HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells

Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells, can be taken up by various uninfected cells including neurons and by dysregulating several biological events induces cell injury and death. In earlier studies, we demonstrated that treatment of neuronal cells with Tat affects the nerve growth factor (NGF) signaling pathway involving MAPK/ERK. Here we demonstrate that a decrease in the level of Egr-1, one of the targets for MAPK, by Tat has a negative impact on the level of p35 expression in NGF-treated neural cells. Further, we demonstrate a reduced level of Egr-1 association with the p35 promoter sequence in NGF-treated cells expressing Tat. As p35, by associating with Cdk5, phosphorylates several neuronal proteins including neurofilaments and plays a role in neuronal differentiation and survival, we examined kinase activity of p35 complexes obtained from cells expressing Tat. Results from H1 kinase assays showed reduced activity of the p35 complex from Tat-expressing cells in comparison to that from control cells. Accordingly, the level of phosphorylated neurofilaments was diminished in Tat-expressing cells. Similarly, treatment of PC12 cells with Tat protein or supernatant from HIV-1 infected cells decreased kinase activity of p35 in these cells. These observations ascribe a role for Tat in altering p35 expression and its activity that affects phosphorylation of proteins involved in neuronal cell survival.     

Dysregulation of CXCL9 and reduced tumor growth in Egr-1 deficient mice

Advances in the treatment of chronic lymphocytic leukemia (CLL) have improved initial overall response (OR) rates, complete response (CR) rates and progression free survival (PFS). Despite these advances, CLL remains incurable with standard therapies. Thus, there remains a need for more effective therapies in both the upfront and relapsed setting, particularly for patients with high-risk cytogenetic abnormalities such as del(11q22) and del(17p13). The 2008 American Society of Hematology (ASH) Annual Meeting featured several presentations which highlighted the ongoing clinical advances in CLL. The benefit of adding rituximab to purine analog therapy in the upfront setting was demonstrated by a large randomized study which showed that the addition of rituximab to fludarabine and cyclophosphamide (FCR) significantly improved OR, CR and PFS. The improvement in PFS directly resulted from an improved ability to eliminate minimal residual disease (MRD) in the peripheral blood, highlighting the importance of MRD eradication. However, a multi-center study suggested that the high CR rates to chemoimmunotherapy regimens such as FCR obtained in academic centers may not be reproducible when the same regimens are given in the community setting. The immunomodulatory drug lenalidomide is active in relapsed high-risk CLL, but two studies of lenalidomide in previously untreated CLL patients failed to achieve a CR and were associated with significant tumor lysis, tumor flare and hematologic toxicity. In the relapsed setting, a combination study of the bifunctional alkylator bendamustine and rituximab (BR) demonstrated a high OR rate in patients with del(11q22) and del(17p13), indicating that further studies to define's bendamustine activity are warranted in high-risk CLL. Similarly, the CDK inhibitor flavopiridol demonstrated significant clinical activity and durable remissions in heavily treated, refractory CLL patients with high-risk cytogenetic features and bulky lymphadenopathy. The monoclonal anti-CD20 antibody ofatumumab appeared to be superior to rituximab in relapsed CLL patients with bulky nodal disease or high-risk cytogenetic features. Ongoing studies of these agents and other novel therapeutic agents in clinical development hold forth the promise that treatment options for CLL patients will continue to expand and improve.     

Induction of OGG1 gene expression by HIV-1 Tat

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by up-regulating OGG1 as a feed-forward mechanism.     

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.     

V-sis induces Egr-1 expression by a pathway mediated by c-Ha-ras

The early growth response gene, Egr-1, is up-regulated transiently by mitogens and many other simuli in all cells tested. Using NIH3T3 cells conditionally expressing v-sis from a metallothionein promoter, we show that the addition of Zn²⁺ stimulates the production of PDGF-B(v-sis) and elicits the expression of Egr-1 in a dose-dependent and time-regulated manner. The signal is likely independent of protein kinase C, but depends on tyrosine kinase and other kinase activities and is mediated by c-Ha-Ras since the presence of dominant-negative mutants of Ras and Raf abrogates the induction of Egr-1 expression by Zn²⁺. Transiently activated Ras expression in NIH3T3 cells also stimulates the transient expression of Egr-1, but cells that constitutively express Rass do not have elevated levels of Egr-1. Transient assays also demonstrated that Zn²⁺ or activated Ras expression stimulated the activity of a 950 bp Egr-1 promoter-reporter gene construct and this is abrogated in the presence of mutant Ras and Raf. The accumulated data show that Egr-1 gene expression is regulated by multiple mechanisms, as would be needed for putative role in Cell proliferation, in suppression of transformation and in differentiation.     

Developmental expression of the putative transcription factor Egr-1 suggests that Egr-1 and c-fos are coregulated in some tissues

We have investigated developmental expression of the gene Egr-1, which encodes a protein containing three zinc fingers. Egr-1 like c-fos is a serum inducible, early response gene, which is co-induced with c-fos in a variety of quite different situations. A single 3.7-kb RNA was detected throughout fetal mouse development, which increased in absolute levels in total fetal RNA from 9.5 to 12.5 days post coitum (p.c.). In situ hybridization to 14.5- and 17.5-day p.c. fetal tissues demonstrated Egr-1 accumulation at several specific sites. These included mesenchymal components of the developing tooth germs and salivary and nasal glands; an ectodermally derived component of the whisker pad and developing muscle, cartilage, and bone. Expression of Egr-1 in cartilage and bone showed a strikingly similar expression to previously published reports of c-fos in these tissues. High levels of Egr-1 RNA was observed at the perichondrial interface of opposing cartilaginous elements and in interstitial cells that lie in between. Bone expression was observed in membranous bone of the head, alveolar bone around the tooth germs, and at periosteal and endochondral ossification sites in the limb bones. Our data support the idea that Egr-1 and c-fos may be coregulated in vivo and together may regulate normal development of the skeleton.     

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.     

Involvement of Inositol 1,4,5-Trisphosphate-Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV-1 Protein Tat

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.     

Dysregulation of macrophage-secreted cathepsin B contributes to HIV-1-linked neuronal apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1(ADA) infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.     

Egr-1 expression in surface Ig-mediated B cell activation. Kinetics and association with protein kinase C activation

We have studied the expression of an immediate/early type gene, Egr-1, in murine B lymphocyte responses to Ag receptor-generated signals. The Egr-1 gene encodes a zinc finger protein with sequence-specific DNA binding activity and is believed to act as an intracellular "third messenger," to couple receptor-generated signals to activation-associated changes in gene expression. We show here that Egr-1 mRNA expression is rapidly and transiently (returning to basal levels by 6 h) induced after receptor crosslinking with anti-receptor antibodies. Egr-1 protein expression is more prolonged, maintaining detectable levels through 12 h. The induction of Egr-1 is a primary response to Ag receptor signaling, as it is independent of new protein synthesis and is inhibited by actinomycin D. We have also examined the linkage of Egr-1 to known signaling pathways associated with G0 to G1 transition by these cells in response to signals generated through the B cell Ag receptor. Egr-1 mRNA was not induced after elevation of intracellular free Ca2+. In contrast, the pharmacologic agents PMA and SC-9, which directly activate protein kinase C, both cause marked increases in Egr-1 mRNA levels with the same kinetics as observed after anti-receptor antibody stimulation. Further, the protein kinase C inhibitors H7, sangivamycin, and staurosporin block anti-receptor antibody-induced expression of Egr-1, thus, B cell Ag receptor-linked Egr-1 expression is likely coupled to the protein kinase C component of transmembrane signaling. Preliminary promoter mapping studies are consistent with this conclusion, because both PMA and anti-receptor antibody act through the same or overlapping cis-regulatory elements.