Kiho Cho

Endogenous retroviruses in systemic response to stress signals.

Infection of germline cells with retroviruses initiates permanent proviral colonization of the germline genome. The germline-integrated proviruses, called endogenous retroviruses (ERVs), are inherited to offspring in a Mendelian order and belong to the transposable element family. Endogenous retroviruses and other long terminal repeat retroelements constitute ~8% and ~10% of the human and mouse genomes, respectively. It is likely that each individual has a distinct genomic ERV profile. Recent studies have revealed that a substantial fraction of ERVs retains the coding potentials necessary for virion assembly and replication. There are several layers of potential mechanisms controlling ERV expression: intracellular transcription environment (e.g., transcription factor pool, splicing machinery, hormones), epigenetic status of the genome (e.g., proviral methylation, histone acetylation), profile of transcription regulatory elements on each ERVs promoter, and a range of stress signals (e.g., injury, infection, environment). Endogenous retroviruses may exert pathophysiologic effects by infection followed by random reintegration into the genome, by their gene products (e.g., envelope, superantigen), and by altering the expression of neighboring genes. Several studies have provided evidence that ERVs are associated with a range of pathogenic processes involving multiple sclerosis, systemic lupus erythematosus, breast cancer, and the response to burn injury. For instance, the proinflammatory properties of the human ERV-W envelope protein play a central role in demyelination of oligodendrocytes. As reviewed in this article, recent advances in ERV biology and mammalian genomics suggest that ERVs may have a profound influence on various pathogenic processes including the response to injury and infection. Understanding the roles of ERVs in the pathogenesis of injury and infection will broaden insights into the underlying mechanisms of systemic immune disorder and organ failure in these patients.

Differential regulation of c-jun expression in liver and lung of mice after thermal injury.

In addition to skin injury, burns may also damage distant organs. Understanding the mechanisms of distant organ injury will substantially improve the survival of burn patients. Transcription factors are the major regulators of gene expression in response to most types of injury. C-Jun, which is a part of the activator protein-1 transcription factor complex, is one of the major immediate-early response genes, which is rapidly induced after injury. The expression of c-Jun in mouse liver and lung at different time points (3 h to 29 days) after thermal injury was examined by using Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blot, and immunohistochemistry. Rapid induction of c-Jun mRNA and protein was observed in the liver 3 h after an 18% TBSA burn. C-Jun expression returned to basal levels within 3 days after injury. In contrast to the up-regulation observed in liver, lungs from the same mice expressed c-Jun constitutively throughout the same time points. The finding that thermal injury leads to up-regulation of c-Jun in liver but not lungs suggests that either the liver has a lower threshold for early response to injury or that different cellular events exist when each organ is stressed.

Involvement of CD14 and toll-like receptor 4 in the acute phase response of serum amyloid A proteins and serum amyloid P component in the liver after burn injury.

Acute phase proteins such as serum amyloid A proteins (SAAs) and serum amyloid P component (SAP) are induced in the liver after various insults (e.g., infection, injury). The cellular and molecular mechanisms controlling the expression of these acute phase proteins may be specifically designed for different insults. The roles of two central molecules of the lipopolysaccharide (LPS)-mediated inflammation pathway (CD14 and toll-like receptor 4 [Tlr4]) were investigated for the regulation of SAAs and SAP in the liver of mice after an 18% total body surface area burn injury. RT-PCR analysis revealed a subtype- and time-dependent induction of SAA mRNAs between 3 h and 3 days, while there was a peak induction of SAP mRNA at day 1. Marked elevations of SAA and SAP protein levels at day 1 supported the mRNA data. Furthermore, a differential regulation of SAAs and SAP mRNAs was noted between CD14 knockout (KO) and their control mice after injury. SAA protein was induced to a lesser degree after injury in C3H/HeJ (Tlr4-defective) mice than in their control mice. In addition, in both CD14 KO and C3H/HeJ mice, the induction of SAP protein was significantly reduced compared with respective controls. These data provide evidence that CD14 and Tlr4 participate, at least in part, in a cascade of signaling events that control the immediate-early and differential induction of SAAs and SAP in the liver after injury. They also suggest that LPS may be one of the initial inducing agents associated with these acute phase responses in the liver after injury.

CLONING AND CHARACTERIZATION OF RHESUS CYTOMEGALOVIRUS GLYCOPROTEIN B

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques is an important model to investigate critical issues of cytomegalovirus biology. To better understand host immunological responses to viral glycoproteins, the glycoprotein B (gB) gene of RhCMV was molecularly cloned, sequenced and characterized. Transcription analysis revealed that RhCMV gB was transcribed as a late gene. The RhCMV gB gene encoded a predicted protein of 854 amino acids that was 60% identical/75% similar to the human CMV (HCMV) gB protein. The region of HCMV gB proposed to be responsible for virus binding to host cells, fusion and cell-to-cell spread was the most highly conserved region with RhCMV gB (74% identity/85% similarity). Conserved elements included 11 of 12 cysteine residues, 14 of 16 potential N-linked glycosylation sites and cross-reactive epitopes. Metabolic labelling experiments demonstrated that RhCMV gB was proteolytically processed similarly to HCMV gB. These results are critical for investigating virus-host relationships in CMV-infected primates.

Genome-wide expression profiles of endogenous retroviruses in lymphoid tissues and their biological properties

Endogenous retroviruses (ERVs) constitute approximately 8-10% of the human and mouse genome. Some autoimmune diseases are attributed to the altered expression of ERVs. In this study, we examined the ERV expression profiles in lymphoid tissues and analyzed their biological properties. Tissues (spleen, thymus, and lymph nodes [axillary, inguinal, and mesenteric]) from C57BL/6J mice were analyzed for differential murine ERV (MuERV) expression by RT-PCR examination of polymorphic U3 sequences. Each tissue had a unique profile of MuERV expression. A genomic map identifying 60 putative MuERVs was established using 22 unique U3s as probes and their biological properties (primer binding site, coding potential, transcription regulatory element, tropism, recombination event, and integration age) were characterized. Interestingly, 12 putative MuERVs retained intact coding potentials for all three polypeptides essential for virus assembly and replication. We suggest that MuERV expression is differentially regulated in conjunction with the transcriptional environment of individual lymphoid tissues.

Enhanced steroid response of a human glucocorticoid receptor splice variant.

ABSTRACT Glucocorticoids remain a recommended therapy in advanced septic shock despite the often unpredictable response, and our understanding of the mechanisms regulating the steroid and stress response remains limited. Since the initial sequencing of the human glucocorticoid receptor &agr; and &bgr; gene (hGR&agr; and hGR&bgr;), only three additional splice variants have been identified—all of which have been postulated to contribute to steroid resistance. During a survey of 97 healthy humans’ blood, we identified two novel hGR splice isoforms (hGR-S1 and hGR-S1(−349A) retaining intron H between exons 8 and 9. Human GR-S1(−349A) contained a base deletion causing an early termination and a truncated protein of 118 amino acids, whereas hGR-S1 had an early termination occurring within intron H and resulted in a 745–amino acid protein. Both isoforms had decreased transactivation potentials compared with hGR&agr; when tested in the absence of exogenous steroids. However, after treating with exogenous steroids, dose-response studies showed hGR-S1(−349A) had a substantial augmentation in activity at higher concentrations of hydrocortisone and methylprednisolone when compared with hGR&agr;, whereas hGR-S1 did not. Removal of the 3′ untranslated region (3′UTR) of the hGR-S1(−349A) mRNA sequence resulted in a loss of the augmented response. The isoform hGR-S1(−349A) augments the response to steroids, and this significant response appears to be critically regulated by the 3′UTR. The identification and evaluation of these unique hGR isoforms helps further the understanding of the complex genetic regulation of the stress and steroid response.

Injury-associated induction of two novel and replication-defective murine retroviral RNAs in the liver of mice

Injury can alter the expression of numerous genes in affected tissues as well as in distant organs. The mouse genome harbors numerous copies of endogenous murine leukemia virus (MuLV)-related retroviral sequences. Mouse liver tissues harvested after burn injury were subjected to RT-PCR analysis to investigate the regulation of MuLV-related sequences using a primer set capable of amplifying the full-length transcript. A doublet of approximately 5-kb was transiently up-regulated at 3 and 6 h after injury. Sequence analyses revealed that these are novel defective endogenous retroviral sequences (MuLV(LI-8) and MuLV(LI-12)), which are predominantly characterized by major deletions in pol and env genes. The MuLV(LI-8) clone is 4.85 kb long and the deduced gag polypeptide sequence was almost identical to a previously reported replication-defective retroviral sequence associated with immunesuppression. In the MuLV(LI-12) clone of 5.06 kb, there were two truncated gag open reading frames (ORFs) and 1 pol ORF fused to the C-terminus of the env p15E. Furthermore, the ORFs for the unique gag p12 presumed to be responsible for the immunesuppression were present in both clones. These novel replication-defective MuLVs may participate in the pathogenesis of distant organs after injury.

Alterations in the expression and modification of histonesin the liver after injury

Chromatin remodeling plays a key role in the transcriptional activation of regulatory factors in the liver in response to a variety of stress signals. The effects of burn injury on histone expression and its modification were investigated in this study. Liver tissues collected after a flame burn injury were subjected to RT-PCR and Western blot analyses of histone regulation. There was a marked induction of histone H3-D variant mRNA at 3 and 6 h. In contrast, histone H2A.2 variant mRNA had a downregulation at 3 days. No apparent changes were noted in other histone variants examined. Western blot analysis revealed a downregulation of all 5 histone subtypes (H1, H2A, H2B, H3, and H4) at 1 day and there was a subsequent induction of H1 and H2A subtypes at 3 days after injury. There was an induction of modified forms (phospho-, acetyl-, and dimethyl-) of histone H3 subtype at day 3. Furthermore, a transient elevation in PCNA (proliferating cell nuclear antigen) levels was apparent in the liver at day 3, which parallels the induction of phospho-histone H3, which is a mitosis marker. These findings suggest that histones participate in a cascade of events associated with phenotypic alterations in the liver after injury.

Cloning and characterization of endogenous retroviruses associated with postinjury stress signals in lymphoid tissues

Endogenous retroviruses (ERVs) constitute a significant fraction of the mouse and human genomes, ∼10% and ∼8%, respectively, and they are transmitted to offsprings in a Mendelian fashion. Recent reports implicated that certain ERVs participate in a range of disease processes. In this study, we examined injury-elicited changes in murine ERV (MuERV) expression in lymphoid tissues and characterized biological properties of the putative MuERVs isolates. Female C57BL/6J mice were subjected to ∼18% total-body-surface-area burn injury. Four different lymphoid tissues (blood, bone marrow, spleen, and thymus) were collected at 24 h for reverse transcriptase-polymerase chain reaction analysis of MuERV expression by amplifying the 3′ U3 regions. Within each tissue examined, there was a unique pattern of injury-elicited changes in MuERV expression. From the 17 unique MuERV U3 clones isolated from all four tissues, nine were derived from injury-induced MuERVs, four from injury repressed, and four from no change. A survey of the C57BL/6J genome using all 17 U3 clones as probes produced 26 pertinent putative MuERVs, of which five were presumed to retain intact coding potentials for essential polypeptides. Biological properties (genomic location, tropism, transcriptional potential, coding potential, primer-binding site, recombination, and integration age) of each putative MuERV were characterized, and their relevance to injury response was discussed. The findings from this study suggest that injury-elicited stress signals either induce or repress specific MuERV populations in a lymphoid tissue-specific and probably cell type-specific manner. It warrants a further investigation into the roles of the injury-responsive MuERVs in postinjury pathogenic processes of the immune system.

Downregulation Of Nf-κb Activity Associated With Alteration In Proliferative Response In The Spleen After Burn Injury

Alterations in proliferation status and cellular composition of immune organs are among key events in the modulation of immune function after burn injury. Nuclear factor (NF)-κB is a transcription factor that plays a pivotal role in the response to injury as well as immune cell differentiation and proliferation. In this study, we investigated the effects of burn injury on the activity of NF-κB and its association with cellular proliferation in the spleen. Western analysis of whole spleen tissues of mice after 18% burn injury revealed a marked reduction in nuclear NF-κB rel A protein expression 3 to 21 days after injury when there was an increase in proliferative activity in the red pulp of the spleen after injury as indicated by an increase in proliferating cell nuclear antigen (PCNA). In the splenic B cells, however, the down-regulation of NF-κB rel A was associated with decreased PCNA expression as well as IκBα and phosphorylated IκBα. In contrast, no significant change in NF-κB rel A or PCNA expression was observed for splenic T cells. These data suggest that there is a differential regulation of NF-κB and proliferative activity in the splenic cell subsets after burn injury. Furthermore, the regulation of NF-κB may be linked to the proliferative changes seen in the spleen after burn injury.