Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1 and NF-kappaB activation.

Liver transplantation is the only therapeutic strategy for many inherited and acquired diseases. The formation of reactive oxygen species following ischemia/reperfusion is a cause of hepatocellular injury during transplantation. This report describes the therapeutic application of mitochondrial superoxide dismutase gene transfer to the liver for acute ischemia/reperfusion injury. Recombinant adenoviral expression of mitochondrial superoxide dismutase in mouse liver prior to lobar ischemia/reperfusion significantly reduced acute liver damage and associated redox activation of both NF-kappaB and AP1. These immediate early transcription factors represent common pathways by which cells respond to environmental stress. This work provides the foundation for redox-mediated gene therapies directed at ameliorating ischemia/reperfusion injury and associated acute rejection in orthotopic liver transplantation.

The antiandrogen enzalutamide downregulates TMPRSS2 and reduces cellular entry of SARS-CoV-2 in human lung cells.

SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide-a well-tolerated drug widely used in advanced prostate cancer-reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

NF-kappaB-independent sensitization of glioblastoma cells for TRAIL-induced apoptosis by proteasome inhibition.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key regulator of stress-induced transcriptional activation and has been implicated in mediating primary or acquired apoptosis resistance in various cancers. In the present study, we therefore investigated the role of NF-kappaB in regulating apoptosis in malignant glioma, a prototypic tumor refractory to current treatment approaches. Here, we report that constitutive NF-kappaB DNA-binding activity was low or moderate in eight different glioblastoma cell lines compared to Hodgkin's lymphoma cells, known to harbor aberrant constitutive NF-kappaB activity. Specific inhibition of NF-kappaB by overexpression of inhibitor of kappaB (IkappaB)alpha superrepressor did not enhance spontaneous apoptosis of glioblastoma cells. Also, overexpression of IkappaBalpha superrepressor had no significant impact on apoptosis induced by two prototypic classes of apoptotic stimuli, that is, chemotherapeutic drugs or death-inducing ligands such as TNF-related apoptosis inducing ligand (TRAIL), which are known to trigger NF-kappaB activation as part of a cellular stress response. Similarly, inhibition of NF-kappaB by the proteasome inhibitor MG132 did not increase doxorubicin (Doxo)-induced apoptosis of glioblastoma cells, although it prevented DNA binding of NF-kappaB complexes in response to Doxo. Interestingly, proteasome inhibition significantly sensitized glioblastoma cells for TRAIL-induced apoptosis. These findings indicate that the characteristic antiapoptotic function of NF-kappaB reported for many cancers is not a primary feature of glioblastoma and thus, specific NF-kappaB inhibition may not be effective for chemosensitization of glioblastoma. Instead, proteasome inhibitors, which enhanced TRAIL-induced apoptosis in an NF-kappaB-independent manner, may open new perspectives to increase the efficacy of TRAIL-based regimens in glioblastoma, which warrants further investigation.

CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver.

The success of orthotopic liver transplantation is dependent on multiple factors including MHC tissue compatibility and ischemic/reperfusion injury. Ischemic/reperfusion (I/R) injury in the liver occurs in a biphasic pattern consisting of both acute phase (oxygen free radical mediated) and subacute phase (neutrophil-mediated) damage. Although numerous studies have given insights into the process of neutrophil recruitment after I/R injury to the liver, the exact mechanism that initiates this subacute response remains undefined. Using a T cell-deficient mouse model, we present data that suggests that T-lymphocytes are key mediators of subacute neutrophil inflammatory responses in the liver after ischemia and reperfusion. To this end, using a partial lobar liver ischemia model, we compared the extent of reperfusion injury between immune competent BALB/c and athymic nu/nu mice. Studies evaluating the extent of liver damage as measured by serum transaminases (GPT) demonstrate similar acute (3-6 h) post-I/R responses in these two mouse models. In contrast, the subacute phase (16-20 h) of liver injury, as measured by both serum GPT levels and percent hepatocellular necrosis, was dramatically reduced in T cell-deficient mice as compared with those with an intact immune system. This reduction in liver injury seen in nu/nu mice was associated with a 10-fold reduction in hepatic neutrophil infiltration. Adoptive transfer of T cell-enriched splenocytes from immune competent mice was capable of reconstituting the neutrophil-mediated subacute inflammatory response within T cell-deficient nu/nu mice. Furthermore, in vivo antibody depletion of CD4(+) T-lymphocytes in immune competent mice resulted in a reduction of subacute phase injury and inflammation as measured by serum GPT levels and neutrophil infiltration. In contrast, depletion of CD8(+) T-lymphocytes had no effect on these indexes of subacute inflammation. Kinetic analysis of T cell infiltration in the livers of BALB/c mice demonstrated a fivefold increase in the number of hepatic CD4(+) T-lymphocytes within the first hour of reperfusion with no significant change in the number of CD8(+) T-lymphocytes. In summary, these results implicate CD4(+) T-lymphocytes as key regulators in initiating I/R-induced inflammatory responses in the liver. Such findings have implications for therapy directed at the early events in this inflammatory cascade that may prove useful in liver transplantation.

The yeast TEL1 gene partially substitutes for human ATM in suppressing hyperrecombination, radiation-induced apoptosis and telomere shortening in A-T cells.

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.

Expression of the recessive glomerulosclerosis gene Mpv17 regulates MMP-2 expression in fibroblasts, the kidney, and the inner ear of mice.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

Overexpression of human catalase inhibits proliferation and promotes apoptosis in vascular smooth muscle cells.

The role of reactive oxygen species, such as superoxide anions (O(2). (-)) and hydrogen peroxide (H(2)O(2)), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H(2)O(2), rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene, beta-galactosidase (AdLacZ). Infection with AdCat resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat, cellular catalase activity was increased by 50- to 100-fold, and intracellular H(2)O(2) concentration was reduced, as compared with control. Infection with AdCat reduced [(3)H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.37+/-0.09 disintegrations per minute per cell [AdLacZ] versus 0.22+/-0.08 disintegrations per minute per cell [AdCat], P<0.05). Five days after infection with 100 MOI of AdCat, cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 780+/-8413 [AdCat], P<0.05 versus 233 700+/-3032 [noninfected] or 222 410+/-5332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCat as compared with control. Infection with AdCat resulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H(2)O(2) importantly modulates survival and proliferation of vascular smooth muscle cells.

Therapeutic approaches for ischemia/reperfusion injury in the liver.

Organ injury caused by transient ischemia followed by reperfusion is associated with a number of clinically and environmentally induced conditions. Ischemia/reperfusion (I/R) conditions arise during surgical interventions such as organ transplantation and coronary bypass surgery, and in diseases such as stroke and cardiac infarct. The destructive effects of I/R arise from the acute generation of reactive oxygen species subsequent to reoxygenation, which inflict direct tissue damage and initiate a cascade of deleterious cellular responses leading to inflammation, cell death, and organ failure. This review summarizes existing and potential approaches for treatment that have been developed from research using model systems of I/R injury. Although I/R injury in the liver is emphasized, other organ systems share similar pathophysiological mechanisms and therapeutic approaches. We also review current knowledge of the molecular events controlling cellular responses to I/R injury, such as activation of AP-1 and NF-kappaB pathways. Therapeutic strategies aimed at ameliorating I/R damage are focused both on controlling ROS generated at the time of oxygen reperfusion and on intervening in the activated signal transduction cascades. Potential therapies include pharmacological treatment with small molecules, antibodies to cytokines, or free-radical scavenging enzymes, such as superoxide dismutase or catalase. Additionally, the use of gene therapy approaches may significantly contribute to the development of strategies aimed at inhibiting of I/R injury.

Redox gene therapy protects human IB-3 lung epithelial cells against ionizing radiation-induced apoptosis.

Toxicity to nontumor-derived tissue has proven to be a significant obstacle in achieving therapeutic levels of gamma irradiation in the treatment of cancer. The formation of reactive oxygen species (ROS) such as superoxide radicals (O2-) following irradiation is thought to be a major determinant of cellular damage. To this end, we describe the generation of two recombinant adenoviral vectors expressing the radical-scavenging enzymes MnSOD and CuZnSOD to test therapeutic strategies of radioprotection. Using a human lung epithelial cell line (IB-3), we have demonstrated that infections with both Ad.CMVMnSOD or Ad.CMVCuZnSOD significantly increase both the levels of SOD protein and enzymatic activity as compared to control cells. This increase in SOD expression reduced the level of apoptosis at 72 hr post-irradiation by 50% as compared to mock- or Ad.CMVLacZ-infected cells. Such studies provide the foundation for radioprotective gene therapies in the treatment of cancer.

Age-dependent hypertension in Mpv17-deficient mice, a transgenic model of glomerulosclerosis and inner ear disease.

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

Gene therapy for colon cancer.

The enormous number of newly diagnosed cases of colorectal cancer that occur each year and the lack of agents that are highly effective for all patients underscore the need for novel approaches to combating the disease. Gene therapy as a developing treatment modality is already well established, with a number of trials ongoing and a vast range of other approaches being assessed in animal and cell culture experiments. In this brief review, we have discussed five gene therapy trials in colon carcinoma that are ongoing or in the approval process in the United States. The gene therapy approaches being employed can be divided into three major categories: (1) enzyme/prodrug systems (HSVtk/ganciclovir; CD/5-fluorocytosine); (2) tumor suppressor gene replacement therapy with wild-type p53; and (3) immune-gene therapy which is based on cytokine or tumor antigen expression to induce tumor immunity (e.g., CEA). Replication-deficient recombinant adenoviral vectors are predominantly used for colon cancer gene therapy, because they can be produced at high titer and they readily infect a number of different cell types. One trial uses polynucleotide therapy for antitumor immunization with intramuscular injection. All of these studies are phase I trials, principally designed to evaluate safety, but they will also provide data on gene delivery. Some trials may provide some insight into potential therapeutic effects. We have alluded to some of the concerns on toxicity related to the use of adenovirus, risks and side effects from transgenes, lack of tumor-specificity of transgene expression, and potential problems with efficient gene delivery to solid tumors. The clinical trials, however, will provide insight that will inform design of future studies with respect to dose, form, and frequency of administration, as well as to the value of biologic and clinical endpoints. The molecular analysis of the fundamental basis of colon cancer has moved at a remarkable pace and that progress seems set to continue. Thus, the basic foundations for gene therapy are undoubtedly in place: a clinical need; growing understanding of basic tumor biology; and ever-improving delivery systems. The field is at a very early stage in its evolution, and one concern is that the considerable hurdles that must be overcome are seen as examples of the failure of cancer gene therapy; however, we believe these challenges will be overcome. The authors also believe that colon cancer gene therapy is likely to take new directions, such as use as adjuvant to radical surgery, rather than attempts to treat end-stage disease when the liver is replaced by metastases. Other new directions might include prophylactic gene-based immunization against a panel of well-characterized tumor antigens, at least for persons shown to be at high risk of colon cancer because of genetic or other predisposition. A marriage between gene therapy approaches and conventional anticancer treatments such as radiotherapy and chemotherapy also seems likely. There is already evidence of this move with demonstration of synergism between p53 replacement and radiotherapy and chemotherapy. It is also likely that therapies will be developed that combine elements from the cancer gene therapies discussed previously, namely, suicide gene transfer, immune modulation, and modulation of defective cancer genes. Perhaps one of the main concerns is not that researchers in cancer gene therapy want to walk before they can run, but that the public and government agencies believe they can. The next 10 years will be an interesting time in the development of novel treatments against colon cancer.

Loss of auditory function in transgenic Mpv17-deficient mice.

The transgenic mouse strain Mpv17 develops severe morphological degeneration of the inner ear and nephrotic syndrome at a young age (Meyer zum Gottesberge et al., 1996; Weiher et al., 1990). The audiograms (1-32 kHz) of Mpv17-negative mice were determined from auditory brain stem responses in young (2 months) and old (7 months) animals. Audiograms of age-matched wild-type mice with the same genetic background, but wild-type at the Mpv17 locus, were also determined. Furthermore, young Mpv17-negative mice that carried a human Mpv17 homologue gene were studied. NMRI mice served as a reference for normal hearing. Mpv17-negative mice suffer from severe sensorineural hearing loss as early as 2 months after birth. In the old Mpv17-negative mice no responses could be elicited at all. The 2 month old wild-type mice had normal audiograms, at 7 months only high threshold responses were seen. The poor audiograms of the Mpv17-negative mice are assumed to be the functional correlate of the morphological degeneration of the cochlea described earlier (Meyer zum Gottesberge et al., 1996). The finding that 2 out of 4 Mpv17-negative mice with the human Mpv17 gene had normal audiograms, shows that the gene inactivation can be functionally compensated by the human Mpv17 gene product.

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects.

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL(DR5), had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL(DR4) when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments.

MnSOD protects colorectal cancer cells from TRAIL-induced apoptosis by inhibition of Smac/DIABLO release.

The mitochondrial enzyme manganese superoxide dismutase (MnSOD) has been shown to have two faces with regard to its role in tumor development. On the one side, it is well documented that overexpression of MnSOD slows down cancer cell growth, whereas on the other side MnSOD also has a metastasis-promoting activity. We set out to examine the role of MnSOD in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, thought to be a first-line tumor surveillance mechanism and failure to undergo apoptosis might contribute to metastasis formation. We show that overexpression of MnSOD at moderate levels is able to protect cells from TRAIL-induced apoptosis. While caspase-8 activation and Bid cleavage were not affected by MnSOD, we detected a marked decrease in caspase-3 activation pointing to a mitochondrial resistance mechanism. Indeed, we found that MnSOD-overexpressing cells showed reduced cytochrome c and no Smac/DIABLO release into the cytosol. The resulting lack of X-linked inhibitor of apoptosis (XIAP) inhibition by cytosolic Smac/DIABLO most likely caused the TRAIL resistance as RNAi against XIAP-rescued caspase-3 activity and TRAIL sensitivity. Our results show that reactive oxygen species are involved in TRAIL-induced Smac/DIABLO release and in TRAIL-triggered apoptosis. Hence, high levels of MnSOD, which decompose and neutralize these reactive oxygen species, might contribute to metastasis formation by allowing disseminated tumor cells to escape from TRAIL-mediated tumor surveillance. As part of TRAIL regimens, adjuvant treatment with XIAP inhibitors in the form of Smac/DIABLO mimetics or MnSOD inhibitors might be able to break TRAIL resistance of malignant tumor cells.

[Gene therapy strategies for colorectal cancer]. / Gentherapeutische Strategien gegen das kolorektale Karzinom.

Current advances in CRC treatment have led to significant but slight improvements in patient survival with curative outcomes only seen in earlier stage cancers. Consequently, much effort is being put into developing completely novel therapies that fulfil a number of criteria including greater efficacy and fewer side effects. Many of these conditions are met by the wide range of gene therapy strategies currently in pre-clinical or clinical trial phases. Gene therapy approaches may be broadly broken down into three main areas: Following a few tragic events in the context of clinical gene therapy studies, safety is currently the prime concern. Further progress in the field is expected from the combination of the described approaches with conventional treatment modalities.

NF-kappaB kinetics predetermine TNF-alpha sensitivity of colorectal cancer cells.

BACKGROUND: Tumour necrosis factor (TNF)-alpha has considerable anti-tumour activity and may have potential as a treatment for metastatic colorectal cancer. However, TNF-alpha responses in patients and cell lines are variable and TNF-alpha treatment is associated with dose limiting clinical toxicity. Activation of NF-kappaB is protective against TNF-alpha induced cell death, and this may explain tumour resistance. METHODS: In order to provide further understanding of determinants of TNF-alpha responses, we studied TNF-alpha induced NF-kappaB activation and variable tumour responses. We analysed the kinetics of TNF-alpha induced NF-kappaB activation in colorectal cancer cells and determined whether it is possible to sensitize colorectal tumour cells to TNF-alpha by modulation of NF-kappaB signalling. RESULTS: We demonstrated that sustained NF-kappaB activation exceeding 16 h was observed in HRT18 and SW480 cells and was associated with TNF-alpha resistance. In contrast, transient NF-kappaB activation in HCT116 cells was associated with sensitivity to cytotoxic TNF-alpha effects, suggesting that NF-kappaB kinetics may have utility as clinical marker of TNF-alpha tumour resistance. Despite variable TNF-alpha responses and NF-kappaB kinetics, all three colorectal cancer cell lines were highly sensitive to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) which induced only transient NF-kappaB activation. This further supports the notion of a pre-determined NF-kappaB response influencing receptor-mediated cell death. We also show that stable transfection and adenoviral-mediated expression of IkappaB(A32/36) can be used to confer TNF-alpha sensitivity to colorectal tumour cells previously resistant. CONCLUSIONS: These findings indicate that a combined approach using gene therapy and recombinant TNF-alpha merits further appraisal. Furthermore, the kinetics of the TNF-alpha response could be determined using a 'test-dose' to indicate whether individual patients might benefit from this gene therapy approach.

Reactive oxygen species in oncogenic transformation.

Ever since ROS (reactive oxygen species) were shown to meet the criteria of true signalling molecules, such as regulated production and a specific biological function, many efforts have been made to understand the precise role of ROS. The function of ROS in pathological mechanisms is taking a more and more central role in various fields of biomedical research, including neurobiology, cardiology and cancer. An elevated oxidative status has been found in many types of cancer cells, and the introduction of chemical and enzymological antioxidants can inhibit tumour cell proliferation, pointing to a critical role of ROS in mediating loss of growth control. The present review describes ROS-regulated mechanisms that are associated with cancer and tumour invasiveness. The cellular processes that are linked to these ROS functions are mitogenic signalling and cell motility, while ROS have also been implicated in apoptosis and cellular senescence, two mechanisms regarded as being anti-tumorigenic. This "two-faced" character of free radicals will be discussed and placed in the context of the physiological conditions of the tumour cell, the different molecular backgrounds, and the specific ROS. More detailed understanding of the signalling pathways regulated by ROS in tumour cells will open up new prospects for chemo- or gene-therapeutic interventions.

Functional rescue of the glomerulosclerosis phenotype in Mpv17 mice by transgenesis with the human Mpv17 homologue.

The germ line insertion of a defective retrovirus into the Mpv17 gene of mice is associated with a recessive phenotype. Mice homozygous for the integration develop glomerulosclerosis at a young age. The phenotype resembles human glomerulosclerosis in its physiological parameters as well as in histology. A human homologue of the Mpv17 gene has been identified, isolated and analyzed. We here show that this gene, which has a role in the production of reactive oxygen species, can rescue the phenotype of Mpv17 deficient mice when introduced by transgenesis. This provides formal proof for the hypothesis that the phenotype is caused by the loss of function of the Mpv17 gene. It also provides evidence for the functional conservation of the Mpv17 gene in mammals and points to a potential role of this gene in human kidney disease.

Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor kappaB independently of IkappaB degradation.

For many inherited and acquired hepatic diseases, liver transplantation is the only possible therapeutic strategy. Ischemia/reperfusion (I/R) damage to donor tissue is thought to be one component that may play a role in the decline of posttransplant tissue function and ultimately rejection. The transcription factors, AP-1 and nuclear factor kappaB (NF-kappaB), play important roles in the acute cellular responses to tissue damage, as well as the inflammatory phase following I/R. We have found that the DNA binding activity of AP-1 was dramatically increased following warm ischemia at 1 to 3 hours postreperfusion. Induced DNA binding activity was composed of predominately c-Jun and JunD hetero- and homodimers as determined by electrophoretic mobility supershift assays. This increase in AP-1 activity occurred in the absence of significant changes in the steady-state protein levels of c-Jun and JunB. Maximal activation of Jun amino-terminal kinase ( JNK) occurred within the 25 to 30 minutes postreperfusion, just before the peak in AP-1 DNA binding. These findings suggest that phosphorylation may play an important role in regulating AP-1 transcriptional complexes. Furthermore, JunD protein levels slightly increased at 3 hours postreperfusion, concordant with changes in AP-1 DNA binding activity. The activation of NF-kappaB at 1 hour postreperfusion was independent of proteolytic degradation of IkappaB- or IkappaB-beta. This activation of NF-kappaB DNA binding activity in the nucleus was preceded by an increase in tyrosine phosphorylation of IkappaB-. These studies suggest that JNK, IkappaB tyrosine kinase, and JunD are potential targets for therapeutic intervention during liver I/R injury.