing ~50,000 annotated transcripts.
Identification of genes differentially affected by K13
List of most differentially regulated genes in 4OHT-treated K13 ER-HUVECs.
chemokine (C-C motif) ligand 2
chemokine (C-C motif) ligand 20
chemokine (C-C motif) ligand 5
chemokine (C-X-C motif) ligand 10
vascular cell adhesion molecule 1
selectin E (endothelial adhesion molecule 1)
superoxide dismutase 2, mitochondrial
chemokine (C-X-C motif) ligand 3
chemokine (C-X-C motif) ligand 2
chromosome 15 open reading frame 48
solute carrier family 7 (cationic amino acid transporter)
chemokine (C-X3-C motif) ligand 1
intercellular adhesion molecule 1 (CD54)
tumor necrosis factor, alpha-induced protein 6
interleukin 6 (interferon, beta 2)
colony stimulating factor 2 (granulocyte-macrophage)
interferon stimulated exonuclease gene 20 kDa
chemokine (C-X-C motif) ligand 5
laminin, gamma 2
tumor necrosis factor, alpha-induced protein 3
matrix metallopeptidase 10 (stromelysin 2)
tumor necrosis factor, alpha-induced protein 2
Epstein-Barr virus induced gene 3
tumor necrosis factor (ligand) superfamily, member 13b
phospholipase A1 member A
GTP cyclohydrolase 1 (dopa-responsive dystonia)
nuclear factor of kappa light polypeptide gene enhancer, alpha
proteasome (prosome, macropain) subunit, beta type, 9
Chemokine (C-X-C motif) 1
histone cluster 2, H2aa3
HLA-G histocompatibility antigen, class I, G
hydroxysteroid (11-beta) dehydrogenase 1
chemokine (C-X-C motif) receptor 7
major histocompatibility complex, class I, B
nuclear receptor coactivator 7
myxovirus (influenza virus) resistance 2 (mouse)
radical S-adenosyl methionine domain containing 2
TNFAIP3 interacting protein 1
tissue factor pathway inhibitor 2
receptor (chemosensory) transporter protein 4
baculoviral IAP repeat-containing 3
interleukin 7 receptor /// interleukin 7 receptor
transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)
papilin, proteoglycan-like sulfated glycoprotein
Major histocompatibility complex, class I, F
myosin, light chain kinase
extracellular link domain containing 1
chemokine (C-C motif) ligand 14
ADAM metallopeptidase with thrombospondin type 1 motif, 18
LIM domain binding 2
dual specificity phosphatase 4
ribonuclease, RNase A family, 1 (pancreatic)
periostin, osteoblast specific factor
insulin-like growth factor binding protein 5
regulator of G-protein signalling 5
uroplakin 1B /// regulator of G-protein signalling 5
regulator of G-protein signalling 4
latent transforming growth factor beta binding protein 2
C-X-C chemokine receptor type 4
Identification of K13-modulated biologically relevant networks
In order to find biological interactions among these 174 genes, pathway analysis was carried out by the Ingenuity Pathway Analysis (IPA) software tool. It was found that 156 of the 174 genes mapped to genetic networks as defined by the IPA tool (Additional File 3). These networks are based on known functional interactions between the gene products as described in the literature. The tool then associates these networks with known biological pathways. Eight networks were affected significantly by K13 expression as they had more of the identified genes present than would be expected by chance (score of >20). These networks were associated with important cancer-related cellular events such as cell death, cellular growth and proliferation, cellular movement, immune response, inflammatory diseases, immune responses and hematological diseases (Additional File 3). The pathway (#1), which is associated with cell death, inflammatory diseases and immunological diseases, was identified as the most significantly influenced by K13. This pathway contained 27 genes with a highly significant score of 51. Consistent with the known ability of K13 to activate the NF-κB pathway, the IPA identified the NF-κB pathway as a key pathway linked to the gene networks perturbed by K13 activity in HUVECs (Additional File 4). Additionally, pathway analysis by Gene Spring GX10 identified NF-κB as the major pathway linked to K13 activity (p < 0.05).
Gene Ontology of vFLIP K13 affected genes.
Molecular Function and Disease
5.18E-22 - 1.76E-05
8.19E-19 - 1.83E-05
8.38E-16 - 1.22E-05
Hematological System Development and Function
8.94E-16 - 1.83E-05
2.64E-15 - 1.83E-05
Cell-To-Cell Signaling and Interaction
3.71E-15 - 1.83E-05
2.98E-14 - 9.68E-06
Immune and Lymphatic System Development and Function
2.68E-13 - 1.76E-05
Connective Tissues Disorders
3.99E-13 - 7.43E-06
Skeletal and Muscular Disorders
3.99E-13 - 7.43E-06
Cellular Growth and Proliferation
1.64E-12 - 1.45E-05
1.27E-11 - 1.87E-05
2.47E-09 - 1.87E-05
4.14E-11 - 1.76E-05
1.73E-10 - 7.56E-06
3.41E-10 - 1.70E-05
4.29E-10 - 1.79E-05
2.34E-09 - 1.83E-05
4.54E-09 - 1.44E-05
3.20E-08 - 1.61E-06
3.29E-08 - 3.29E-08
Confirmation of microarray results by a multiplex cytokine assay (Luminex) and comparative analysis of chemokines and cytokines induced by K13 and KSHV
Luminex-based multiplex cytokine assay showing the expression of chemokines and cytokines in the supernatants of HUVEC Vector and K13-ER cells with and without 4OHT treatment and following infection with KSHV.
Vector Mock treated
Vector + 4OHT
Fold change K13-ER
Fold change KSHV
Fold change based on Microarray data
K13 stimulates the promoter of CXCL10/IP-10 via NF-κB activation
Differential modulation of lymphatic differentiation genes by K13 and KSHV
K13 is one of the few KSHV-encoded latent proteins and is consistently expressed in the KS spindle cells, the hallmark of KS lesions . Ectopic expression of K13 in human vascular endothelial cells is sufficient to make them acquire a spindle-shaped morphology that is associated with NF-κB activation and increased production of a number of genes known to be upregulated in KSHV-infected cells [21, 22]. In this study, we provide a comprehensive picture of global transcriptional changes induced by K13 in HUVECs. Our results provide the starting point for future detailed analysis of the K13-induced genes in the pathogenesis of KS.
As K13-induced spindle cell transformation of HUVECs is accompanied by their loss of proliferating potential, it is not possible to generate stable cultures of long-term proliferating HUVECs expressing K13 for the purpose of global gene expression analysis. To circumvent this problem, we took advantage of our previously characterized HUVEC-K13-ERTAM model system in which the K13 activity is dormant in the absence of 4OHT treatment . The HUVEC-K13-ERTAM maintains their normal cobblestone appearance and are indistinguishable from the control vector-expressing cells in their appearance and growth characteristics in the absence of 4OHT treatment. The lack of leakiness in this system was further confirmed by our microarray and qRT-PCR analysis, which revealed no significant difference in the gene expression profile between the vector and K13-ERTAM-expressing HUVECs in the absence of 4OHT. While 4OHT treatment significantly changed the expression of a number of genes in K13-ERTAM cells, it had no major effect on gene expression in the control vector cells, thereby confirming that that 4OHT treatment had no major effect on gene expression on its own. Another advantage of the use of this inducible model system is that allowed us to compare the effect of K13 activity on gene expression in the same cell population, thereby avoiding any artifacts due to cell-to-cell (clonal) variation.
It has been proposed that KSHV infection plays a major role in the recruitment of inflammatory cells to the KS lesions by upregulating the expression of chemotactic chemokines and, consistent with this notion, latent infection of vascular endothelial cells with KSHV has been known to upregulate the expression of several cellular chemokines, such as IL-8, GRO-1, MCP-1, NAP-2, CCL5/RANTES and CXCL16 [32, 15, 21, 18, 33, 22]. The chemokines induced by KSHV infection, such as IL-8, have been also postulated to contribute to neoangiogenesis characteristic of KS lesions. Our analysis revealed that chemokines were the most upregulated genes upon induction of K13 activity in HUVECs, which is consistent with the previous studies [21, 22] and two recent studies that were published while this manuscript was under review [34, 35]. Notable chemokines genes whose expression was increased significantly by K13 included CCL2 (71 fold), CCL20 (53 fold), CCL5/RANTES (48 fold), CXCL10 (47 fold), CCL3 (33 fold), IL8 (31 fold), CX3CL1 (22 fold), CXCL1 (7) and CXCL5 (13 fold). In addition, expression of genes encoding a number of cytokines (e.g. IL1a, IL6, IL15, IL32, CSF1, CSF2, CSF3 and EBI3) and TNF family ligands (TNFSF13B and Lymphotoxin beta) was significantly increased upon induction of K13 activity. Since chemokines and cytokines collectively represented the most upregulated genes in K13 expressing HUVECs, we confirmed their increased secretion in the supernatant of 4OHT treated K13-ERTAM cells. More importantly, a comparison with KSHV-infected cells revealed that ectopic expression of K13 is sufficient to induce most chemokines/cytokines that are induced by KSHV infection. Since K13 is one of the few KSHV genes that are expressed in latently-infected KS spindle cells, the above results support the hypothesis that K13, either alone or in combination with other viral genes, plays a key role in the up-regulation of chemokines and cytokines and subsequent recruitment of inflammatory cells to the KS lesions.
We observed significant upregulation of adhesion molecules (e.g. ICAM-1, VCAM-1 and E-Selectin), MHC-I, TAP1, TAP2 and tapasin upon induction of K13 activity, which is consistent with recent reports [22, 36]. Increased expression of adhesion molecules on vascular endothelial cells expression by K13 might synergize with chemokines to promote the recruitment of inflammatory and blood cells into KS lesions. On the other hand, increased MHC-I expression has been postulated to ensure controlled viral dissemination during latency by promoting cytotoxic T lymphocyte (CTL) proliferation . K13-induced upregulation of adhesion molecules and MHC-1 molecules during natural infection with KSHV may be modulated by concomitant expression of viral lytic proteins, such as the K5 gene product and vIRF1, which have been shown to down-regulate the expression ICAM-1 and MHC-I molecules [36, 37].
Infection with KSHV has been reported to contribute to neoangiogenesis, another characteristic feature of KS lesions, by upregulating the expression of several genes involved in the control of vascular modeling and angiogenesis, such as VEGF-A, VEGF-C, angiopoietin-related protein 4, thrombomodulin, and matrix metalloproteinase (MMP-1) . The expression of VEGF-A, VEGF-C and angiopoietin-related protein 4 was not significantly increased, and expression of thrombomodulin was decreased 2-fold upon induction of K13 activity. Additionally, the expression of tissue factor pathway inhibitor 2 (TFPI2), a gene which is a negative inhibitor of aberrant angiogenesis associated with tumor development was increased 5-fold by K13 . However, the expression of semaphorin 3C (SEMA3C) and TNF-alpha-induced protein 2 (TNFAIP2), two invasion and angiogenic factors [39–41], was induced 2 and 21 fold upon activation of K13 activity, respectively. Similarly, the expression of matrix metallopeptidase 10 (MMP10 or Stromolysin 2), an enzyme implicated in the breakdown of extracellular matrix during tumor invasion, metastases and angiogenesis , was highly induced (11 fold) upon induction of K13 activity. Finally, K13 activity strongly (21 fold) induced the expression of TNF alpha-induced protein 6 (TNFAIP6, also known as TSG-6), a member of the Link module superfamily that regulates extracellular matrix remodeling and inflammatory response . Thus, K13 activity may contribute to neoangiogenesis in KS lesions via increased production of angiogenic factors, such as IL-8, SEMA3C and TNFAIP2, and to invasion and metastases by stimulating extracellular matrix remodeling through increased production of MMP10 and TNFAIP6.
K13 strongly induced two stress response genes, cyclooxygenase-2 (COX-2) and manganese superoxide dismutase (SOD2), which have been reported previously to be strongly induced by KSHV infection . COX-2 is an angiogenic stress response gene that was recently shown to facilitate latent KSHV gene expression and the establishment and maintenance of latency . SOD2 plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species , and may promote survival of KSHV-infected cells. Furthermore, while this manuscript was under review, an independent study reported upregulation of SOD2 expression by K13 in vascular endothelial cells, which correlated with decreased intracellular superoxide accumulation and increased resistance to superoxide-induced death . Two other anti-apoptotic genes, baculoviral IAP repeat containing 3 (BIRC3/cIAP2) and BCl2-related protein A1 (BCL2A1), were also strongly upregulated upon induction of K13 activity in our study, and may contribute to the survival of KSHV-infected cells.
The genes belonging to the interferon response pathway represented another class of genes whose expression was upregulated upon induction of K13 activity. Notable genes in this class included interferon γ-inducible protein 30 (IFI30), 28 kDa interferon responsive protein (IFRG28), interferon stimulated exonuclease gene 20 kDa (ISG20), guanylate binding protein 1 (GBP1), interferon induced transmembrane protein 1 (IFITM1), interferon regulatory factor-1, -2 and -7, interferon-induced protein 35 (IFI35), interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), interferon omega 1, interferon induced with helicase C domain 1 (IFIHI), and interferon, beta 1.
K13 is a powerful activator of the NF-κB pathway and others and we have previously reported that K13-induced upregulation of proinflammatory cytokines in vascular endothelium cells is associated with NF-κB activation and can be blocked by genetic and pharmacological inhibitors of this pathway [21, 22]. Consistent with the ability of K13 to activate NF-κB, most of the genes induced by K13 in the present study are known targets of the NF-κB pathway [46, 47], including NFKBIA (IκBα), which is not only a direct target gene of the NF-κB pathway but also a key negative regulator of this pathway. The involvement of the NF-κB pathway in K13-induced transcriptional activation of chemokine genes was further supported by our studies using the CXCL10 promoters. It needs to be noted, however, that the transcriptional activation of genes is usually complex and it is conceivable that NF-κB pathway cooperates with other signaling pathways in the transcriptional activation of some genes.
Finally, we observed that K13 also down-regulated the expression of several genes in HUVECs. In contrast to the upregulated genes, the down-regulated genes were diverse and did not belong to particular functional class. Nevertheless, these genes are known to act as tumor suppressor (e.g. ADAMTS18 and Periostin) [48, 49], apoptosis-inducer (e.g. insulin like growth factor binding protein 5) , and regulator of vascular integrity (regulator of G-protein signaling 5) , suggesting that their down-regulation by K13 may have a causal role in the pathogenesis of KS lesions.
Our microarray and multiplex cytokine analyses demonstrate that K13 activity may contribute to the upregulation of a majority of genes induced in HUVECs that are latently infected with KSHV. These results are consistent with the notion that K13, a powerful NF-κB activator, is one of the few KSHV genes that are expressed in latently-infected cells. However, interestingly, K13, by itself, can not mimic the effect of KSHV infection in inducing lymphatic reprogramming of blood vascular endothelial cells. Thus, lymphatic differentiation by KSHV may require supplementation by other viral gene activities.
Human herpesvirus 8
Kaposi's sarcoma-associated herpesvirus
viral Fas-associated death domain-like IL-1β-converting enzyme inhibitory protein
Inhibitor of κB
Primary effusion lymphoma
Nuclear Factor kappa B
Murine stem cell virus
Latency-associated nuclear antigen
Lymphatic vessel endothelial hyaluronan receptor 1
prospero-related homeobox 1
Vascular endothelial growth factor receptor 3.
This work was supported by grants from the National Institutes of Health (CA85177 and CA124621) and the Mario Lemieux Foundation. We thank Dr. Dan Muruve for the CXCL10 reporter construct, Dr. Siddhartha Kar for a critical reading of the manuscript and Dr. Ansuman Chattopadhyay for help with the analysis of the data.
- Dourmishev LA, Dourmishev AL, Palmeri D, Schwartz RA, Lukac DM: Molecular genetics of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) epidemiology and pathogenesis. Microbiol Mol Biol Rev. 2003, 67: 175-212. 10.1128/MMBR.67.2.175-212.2003. table of contentsPubMedPubMed CentralView Article
- Ensoli B, Gallo RC: AIDS-associated Kaposi's sarcoma: a new perspective of its pathogenesis and treatment. Proc Assoc Am Physicians. 1995, 107: 8-18.PubMed
- Ensoli B, Sturzl M: Kaposi's sarcoma: a result of the interplay among inflammatory cytokines, angiogenic factors and viral agents. Cytokine Growth Factor Rev. 1998, 9: 63-83. 10.1016/S1359-6101(97)00037-3.PubMedView Article
- Flore O, Rafii S, Ely S, O'Leary JJ, Hyjek EM, Cesarman E: Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus. Nature. 1998, 394: 588-592. 10.1038/29093.PubMedView Article
- Moses AV, Fish KN, Ruhl R, Smith PP, Strussenberg JG, Zhu L, Chandran B, Nelson JA: Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8. J Virol. 1999, 73: 6892-6902.PubMedPubMed Central
- Ciufo DM, Cannon JS, Poole LJ, Wu FY, Murray P, Ambinder RF, Hayward GS: Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures. J Virol. 2001, 75: 5614-5626. 10.1128/JVI.75.12.5614-5626.2001.PubMedPubMed CentralView Article
- Carroll PA, Brazeau E, Lagunoff M: Kaposi's sarcoma-associated herpesvirus infection of blood endothelial cells induces lymphatic differentiation. Virology. 2004, 328: 7-18. 10.1016/j.virol.2004.07.008.PubMedPubMed CentralView Article
- Hong YK, Foreman K, Shin JW, Hirakawa S, Curry CL, Sage DR, Libermann T, Dezube BJ, Fingeroth JD, Detmar M: Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus. Nat Genet. 2004, 36: 683-685. 10.1038/ng1383.PubMedView Article
- Wang HW, Trotter MW, Lagos D, Bourboulia D, Henderson S, Makinen T, Elliman S, Flanagan AM, Alitalo K, Boshoff C: Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet. 2004, 36: 687-693. 10.1038/ng1384.PubMedView Article
- Chaudhary PM, Jasmin A, Eby MT, Hood L: Modulation of the NF-kappa B pathway by virally encoded death effector domains-containing proteins. Oncogene. 1999, 18: 5738-5746. 10.1038/sj.onc.1202976.PubMedView Article
- Liu L, Eby MT, Rathore N, Sinha SK, Kumar A, Chaudhary PM: The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex. J Biol Chem. 2002, 277: 13745-13751. 10.1074/jbc.M110480200.PubMedView Article
- Matta H, Chaudhary PM: Activation of alternative NF-kappa B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein (vFLIP). Proc Natl Acad Sci USA. 2004, 101: 9399-9404. 10.1073/pnas.0308016101.PubMedPubMed CentralView Article
- Sun Q, Matta H, Chaudhary PM: The human herpes virus 8-encoded viral FLICE inhibitory protein protects against growth factor withdrawal-induced apoptosis via NF-kappa B activation. Blood. 2003, 101: 1956-1961. 10.1182/blood-2002-07-2072.PubMedView Article
- Sun Q, Zachariah S, Chaudhary PM: The human herpes virus 8-encoded viral FLICE-inhibitory protein induces cellular transformation via NF-kappaB activation. J Biol Chem. 2003, 278: 52437-52445. 10.1074/jbc.M304199200.PubMedView Article
- Sun Q, Matta H, Lu G, Chaudhary PM: Induction of IL-8 expression by human herpesvirus 8 encoded vFLIP K13 via NF-kappaB activation. Oncogene. 2006, 25: 2717-2726. 10.1038/sj.onc.1209298.PubMedView Article
- Chugh P, Matta H, Schamus S, Zachariah S, Kumar A, Richardson JA, Smith AL, Chaudhary PM: Constitutive NF-kappaB activation, normal Fas-induced apoptosis, and increased incidence of lymphoma in human herpes virus 8 K13 transgenic mice. Proc Natl Acad Sci USA. 2005, 102: 12885-12890. 10.1073/pnas.0408577102.PubMedPubMed CentralView Article
- Guasparri I, Keller SA, Cesarman E: KSHV vFLIP is essential for the survival of infected lymphoma cells. J Exp Med. 2004, 199: 993-1003. 10.1084/jem.20031467.PubMedPubMed CentralView Article
- Xu Y, Ganem D: Induction of chemokine production by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells. J Gen Virol. 2007, 88: 46-50. 10.1099/vir.0.82375-0.PubMedView Article
- Zhao J, Punj V, Matta H, Mazzacurati L, Schamus S, Yang Y, Yang T, Hong Y, Chaudhary PM: K13 blocks KSHV lytic replication and deregulates vIL6 and hIL6 expression: a model of lytic replication induced clonal selection in viral oncogenesis. PLoS One. 2007, 2: e1067-10.1371/journal.pone.0001067.PubMedPubMed CentralView Article
- Ye FC, Zhou FC, Xie JP, Kang T, Greene W, Kuhne K, Lei XF, Li QH, Gao SJ: Kaposi's sarcoma-associated herpesvirus latent gene vFLIP inhibits viral lytic replication through NF-kappaB-mediated suppression of the AP-1 pathway: a novel mechanism of virus control of latency. J Virol. 2008, 82: 4235-4249. 10.1128/JVI.02370-07.PubMedPubMed CentralView Article
- Matta H, Surabhi RM, Zhao J, Punj V, Sun Q, Schamus S, Mazzacurati L, Chaudhary PM: Induction of spindle cell morphology in human vascular endothelial cells by human herpesvirus 8-encoded viral FLICE inhibitory protein K13. Oncogene. 2007, 26: 1656-1660. 10.1038/sj.onc.1209931.PubMedView Article
- Grossmann C, Podgrabinska S, Skobe M, Ganem D: Activation of NF-kappaB by the latent vFLIP gene of Kaposi's sarcoma-associated herpesvirus is required for the spindle shape of virus-infected endothelial cells and contributes to their proinflammatory phenotype. J Virol. 2006, 80: 7179-7185. 10.1128/JVI.01603-05.PubMedPubMed CentralView Article
- Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3: RESEARCH0034-10.1186/gb-2002-3-7-research0034.PubMedPubMed CentralView Article
- Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD: IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol. 2002, 168: 3195-3204.PubMedView Article
- Matta H, Punj V, Schamus S, Mazzacurati L, Chen AM, Song R, Yang T, Chaudhary PM: A nuclear role for Kaposi's sarcoma-associated herpesvirus-encoded K13 protein in gene regulation. Oncogene. 2008, 27: 5243-5253. 10.1038/onc.2008.150.PubMedView Article
- Baxter A, Brough S, Cooper A, Floettmann E, Foster S, Harding C, Kettle J, McInally T, Martin C, Mobbs M, et al: Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors. Bioorg Med Chem Lett. 2004, 14: 2817-2822. 10.1016/j.bmcl.2004.03.058.PubMedView Article
- Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V, et al: NF-kappa B as a therapeutic target in multiple myeloma. J Biol Chem. 2002, 277: 16639-16647. 10.1074/jbc.M200360200.PubMedView Article
- Matta H, Sun Q, Moses G, Chaudhary PM: Molecular genetic analysis of human herpes virus 8-encoded viral FLICE inhibitory protein-induced NF-kappaB activation. J Biol Chem. 2003, 278: 52406-52411. 10.1074/jbc.M307308200.PubMedView Article
- Beckstead JH, Wood GS, Fletcher V: Evidence for the origin of Kaposi's sarcoma from lymphatic endothelium. Am J Pathol. 1985, 119: 294-300.PubMedPubMed Central
- Jussila L, Valtola R, Partanen TA, Salven P, Heikkila P, Matikainen MT, Renkonen R, Kaipainen A, Detmar M, Tschachler E, et al: Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res. 1998, 58: 1599-1604.PubMed
- Sturzl M, Wunderlich A, Ascherl G, Hohenadl C, Monini P, Zietz C, Browning PJ, Neipel F, Biberfeld P, Ensoli B: Human herpesvirus-8 (HHV-8) gene expression in Kaposi's sarcoma (KS) primary lesions: an in situ hybridization study. Leukemia. 1999, 13 (Suppl 1): S110-112. 10.1038/sj.leu.2401323.PubMedView Article
- Naranatt PP, Krishnan HH, Svojanovsky SR, Bloomer C, Mathur S, Chandran B: Host gene induction and transcriptional reprogramming in Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)-infected endothelial, fibroblast, and B cells: insights into modulation events early during infection. Cancer Res. 2004, 64: 72-84. 10.1158/0008-5472.CAN-03-2767.PubMedView Article
- Caselli E, Fiorentini S, Amici C, Di Luca D, Caruso A, Santoro MG: Human herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant protein 1-dependent capillary-like structure formation: role of the IKK/NF-kappaB pathway. Blood. 2007, 109: 2718-2726.PubMed
- Thurau M, Marquardt G, Gonin-Laurent N, Weinlander K, Naschberger E, Jochmann R, Alkharsah KR, Schulz TF, Thome M, Neipel F, Sturzl M: Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide-induced cell death. J Virol. 2009, 83: 598-611. 10.1128/JVI.00629-08.PubMedPubMed CentralView Article
- Sakakibara S, Pise-Masison CA, Brady JN, Tosato G: Gene regulation and functional alterations induced by Kaposi's sarcoma-associated herpesvirus-encoded ORFK13/vFLIP in endothelial cells. J Virol. 2009, 83: 2140-2153. 10.1128/JVI.01871-08.PubMedPubMed CentralView Article
- Lagos D, Trotter MW, Vart RJ, Wang HW, Matthews NC, Hansen A, Flore O, Gotch F, Boshoff C: Kaposi sarcoma herpesvirus-encoded vFLIP and vIRF1 regulate antigen presentation in lymphatic endothelial cells. Blood. 2007, 109: 1550-1558. 10.1182/blood-2006-05-024034.PubMedView Article
- Ishido S, Choi JK, Lee BS, Wang C, DeMaria M, Johnson RP, Cohen GB, Jung JU: Inhibition of natural killer cell-mediated cytotoxicity by Kaposi's sarcoma-associated herpesvirus K5 protein. Immunity. 2000, 13: 365-374. 10.1016/S1074-7613(00)00036-4.PubMedView Article
- Ivanciu L, Gerard RD, Tang H, Lupu F, Lupu C: Adenovirus-mediated expression of tissue factor pathway inhibitor-2 inhibits endothelial cell migration and angiogenesis. Arterioscler Thromb Vasc Biol. 2007, 27: 310-316. 10.1161/01.ATV.0000254147.89321.cf.PubMedView Article
- Herman JG, Meadows GG: Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells. Int J Oncol. 2007, 30: 1231-1238.PubMed
- Beckers J, Herrmann F, Rieger S, Drobyshev AL, Horsch M, Hrabe de Angelis M, Seliger B: Identification and validation of novel ERBB2 (HER2, NEU) targets including genes involved in angiogenesis. Int J Cancer. 2005, 114: 590-597. 10.1002/ijc.20798.PubMedView Article
- Sarma V, Wolf FW, Marks RM, Shows TB, Dixit VM: Cloning of a novel tumor necrosis factor-alpha-inducible primary response gene that is differentially expressed in development and capillary tube-like formation in vitro. J Immunol. 1992, 148: 3302-3312.PubMed
- Chang S, Young BD, Li S, Qi X, Richardson JA, Olson EN: Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell. 2006, 126: 321-334. 10.1016/j.cell.2006.05.040.PubMedView Article
- Milner CM, Day AJ: TSG-6: a multifunctional protein associated with inflammation. J Cell Sci. 2003, 116: 1863-1873. 10.1242/jcs.00407.PubMedView Article
- Sharma-Walia N, Raghu H, Sadagopan S, Sivakumar R, Veettil MV, Naranatt PP, Smith MM, Chandran B: Cyclooxygenase 2 induced by Kaposi's sarcoma-associated herpesvirus early during in vitro infection of target cells plays a role in the maintenance of latent viral gene expression. J Virol. 2006, 80: 6534-6552. 10.1128/JVI.00231-06.PubMedPubMed CentralView Article
- Landis GN, Tower J: Superoxide dismutase evolution and life span regulation. Mech Ageing Dev. 2005, 126: 365-379. 10.1016/j.mad.2004.08.012.PubMedView Article
- Tian B, Nowak DE, Brasier AR: A TNF-induced gene expression program under oscillatory NF-kappaB control. BMC Genomics. 2005, 6: 137-10.1186/1471-2164-6-137.PubMedPubMed CentralView Article
- Tian B, Nowak DE, Jamaluddin M, Wang S, Brasier AR: Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling. J Biol Chem. 2005, 280: 17435-17448. 10.1074/jbc.M500437200.PubMedView Article
- Jin H, Wang X, Ying J, Wong AH, Li H, Lee KY, Srivastava G, Chan AT, Yeo W, Ma BB, et al: Epigenetic identification of ADAMTS18 as a novel 16q23.1 tumor suppressor frequently silenced in esophageal, nasopharyngeal and multiple other carcinomas. Oncogene. 2007, 26: 7490-7498. 10.1038/sj.onc.1210559.PubMedPubMed CentralView Article
- Kim CJ, Yoshioka N, Tambe Y, Kushima R, Okada Y, Inoue H: Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells. Int J Cancer. 2005, 117: 51-58. 10.1002/ijc.21120.PubMedView Article
- Marshman E, Green KA, Flint DJ, White A, Streuli CH, Westwood M: Insulin-like growth factor binding protein 5 and apoptosis in mammary epithelial cells. J Cell Sci. 2003, 116: 675-682. 10.1242/jcs.00263.PubMedView Article
- Bondjers C, Kalen M, Hellstrom M, Scheidl SJ, Abramsson A, Renner O, Lindahl P, Cho H, Kehrl J, Betsholtz C: Transcription profiling of platelet-derived growth factor-B-deficient mouse embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells. Am J Pathol. 2003, 162: 721-729.PubMedPubMed CentralView Article
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/2/50/prepub
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