Lee K. Adamson

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.

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.

Alterations in the Levels of Metallothionein and Metals in the Liver, and Unique Serum Liver Enzyme Response in Metallothionein Knock-Out Mice after Burn Injury

Objectives: Metallothionein (MT) is a small cysteine-rich protein that sequesters and distributes metal ions. Its overexpression stimulates cell proliferation and inhibits apoptosis. We investigated the effects of burn injury on MT expression and metal localization. We also sought to determine roles of MT in the pathophysiologic alterations in the liver after injury. Methods: Mice (C57BLKS/J, MT-I/II knock-out, KO, and wild-type control mice) were subjected to an 18% burn. Liver tissues harvested after injury were analyzed for the MT expression and the levels of zinc, copper, manganese, and iron. Levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were measured in serum samples from MT-I/MT-II KO mice and controls after injury. Results: Transient induction of MT-I and MT-II mRNAs was observed 3–6 h after injury, while MT-I/MT-II protein peaked on day 1. The induction was localized to hepatocytic nuclei. The intrahepatic levels of zinc, copper, and iron were transiently elevated on day 1, when a downregulation of manganese was evident. Interestingly, only the serum levels of aspartate aminotransferase were significantly augmented in MT-I/MT-II KO mice compared to controls after injury. Conclusions: These data suggest that MT and metals may participate in the pathogenesis of the liver after burn injury.

Regulation of murine endogenous retroviruses in the thymus after injury

We recently reported the induction of murine endogenous retroviruses (murine AIDS-related) in several distant organs of mice after burn injury. The regulation of endogenous retroviruses in response to burn injury was further investigated in the thymus. Female C57BLKS/J mice were subjected to 18% total body surface area flame burn injury. Thymus tissues collected at several time points (3 h to 7 days) were analyzed for the expression of subgenomic transcripts of murine endogenous retroviruses by RT-PCR. Interestingly, a novel 1.7-Kb subgenomic transcript and a recently described 1.1 Kb subgenomic transcript of murine endogenous retroviruses were transiently down-regulated in the thymus at day 1 after injury. The 1.7 Kb transcript has a coding potential for a truncated form of the envelope protein (total 214 amino acids) with a deletion of 418 amino acids near the C-terminus. The second transcript of 1.1 Kb has an open reading frame for the C-terminal transmembrane domain of the envelope protein including the p2E protein (R peptide). These data suggest the pathophysiologic effects of burn injury on the differential expression of murine endogenous retroviruses in the thymus after injury.

Injury-associated differential regulation of histone expression and modification in the thymus of mice.

One of the key events in the regulation of gene expression is chromatin remodeling involving histone regulation. We investigated the effects of burns on the expression of histone that might be associated with altered molecular and pathological profiles in the thymus. A markedly decreased expression of histone variant H2A.1 mRNA was identified in the thymus after burn during a differential display experiment. Subsequently, we examined the histone expression (mRNA and protein) and posttranslational modification in the thymus after burn. Also, changes in proliferating cell nuclear antigen (PCNA), a central molecule in chromatin assembly, was examined. Reverse-transcription polymerase chain reaction analysis revealed a transient decrease in the expression of several histone variants (H2A.1, H1(r1), H3-B, H3-1, and H4-D) mRNAs in the thymus at 1 day after burn. A decrease in histone subtypes H2A, H2B, H3, and H4, but not H1, was demonstrated 1 and 3 days after burn according to the results of Western blot. Furthermore, there were different levels of decreases in acetylated and dimethylated forms of histone H3 1 and 3 days after burn. In addition, decreased levels of PCNA were evident in the thymus 1 day after burn. Changes in the expression of histones and PCNA may reflect mere decrease in proliferating cells and/or a reorganization of the chromatin structure associated with altered transcriptional activities, eventually contributing to the phenotypic changes in the thymus after burn.

Direct quantification of autologous serum albumin leakage after burn injury in mice

The current technique determining the extent of capillary leakage after injury is to measure the leakage of dye-labeled foreign albumin. A recent report, however, demonstrated that albumin leakage is dependent upon the type of fluorescent dye used for labeling. We chose to develop and test a technique for determining the extent of vascular albumin leakage after burn injury without the use of dyes. Skin and blood samples were harvested at 3h and 7 days after burn injury in mice. Total skin lysates and extracts as well as sera were analyzed for albumin leakage. Coomassie staining and Western blot analyses of skin preparations followed by the densitometric measurement revealed increased levels of albumin, suggesting that the leakage of serum albumin started within 3h after burn injury. Instead of employing dye-labeled foreign albumin, the use of SDS-polyacrylamide gel electrophoresis of tissue extracts followed by Coomassie staining will allow for a simple and direct quantification of autologous albumin leakage due to burn as well as other types of injury.

BURN INJURY INDUCES AN INHIBITORY SIGNAL IN THE LUNG SMAD PATHWAY.: 3

Smad signaling mediates the cellular response to transforming growth factor-beta (TGF-beta). We hypothesize that variations in Smad signaling modify the response to TGF-beta signaling in the lung after injury. C57BLKS/J mice were subjected to an 18% surface area burn injury, sacrificed at specific time points and their lung tissue was harvested. Lung TGF-beta1 expression, as determined by RT-PCR, ELISA and PAI/Luciferase assay, was not affected by injury. Western blots for Smad2/3 and Smad4 on nuclear fractions revealed decreased Smad2, Smad3, and Smad4 protein levels at 3h, while their total cellular levels did not differ from control mice. Smad7 protein increased transiently at 3 h. Correlating with Smad inhibition, transcription in type I alpha-2 collagen was also transiently depressed. By RT-PCR, Smad3 and Smad7 mRNAs decreased at 3 h, while Smad2 and Smad4 mRNA levels remained constitutive. Burn injury did not alter lung TGF-beta1 expression but caused Smad inhibition through decreased nuclear translocation of Smad2, Smad3, and Smad4, and upregulated Smad7. Transcription was not the key regulatory step in Smad protein expression, as transient decreases in Smad3 and Smad7 mRNA did not correlate with protein levels. It appears that Smad activity may in part attenuate TGF-beta activity after burn injury.