Arch Virol (2012) 157:63–74 DOI 10.1007/s00705-011-1144-3
ORIGINAL ARTICLE
Activating transcription factor 4 (ATF4) is upregulated by human herpesvirus 8 infection, increases virus replication and promotes proangiogenic properties Elisabetta Caselli • Sabrina Benedetti Jessica Grigolato • Arnaldo Caruso • Dario Di Luca
•
Received: 11 August 2011 / Accepted: 6 October 2011 / Published online: 21 October 2011 Ó Springer-Verlag 2011
Abstract Human herpesvirus 8 (HHV-8) triggers proangiogenic behaviour in endothelial cells by inducing monocyte chemoattractant protein 1 (MCP-1) through activation of Nuclear Factor jB (NF-jB). However, NF-jB inhibition still results in partial MCP-1 induction and consequent angiogenesis, suggesting the involvement of another transcriptional pathway. We analysed activating transcription factor 4 (ATF4), since it is central in the cellular response to stress and is involved in angiogenesis. The results show that HHV-8 upregulates ATF4 expression, which in turn promotes HHV-8 infection, and induces MCP-1 production and proangiogenic properties in endothelial cells. By contrast, ATF4 silencing decreases virus replication and inhibits virus-induced MCP-1 production and induction of tube-like structures. Therefore, ATF4 plays a role in HHV-8 replication and associated virusinduced angiogenesis. The elucidation of molecular pathways involved in this process will result in a better understanding of the virus-induced angiogenic process and might help in designing novel therapies to reduce tumour growth.
Introduction Human herpesvirus 8 (HHV-8), also known as KS-associated herpesvirus (KSHV), belongs to the genus Rhadinovirus, subfamily Gammaherpesvirinae of the family E. Caselli (&) S. Benedetti J. Grigolato D. Di Luca Section of Microbiology, Department of Experimental and Diagnostic Medicine, University of Ferrara, via L. Borsari 46, 44100 Ferrara, Italy e-mail:
[email protected] A. Caruso Section of Microbiology, University of Brescia, Brescia, Italy
Herpesviridae. Originally discovered in Kaposi sarcoma (KS) lesions of an AIDS patient [15], HHV-8 is the causative agent of all clinical forms of KS and plays an important role also in the development of primary effusion lymphomas (PEL) [13], multicentric Castleman’s disease [44], and lymphoproliferative disorders affecting HIVinfected patients [3]. KS is a not a true sarcoma, defined as a tumour arising from mesenchymal tissue, but it has several features typical of vascular neoplasms. KS lesions contain abnormally dense and irregular blood vessels that fill with blood cells, giving the tumour its characteristic bruise-like appearance. Several morphological variants of KS are known, including anaplastic, telangiectatic, hyperkeratotic, keloidal, micronodular, pyogenic granulomalike, ecchymotic, and intravascular KS [25], but all of the described forms are characterized by angiogenesis and contain tumour cells of endothelial origin with a characteristically abnormal elongated shape, called spindle cells. Inflammatory cells, including lymphocytes, plasma cells and dendritic cells, are also present in KS lesions [29]. The majority of KS neoplastic cells support a latent HHV-8 infection, and virus lytic replication is limited to a small number of cells [7, 42, 45, 46]. However, latency alone is not sufficient to induce HHV-8 tumorigenesis, and several lines of evidence suggest that ongoing lytic replication is required for neoplastic development [12]. KS tumorigenesis is preceded by the upregulation of several key HHV-8 gene products, including homologues of cellular oncogenes, cytokines, chemokines or chemokine receptors, which promote cellular growth and transformation when abnormally regulated [39, 48]. It has been reported previously by us that HHV-8 infection of endothelial cells triggers proangiogenic behaviour by promoting transcriptional activation and secretion of monocyte chemoattractant protein 1 (MCP-1) through a potent and
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sustained activation of Nuclear Factor jB (NF-jB) [10]. However, mutations or deletions involving NF-jB binding sites located in the MCP-1 enhancer region resulted in only a partial inhibition of HHV-8-induced MCP-1 transcription and capillary-like structure formation, suggesting the involvement of at least another transcriptional pathway [10]. ATF4 (also known as CREB-2, TAXREB67 or C/ATF) has recently been recognized as a central factor used by the cell to respond to multiple stresses, including nutrient depletion, oxidative stress, UV irradiation, reductive stress, exposure to toxins, and hypoxia [2, 38]. The exact molecular events controlling the stress response have not been fully elucidated, but it is known that ATF4 is a common downstream target that integrates signalling from different stimuli. This pathway is collectively referred to as the integrated stress response (ISR) and consequent unfolded protein response (UPR) [27, 28]. Protein misfolding is caused by several factors, including anoxia, hypoxia [1, 6, 16, 20, 24], expression of mutant proteins [33] and infection by viruses, including hepatitis C virus [49], Japanese encephalitis virus [47], bovine diarrhoea virus [31], adenoviruses [24] and human cytomegalovirus [30]. Viruses inducing ER stress are faced with the consequences of UPR activation, which can be detrimental to viral replication (attenuation of translation) but can also be beneficial to viral infection, since ATF4 transcriptionally activates genes involved in re-establishing cellular metabolism (as part of the UPR recovery effort) and translation. As a result, some viruses modulate the UPR, inhibiting the effects that would be detrimental while maintaining those that may be beneficial. ATF4 is also related to hypoxia tolerance and tumour progression [21, 32]. It has been found overexpressed in tumours [1, 5] and is associated with VEGF expression and angiogenesis in different grades of breast cancer [14]. Furthermore, ATF4 has been observed to be an essential mediator of IL8, IL6 and MCP-1 expression in human aortic endothelial cells [23] and to promote VEGF production and resulting angiogenesis [40, 41]. These observations prompted us to investigate the possible relationship between ATF4 and HHV-8-induced angiogenesis. Here, it is shown that in vitro HHV-8 infection upregulates ATF4 expression, which in turn favours HHV-8 replication and induces MCP-1 production in a, NF-jB-independent manner, sustaining virus proangiogenic potential in primary human endothelial cells.
Materials and methods Cells The PEL-derived BC-3 cell line [4], used for HHV-8 production and reactivation, was cultured as described [10].
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For infection and transfection studies, the following human cells were used: primary human umbilical vein endothelial cells (HUVECs), human microdermal endothelial cells (HMECs), the 293 epithelial cell line and the Jurkat T cell line. HUVECs were isolated as described previously [9] and cultured in EGM-2 medium (BioWhittaker,Walkersville, MD) using collagen-coated flasks (Biocoat Collagen, BD Biosciences, Bedford, MA). HMECs were obtained as described previously [18] and cultured in EGM-2 medium. The human 293 cell line (ATCC CRL-1573) was grown in complete DMEM medium plus 10% FCS. The Jurkat T cell line (ATCC TIB-152) was grown in complete RPMI medium plus 10% FCS. HHV-8 infection HHV-8 inoculum was obtained from BC-3 cells stimulated with TPA (20 ng/ml) as described previously [9]. Cell-free purified virions were suspended in PBS containing 1% BSA and stored at -80 °C until use. Virus stock was quantitated by real-time PCR TaqMan assays (qPCR) as described [10], and it contained 59105 copies of viral DNA/ll. Prior to use, the virus stock was treated for 10 min at room temperature with DNase-I and RNase-A to eliminate free viral nucleic acids potentially present in the preparation. Infective titers were determined by counting the number of cells positive for the HHV-8 K8.1 protein by immunofluorescence at 36 h after infection, as described [10]. For in vitro infection experiments, HUVEC, HMEC, 293 or Jurkat cells were seeded at optimal density 24 h before infection and subsequently infected as described [9, 10, 22]. Briefly, 0.59106 cells were infected with 10 ll of purified virus preparation (i.e., 59106 copies of viral DNA, corresponding to an m.o.i. of 10). After 3 h of absorption at 37 °C, the viral inoculum was removed, and cells were washed with PBS and incubated in complete medium for the designed times. Cell samples were harvested at specific time points for subsequent analysis. Control samples were left uninfected or mock-infected with UV-inactivated HHV-8, obtained by exposing the purified inoculum to UV light (200 mJ/cm2) for 30 min, resulting in a virus that was still able to bind and enter the cell but not able to transcribe any virus function [10]. Analysis of virus infection The presence of HHV-8 and transcription in infected cells were evaluated by qPCR and PCR after reverse transcription (RT-PCR), performed on total DNA or RNA, respectively, extracted from cells as described [10]. qPCR reactions were carried out in triplicate on a 7500 PCR System (Applied Biosystems), amplifying the HHV-8 ORF26 gene and using 100 ng of total DNA as a template
Regulation of ATF4 by human herpesvirus 8
[10]. An HHV-8 DNA standard curve was obtained by serial dilution of pCR-ORF26 plasmid, containing a cloned fragment of HHV-8 ORF26 (nucleotides 47127-47556). Human genomic DNA was quantified by amplification of the RNaseP gene. In RT-PCR assays, ORF50 and ORF26 gene transcripts were analyzed, using primers and conditions described previously [9]. Total RNA was extracted from cell pellets using an RNeasy isolation kit (QIAGEN), according to manufacturer’s instructions, and DNA contamination was eliminated by digestion with RNase-free DNase (Roche). After reverse transcription using AMV reverse transcriptase (Ambion), PCR amplifications were performed using an amount of cDNA corresponding to 200 ng of total RNA. Semi-quantitation of PCR products was performed by amplification of serial dilutions of template cDNA. Amplification of the human b-actin transcript was used as an internal control as described [8]. Antigen expression of HHV-8 was evaluated by IFA, performed on 59104 infected cells fixed with cold methanol/acetone (50:50) and incubated with antibodies directed against lytic (ORFK8.1) or latent (ORF73, LANA) antigens as already described [10]. After primary antibody incubation, secondary antibodies (anti-mouse IgG, fluorescein-conjugated, Sigma) were added, and slides were mounted for fluorescence observation using a Nikon Eclipse TE2000-S microscope. Plasmids, cell transfection and reporter assays Plasmid pCG-ATF4, containing the full-length ATF4 coding sequence driven by a CMV promoter, was a generous gift from T. Hai [35]. Plasmids pCR-50sp and pCR-57sp, containing the spliced functional forms of the HHV-8 ORF50 and ORF57 genes, respectively, driven by a CMV promoter, were described previously [8]. Similarly, the reporter plasmids pGL-50pr, pGL-57pr and pGL-T1.1pr, containing the HHV-8 ORF50, ORF57 and T1.1 promoters cloned in the pGL3-Basic reporter plasmid (Promega, Mannheim, Germany) upstream of a promoterless luciferase gene, have already been described [43]. The MCP-1 promoter-luc plasmids, containing the proximal promoter (pGLM-PRM, nt -107 to -60), proximal promoter and distal enhancer (pGLM-ENH, nt -107 to -60 plus nt -2513 to -2742), proximal promoter and distal enhancer in which one (pGLMMA1, pGLM-MA2) or both (pGLMMA1A2) NF-jB sites (A1 and A2) are mutated, have been described previously [10]. To analyze the transcriptional activation of HHV-8 or MCP-1 promoters, HUVEC, Jurkat or 293 cells were cotransfected with 1 lg of each of the promoter-luc constructs together with 0.5 lg pRL-SV40 vector (Promega) as an internal control in the presence of 1 lg of pCG-ATF4 or pCG plasmid, with or without the addition of 50, 100 or 500 ng of pCR-50sp plasmid. Transfections were performed
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using the Nucleofector method (Amaxa, Cologne, Germany) following the manufacturer’s instructions. Efficiency of transfection was 50% in HUVECs and 70% in 293 cells, as determined by transfection with pMax-GFP plasmid (Amaxa). Transfected cells were seeded on 24-well plates and lysed after 30 hours of incubation (Dual-Glo Luciferase Assay System, Promega). Cell lysates were analyzed for luc levels using a Berthold Microplate Luminometer. Analysis of ATF4 transcription and translation ATF4 transcription was analyzed by real-time qPCR after retrotranscription (RT-qPCR), using a specific TaqMan assay (Applied Biosystems, Foster City, CA). An amount of cDNA corresponding to 200 ng of total RNA extracted from infected, uninfected or transfected cells was used in each reaction. Human RNaseP amplification was used as an internal control. Amplifications were carried out in a 50-ll reaction mix and performed in triplicate on a 7500 realtime PCR system (Applied Biosystems). The relative amounts of ATF4 and RNaseP (internal control) mRNAs were determined by analysis of qPCR data, performed by the 2-DDCt method using the Relative Quantitation Study software (Applied Biosystems). Levels of ATF4 mRNA were expressed as fold difference in HHV-8-infected or ATF4-transfected cells compared to mock-infected or -transfected cells (calibrator sample). ATF4 protein expression was evaluated by western blot, using a rabbit polyclonal antibody directed against ATF4CREB2 (Santa Cruz Biotechnology, Inc). Blots were performed using 100 lg of cellular protein lysate, separated by electrophoresis in SDS-polyacrylamide gels (SDS-PAGE), and electrically transferred onto nitrocellulose paper using a transfer buffer consisting of 25 mM Tris, 192 mM glycine and 20% methanol. After 1 h incubation in saturation buffer, consisting of 5% dehydrated non-fat milk in TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), blots were incubated for 90 min with anti-ATF4 antibody diluted 1:200 in TBS-T, then washed three times with TBS-T, and finally incubated for 90 min with an HRPconjugated anti-rabbit antibody (Santa Cruz Biotechnology, Inc) diluted 1:1000 in TBS-T. After three additional washings in TBS-T, blots were developed with the addition of a chemiluminescent HRP substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce), according to the manufacturer’s protocol. Analysis of in vitro cord formation capability An in vitro capillary-like structure formation assay was performed using Reduced Growth Factor Basement Membrane Extract (RGF-BME) (Cultrex; Trevigen, Gaithersburg Gaithersburg, MA), as described [10]. Uninfected, infected
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or transfected HUVECs were cultured (59104 cells per well per cm2) in gelled RGF-BME at 37 °C and 5% CO2 in EBM (no serum or angiogenic factors). Tube formation on BME was analyzed by optical microscopy at different times after cell seeding. Analysis of MCP-1 production MCP-1 was quantitated in supernatants from HHV-8infected, mock-infected or transfected HUVECs, collected at different times after infection. Samples were cleared by centrifugation to eliminate cell debris and stored at -80 °C until use. After thawing, the amount of MCP-1 was determined by specific ELISA (Biosource International, Camarillo, CA), following the manufacturer’s instructions. Each sample was tested in triplicate. Small interfering RNA (siRNA) studies For silencing studies, HUVECs plated at optimal density were transfected with 100 nM (1.35 lg/ml) siRNA targeting human ATF4 or of unrelated negative control siRNA (Ambion) using the RNAiFect technique (QIAGEN) and following manufacturer’s instructions. Effective knockdown of ATF4 transcription was checked by RT-qPCR using the gene expression assay described above (Applied Biosystems). Statistical analysis Statistical significance of the results was evaluated by Student’s t test.
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HHV-8 infection induces a substantial increase of ATF4 transcription and translation (Fig. 1). An increase in transcription was detectable as early as 8 h.p.i. and peaked at 24 h.p.i., when levels of ATF4 mRNA were 10 times higher than the controls. The increase persisted at 48 and 72 h.p.i. but declined slowly. The level of ATF4 protein increased consistently, and this protein accumulated until 72 h.p.i., when it was 6.9-fold more abundant than in mock-infected samples. The increase in ATF4 levels following HHV-8 infection was observed with minimal variations in all the cell types tested (Table 1), indicating that ATF4 upregulation occurs as an early event in HHV-8-infected cells, regardless of the cell type used. Furthermore, UV-irradiated HHV-8 did not induce an increase in the amount of ATF4 (Fig. 1), suggesting that ATF4 upregulation was not a consequence of mere virus binding to the cell, but it was dependent on the expression of viral functions during the early phases of productive infection. The effect of HHV-8 ORF50, an immediate early function with a key role in activating viral replication, on the induction of ATF4 was therefore studied. The same cell types used for infection experiments were transfected with pCR-ORF50 plasmid or pCR empty vector as a control. ATF4 expression was analyzed by RT-qPCR and western blot at 8, 24, 48 and 72 hours posttransfection (h.p.t.). The results show that ORF50 expression induced a 4-fold increase of ATF4 mRNA and a 3-fold increase of ATF4 protein (Fig. 1), whereas neither pCRnor pCR-ORF57-transfected cells showed any increase in ATF4 transcript or protein. Similar results were obtained in all of the cell types tested (Table 2), suggesting that ORF50 is important for ATF4 induction in infected cells. ATF4 increases HHV-8 replication
Results HHV-8 infection increases ATF4 expression The effect of HHV-8 infection on ATF4 expression was studied in different human cell types, including human primary endothelial cells (HUVECs, as a representative of natural targets of HHV-8 in vivo), immortalized microdermal endothelial cells (HMEC), epithelial cells (293 cell line) and T lymphocytes (Jurkat cell line). As reported previously by us, HHV-8 supports a productive infection in all these cell types, followed by the establishment of latency at 5-10 days postinfection (d.p.i.) [9–11, 22]. The efficiency of infection ranged between 15% (293 and Jurkat T cells) and 20% (HMEC and HUVEC), as measured by virus K8.1 antigen expression by IFA. Control samples were uninfected or mock-infected with UV-inactivated HHV-8. The results, obtained by quantitative real-time PCR after retrotranscription (RT-qPCR) and western blot, show that
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Since HHV-8 infection upregulates ATF4 expression, it was analyzed whether the increase of ATF4 was beneficial for virus replication. The same cell types used for HHV-8 infection were transfected with a plasmid encoding the fulllength ATF4 gene (pCG-ATF4) or with the pCG empty vector (control) and subsequently infected with HHV-8. Virus replication and transcription were analyzed at 8, 24 and 48 h.p.i., by qPCR, and RT-PCR or RT-qPCR was performed on ORF50 (immediate early, IE) and ORF26 (late, L) virus genes. As shown in Fig. 2, virus replication strongly increased in pCG-ATF4-transfected cells compared to control pCG-transfected cells. The amount of viral DNA increased between 1 and 2 logs, depending on the cell type, being higher in HUVECs and Jurkat cells (100-fold) compared to 293 and HMEC cells (10 and 40-fold, respectively). Also, viral transcripts increased significantly, as shown by RT-PCR and RT-qPCR results, with about 1 log increase of both IE (ORF50) and L (ORF26) mRNAs
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Fig. 1 HHV-8 infection and ORF50 transfection increase ATF4 expression. (A-B) HUVECs were infected with HHV-8, left uninfected (Control) or infected with UV-inactivated virus (Mock) and collected at the indicated hours post infection (h.p.i.). (C-D) HUVECs were transfected with pCR-ORF50, pCR-ORF57 or pCR empty vector (Control) and collected at the indicated hours post transfection (h.p.t.). ATF4 mRNA was quantitated by RT-qPCR, using a template
of cDNA derived from 200 ng of total RNA. ATF4 protein production was analyzed by western blot, performed on 100 lg of total cell protein lysate. Results are expressed as the mean fold increase of triplicate samples in at least three different assays. Results of densitometric analysis of western blots were plotted and expressed as the mean ± SD of duplicate samples in three different experiments
Table 1 HHV-8 infection induces ATF4 expression in different cell types
Table 2 ORF50 transfection induces ATF4 expression in different cell types
Cell type HUVEC HMEC
ATF4 mRNAa 9.5 ± 0.1
ATF4 proteinb
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ATF4 mRNAa
ATF4 proteinb
9.5 ± 0.1
HUVEC
4.2 ± 0.3
3.1 ± 0.2
HMEC
4.5 ± 0.2
3.2 ± 0.5
9.6 ± 0.2
7.5 ± 0.1
293 epithelial cells
10.1 ± 0.2
8.2 ± 0.2
293 epithelial cells
4.1 ± 0.5
3.0 ± 0.1
Jurkat T cells
10.2 ± 0.1
9.4 ± 0.2
Jurkat T cells
4.0 ± 0.3
2.8 ± 0.2
Results are expressed as fold increase ± SD observed in HHV-8 infected cells compared to control levels (mean value of uninfected and mock-infected cells) and represent the mean of triplicate samples in at least two different experiments
Results are expressed as fold increase ± SD observed in ORF50transfected cells compared to control levels (mean value of untransfected and pCR-transfected cells) and represent the mean of triplicate samples in three different experiments
a
ATF4 transcriptional activation was measured by RT-qPCR at 24 hours postinfection
a
b
ATF4 protein accumulation was measured by western blot at 72 hours postinfection
b
(Fig. 2). The results were confirmed by the analysis of virus expression, performed by immunofluorescence on ORFK8.1 (lytic) and ORF73-LANA (latent) proteins. The number of antigen-expressing cells almost doubled in ATF4-transfected cells compared to control cells (Fig. 2). To test the possibility that ATF-4 activates HHV-8 gene promoters, reporter assays were performed in HUVEC, 293 and Jurkat cells transfected with plasmids encoding the luciferase (luc) gene under the control of HHV-8 ORF50, ORF57 or T1.1 promoters (pGL-50pr, pGL-57pr, pGLT1.1pr). Cultures were cotransfected with pCG-ATF4 plasmid or pCG empty vector (as a negative control) or
with a plasmid coding for ORF50 transactivator (pCR50sp), which was used as a positive transactivation control [43]. As shown in Fig. 3, ATF4 did not activate any of the HHV-8 promoters tested, showing that ATF4 does not have a direct transcriptional effect on HHV-8 promoters.
ATF4 transcriptional activation was measured by RT-qPCR at 24 hours postinfection
ATF4 protein accumulation was measured by western blot at 72 hours postinfection
ATF4 activates the MCP-1 promoter and induces MCP-1 secretion It was reported previously by us that NF-jB inhibitors only partially affect HHV-8-induced production of MCP-1 and its resulting angiogenesis [10], suggesting that another
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TPA treatment caused a significant transcriptional activation in cells transfected with the pGLM-ENH plasmid (5.1-fold) containing both the MCP-1 promoter and enhancer, and deletion/mutation of jB sites resulted in the loss of TPAinduced activation (Fig. 4). Instead, ATF4 transfection resulted in an increase of luc expression independently from the presence of a functional MCP-1 enhancer region. Similar values were in fact observed in cells transfected with plasmids containing or lacking a functional MCP-1 enhancer region, suggesting that ATF4 activates the MCP-1 promoter directly, and that the jB sites in the enhancer region are not needed for ATF4 stimulation. Experiments performed in 293 cells gave similar results (not shown).
factor was involved in MCP-1 activation. The hypothesis of a direct role of ATF4 in inducing MCP-1 transcription was therefore tested. HUVECs were cotransfected with pCG-ATF4 or pCG plasmids and plasmids containing the reporter luc gene cloned downstream of the MCP-1 promoter/enhancer regions: pGLM-PRM (containing the proximal promoter region of the MCP-1 gene), pGLM-ENH (containing both the proximal promoter and the distal enhancer regions), and pGLM-MA1, pGLM-MA2 and pGLM-MA1A2, where one or both NF-jB binding sites (A1 and A2) in the enhancer region are mutated. As a positive control of NF-jB activation, cells were treated with TPA at 12 h.p.t.. As expected,
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Fig. 2 ATF4 increases HHV-8 replication. HUVECs were transfected with pCG-ATF4 or pCG plasmid infected with HHV-8 (24 hours posttransfection, h.p.t.) and collected at the indicated hours post-infection (h.p.i.) to analyze virus replication. (A) Virus genomes were quantitated by a qPCR specific for the ORF26 gene, using 100 ng of total extracted DNA. The results represent the mean ± SD of triplicate samples in three different experiments. (B) Virus transcription was analyzed by semi-quantitative RT-PCR, amplifying ORF50 (IE) and ORF26 (L) mRNAs; serial dilutions of cDNA were used as the template, starting from an amount corresponding to 200 ng of total RNA collected at 72 h.p.i. The b-actin transcript was also amplified as an internal control. In addition, ORF26 mRNA was also quantitated by RT-qPCR. (C) Virus antigen expression was analyzed by IFA using anti-ORFK8.1 and antiLANA mAbs. Results refer to 72 h.p.i. Pictures were taken with a Nikon Eclipse TE2000-S microscope equipped for fluorescence observation. Original magnification, 4X
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accounts for the residual activity that was not blocked by NF-jB inhibitors.
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Given the results obtained, the ability of ATF4 to induce tube formation in HUVECs was investigated. Cells were transfected with pCG-ATF4 or pCG empty vector (negative control), or infected with HHV-8 (positive control) and then seeded on BME at 24 h.p.t./h.p.i. The formation of tubule-like structures was evaluated 24 hours later (Fig. 5). As expected, HHV-8-infected HUVECs formed an extensive network of capillary-like structures, confirming previous results obtained by us [10]. Interestingly, ATF4transfected HUVECs also showed a pro-angiogenic phenotype, although with lower efficiency compared to viral infection, suggesting that high levels of ATF4 can induce a proangiogenic behaviour, even in the absence of viral infection. ATF4 silencing impairs HHV-8 replication and induction of proangiogenic properties
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Transfected plamids Fig. 3 ATF4 does not activate HHV-8 promoters directly. Cells were cotransfected with the indicated amounts of pCG-ATF4 or pCRORF50 plasmids together with reporter plasmids containing the luc gene cloned under the transcriptional control of HHV-8 ORF50 (A), ORF57 (B) or T1.1 (C) gene promoters. Luciferase activity was analyzed 30 hours post-transfection (h.p.t.). Results are expressed as fold activation compared to negative control (value = 1) and represent the mean ± SD of triplicate samples in three different assays
ELISA analysis of the culture supernatant showed that ATF4 activation of the MCP-1 promoter resulted in MCP-1 induction, as shown by the release of up to 1200 pg/ml of MCP-1 at 48 h.p.t. (Fig. 4). As expected, the amount of MCP-1 induced by ATF4 alone was lower than that induced by HHV-8 infection, due to the simultaneous involvement of NF-jB activation [10], and this probably
To verify the role of ATF4 in HHV-8 biology, ATF4 expression was silenced through transfection of small interfering RNA (siRNA) prior to HHV-8 infection. Control cells were transfected with scrambled unrelated siRNA. Effective knockdown of ATF4 transcription was checked by RT-qPCR, and inhibition of ATF4 transcription was 80% at 24 h.p.t. (Fig. 6). Silenced or non-silenced cells were infected with HHV-8, and virus replication was analyzed at 24, 48, and 72 h.p.i. by qPCR (DNA) and RT-qPCR (RNA). As shown in Fig. 6, ATF4 silencing caused a significant impairment of HHV-8 replication, and the amount of viral DNA was approximately 70% less compared to non-silenced cells. Analysis of viral transcripts confirmed these results (not shown). ATF4 silencing also inhibited the production of MCP-1 and the formation of HHV-8-induced tubule-like structures in HUVECs (Fig. 7). MCP-1 production was 40% lower in ATF4-silenced cells than in control cells. As shown in Fig. 7, control cells infected with HHV-8 showed clear proangiogenic behaviour. Instead, the formation of tubulelike structures was impaired in ATF4-silenced cells, confirming that ATF-4 is a mediator of HHV-8 proangiogenic potential. The inhibition was evident at early times postseeding, whereas at later times, HHV-8-infected cells almost completely regained their ability to form tube-like structures, likely due to the decline in siRNA activity and to the parallel virus-induced NF-jB activation (not shown). By contrast, the simultaneous inhibition of NF-jB (by treatment with 5 lM MG132) [10] and ATF4 (by specific
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Fig. 4 ATF4 activates the MCP-1 promoter and induces production of MCP-1. (A) HUVECs were cotransfected with pCG-ATF4 or pCG (Control) plasmids and the indicated luc reporter plasmids: pGLMPRM (containing the enhancerless MCP-1 promoter), pGLM-ENH (containing both MCP-1 promoter and enhancer regions), and pGLMMA1, pGLM-MA2 and pGLM-MA1A2 (containing the MCP-1 promoter and a mutated enhancer region where one or both NF-jB sites are mutated). Positive controls were treated with TPA 100 ng/ml 12 hours post-transfection (h.p.t.). Luciferase activity was analyzed at
Fig. 5 ATF4 induces capillary-like formation in endothelial cells. HUVECs were left untreated, transfected with pCG-ATF4 or pCG plasmids, or infected with HHV-8 and seeded on solidified RGF-BME (59104 cells/well/cm2) at 37 °C and 5% CO2 in EBM (no serum or angiogenic factors). Tube formation was analyzed by optical microscopy at different times after cell seeding. After 24 hours, cells were observed by optical microscopy (Nikon Eclipse TE2000-S microscope). Original magnification, 4X
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30 h.p.t. and is expressed as fold activation relative to the basal level of controls (value = 1). Results represent the mean ± SD of triplicate samples in two different assays. (B) HUVECs were transfected with pCG-ATF4 or pCG (Control) plasmids or infected with HHV-8. MCP-1 release was analyzed by ELISA on culture supernatants collected at 24 or 48 hours postinfection (h.p.i.) or h.p.t.. Results are expressed as concentrations of chemokine in culture medium (pg/ml) and represent the mean ± SD of duplicate samples in three different experiments
HHV-8
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Regulation of ATF4 by human herpesvirus 8
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siRNA) completely abolished the tube formation capability of HUVECs, but this was accompanied by a high level of cell mortality (not shown).
Discussion Kaposi’s sarcoma is a vascular neoplasia characterized by the proliferation of neovascular formations and proliferating spindle cells originating from endothelial cells. HHV-8 is essential for the development of all forms of KS, and its genome is detected in almost all neoplastic cells. Previous results obtained by us showed that the proangiogenic potential of HUVEC cells associated with HHV-8 infection is strictly dependent on MCP-1 secretion [10]. In fact, it was described by us that HHV-8, subsequent to a robust and sustained activation of cellular NF-jB, upregulates the expression of MCP-1. Interestingly, inhibition of MCP-1 activity by specific antibody abolished HUVEC tube formation completely, but inhibition of NF-jB resulted in A
ATF4 silencing
ATF4 transcription (%)
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N°target molecules (ORF26)
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HHV-8 replication 8,00E+04 7,00E+04 6,00E+04 5,00E+04 4,00E+04
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2,00E+04
HHV8/ATF4(-)
1,00E+04 0,00E+00
24 h
48 h
72 h
Time (h.p.i.)
Fig. 6 ATF4 silencing impairs HHV-8 replication. (A) Cells were left untransfected (Control) or transfected with scrambled unrelated siRNA (NC) or with anti-ATF4 siRNA (ATF4(-)). ATF4 transcription was evaluated by RT-qPCR at 24 hours post-transfection (h.p.t.). Results are expressed as percentages relative to the control cells. (B) Untransfected, NC-transfected and ATF4(-)-transfected cells were infected with HHV-8 at 24 h.p.t. Cells samples were collected at the indicated hours postinfection (h.p.i.) for analysis of virus replication by qPCR (ORF26 gene). Results are expressed as the number of target molecules per 100 ng of cellular DNA and represent the mean ± SD of triplicate samples in three different experiments
only a reduction of MCP-1 levels and proangiogenic cell behaviour. In addition, it was reported recently that the activation of CCAAT/enhancer binding protein beta (C/EBPb) is of primary relevance for the induction of MCP-1 expression by HHV-8 de novo infection [17], suggesting the simultaneous involvement of pathways other than the NF-jB pathway. In the search for other factors involved in HHV-8 triggering of MCP-1 production and angiogenesis, attention was focused on ATF4 as a potential candidate for the residual NF-jB-independent production of MCP-1. In fact, ATF4 is an essential mediator of MCP-1 expression in human endothelial cells [23]. In addition, ATF4 is overexpressed in tumours when compared to normal tissues [1, 5] and is related to hypoxia tolerance, tumour progression, VEGF expression and angiogenesis [14, 21, 32, 41]. Therefore, the interplay between HHV-8 infection and ATF4 expression was analysed in different cell types. The results reported here indicate that acute HHV-8 infection upregulates ATF4 expression, both at the transcriptional and translational level, possibly due to the activation of the integrated stress response following virus entry and the beginning of replication. Although this notion is new for HHV-8, it has already been described for other viruses [24, 31, 47, 49], including another human herpesvirus, HCMV [30]. In general, viral infections trigger cellular stress responses, potentially resulting in attenuation of viral replication. In the case of HCMV, the virus induces the UPR but regulates its signal transduction pathway in order to eliminate deleterious effects while maintaining potentially beneficial ones deriving from activation of ATF4 [30]. In this study, something very similar was found for HHV-8. Virus infection activates ATF4, which in turn enhances viral replication. This effect was observed in different cell types, suggesting that the induction of ATF4 is independent of the ability to develop a full productive infection. Transfection with the major viral transactivator, ORF50, partially reproduced the induction of ATF4, whereas treatment with a UV-irradiated virus and transfection with another early gene, ORF57, did not induce an increase in ATF4. This suggests that ORF50 might be responsible, at least in part, for virus induction of ATF4 in infected cells. Interestingly, it was reported previously that expression of HHV-8 latency-associated nuclear antigen (LANA) inhibits the transcriptional activation activity of ATF4 [36], confirming that ATF4 induction is specifically associated to the early steps of acute HHV-8 infection and supporting the hypothesis that ATF4 is an important requisite for viral replication. However, expression of ATF4 has no direct activating effect on the HHV-8 promoters tested, suggesting an indirect action on viral replication, possibly mediated by additional cell or viral factors. In fact,
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MCP-1 (pg/ml)
72
8000
MCP-1 ELISA
6000 4000
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2000
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0 24 h
48 h
HHV8/ATF4(-)
Time (h.p.i.)
N.I.
HHV-8
ATF4(-)
NC
Control
B
Fig. 7 ATF4 silencing inhibits virus-induced MCP-1 production and tubule-like structure formation in HUVECs. (A) Cells were left untransfected (C) or transfected with scrambled unrelated siRNA (NC) or with anti-ATF4 siRNA (ATF4(-)) and then infected with HHV-8 at 24 hours post-transfection (h.p.t.). Cell culture supernatants were collected at the indicated hours postinfection (h.p.i.) for analysis of MCP-1 release by specific ELISA. Results are expressed as pg/ml of chemokine in culture medium and represent the mean ± SD of duplicate samples in three different experiments. (B) Control untransfected, NC-transfected and ATF4(-)-transfected cells were not infected (N.I.) or infected with HHV-8 at 24 h.p.t. After 24 h.p.i., cells were plated on RGF-BME (59104 cells/well/cm2) at 37 °C and 5% CO2 in EBM (no serum or angiogenic factors), and formation of tubule-like structures was evaluated 6 hours post-seeding by optical microscopy (Nikon Eclipse TE2000-S microscope). Original magnification, 4X. The results shown are representative of three different experiments
ATF4, as a member of the ATF/CREB family, can form heterodimers with diverse DNA binding specificity [26]. This is of particular interest in light of recent reports showing that HHV-8 lytic replication is activated by oxidative stress [34, 50] and that signalling mediators triggering production of reactive oxygen species (ROS) are implicated in KS tumour angiogenesis [37], highlighting the carcinogenic potential of oxidative stress in the context of chronic infection. This suggests a potential role for
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ATF4, since ER and oxidative stresses find a common downstream signal in ATF4 [19]. Considering previous results obtained by us showing that HHV-8 induced MCP-1 expression and angiogenesis using both NF-jB-dependent and -independent pathways, it is suggested here that viral induction of ATF4 might be responsible for residual NF-jB-independent MCP-1 production. In fact, ATF4 transfection activates the MCP-1 promoter in the region lacking NF-jB binding sites, it induces production and release of MCP-1, and overexpression of ATF4 promotes proangiogenic activity in primary human endothelial cells. In addition, silencing of ATF4 results in a significant impairment of HHV-8 replication and inhibition of HHV-8-induced tubule-like structure formation in HUVECs. The results reported here show that the distinctive proangiogenic ability of HHV-8, which is strictly dependent on MCP-1, is the result of at least two converging pathways, one supported by NF-jB [10] and one mediated by ATF4. ATF4 is induced by acute HHV-8 infection, possibly by the expression of viral ORF50. Activation of ATF4 promotes HHV-8 replication and allows a full induction of virus-induced proangiogenic properties. Since these in vitro features mimic early-stage tumorigenesis, it is proposed here that ATF4 induction by HHV-8 can contribute to KS tumour development. Acknowledgments This work was supported by the Italian Ministry of University and Scientific Research (PRIN projects), FAR (University of Ferrara) and Istituto Superiore di Sanita` (ISS; AIDS project). We thank Dr. T. Hai (Department of Molecular and Cellular Biochemistry, Neurobiotechnology Center, Ohio State University, Columbus, USA) for providing the pCG-ATF4 plasmid. We thank Annalisa Peverati for excellent technical assistance, and Linda M. Sartor for revising the English manuscript Conflict of interest flicting interests.
The authors declare that they have no con-
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