Regulatory factors and control of anthrax toxin gene expression

Bacillus anthracis plasmid pXO1 carries genes for three anthrax toxin proteins, pag (protective antigen), cya (edema factor), and lef (lethal factor). Expression of the toxin genes is enhanced by two signals: CO$\sb2$/bicarbonate and temperature. The CO$\sb2$/bicarbonate effect requires the presence of pXO1. I hypothesized that pXO1 harbors a trans-acting regulatory gene(s) required for CO$\sb2$/bicarbonate-enhanced expression of the toxin genes. Characterization of such a gene(s) will lead to increased understanding of the mechanisms by which B. anthracis senses and responds to host environments.^ A regulatory gene (atxA) on pXO1 was identified. Transcription of all three toxin genes is decreased in an atxA-null mutant. There are two transcriptional start sites for pag. Transcription from the major site, P1, is enhanced in elevated CO$\sb2$. Only P1 transcripts are significantly decreased in the atxA mutant. Deletion analysis of the pag upstream region indicates that the 111-bp region upstream of the P1 site is sufficient for atxA-mediated increase of this transcript. The cya and lef genes each have one apparent transcriptional start site. The cya and lef transcripts are significantly decreased in the atxA mutant. The atxA mutant is avirulent in mice. The antibody response to all three toxin proteins is significantly decreased in atxA mutant-infected mice. These data suggest that the atxA gene product activates expression of the toxin genes and is essential for virulence.^ Since expression of the toxin genes is dependent on atxA, whether increased toxin gene expression in response to CO$\sb2$/bicarbonate and temperature is associated with increased atxA expression was investigated. I monitored steady state levels of atxA mRNA and AtxA protein in different growth conditions. The results indicate that expression of atxA is not influenced by CO$\sb2$/bicarbonate. Steady state levels of atxA mRNA and AtxA protein are higher at 37$\sp\circ$C than 28$\sp\circ$C. However, increased pag expression at high temperature can not be attributed directly to increased atxA expression.^ There is evidence that an additional factor(s) may be involved in regulation of pag. Expression of pag in strains overproducing AtxA is significantly decreased compared to the wildtype strain. A specific interaction of tagged-AtxA with the pag upstream DNA has not been demonstrated. Furthermore, four proteins in B. anthracis extract can be co-immunoprecipitated with tagged-AtxA. Amino-terminal sequence of one protein has been determined and found highly homologous to chaperonins of GroEL family. Studies are under way to determine if this GroEL-like protein interactions with AtxA and plays any role in atxA-mediated activation of toxin genes. ^

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Systemic root signalling in a belowground, volatile-mediated tritrophic interaction

Plants attacked by leaf herbivores release volatile organic compounds (VOCs) both locally from the wounded site and systemically from non-attacked tissues. These volatiles serve as attractants for predators and parasitoids. This phenomenon is well described for plant leaves, but systemic induction of VOCs in the roots has remained unstudied. We assessed the spatial and temporal activation of the synthesis and release of (E)-β-caryophyllene (EβC) in maize roots upon feeding by larvae of Diabrotica virgifera virgifera, as well as the importance of systemically produced EβC for the attraction of the entomopathogenic nematode Heterorhabditis megidis. The production of EβC was found to be significantly stronger at the site of attack than in non-attacked tissues. A weak, but significant, increase in transcriptional activity of the EβC synthase gene tps23 and a corresponding increase in EβC content were observed in the roots above the feeding site and in adjacent roots, demonstrating for the first time that herbivory triggers systemic production of a volatile within root systems. In belowground olfactometers, the nematodes were significantly more attracted towards local feeding sites than systemically induced roots. The possible advantages and disadvantages of systemic volatile signalling in roots are discussed.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Developmental programming of tumor suppressor gene penetrance

Epidemiological studies have led to the hypothesis that major risk factors for developing diseases such as hypertension, cardiovascular disease and adult-onset diabetes are established during development. This developmental programming hypothesis proposes that exposure to an adverse stimulus or insult at critical, sensitive periods of development can induce permanent alterations in normal physiological processes that lead to increased disease risk later in life. For cancer, inheritance of a tumor suppressor gene defect confers a high relative risk for disease development. However, these defects are rarely 100% penetrant. Traditionally, gene-environment interactions are thought to contribute to the penetrance of tumor suppressor gene defects by facilitating or inhibiting the acquisition of additional somatic mutations required for tumorigenesis. The studies presented herein identify developmental programming as a distinctive type of gene-environment interaction that can enhance the penetrance of a tumor suppressor gene defect in adult life. Using rats predisposed to uterine leiomyoma due to a germ-line defect in one allele of the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, these studies show that early-life exposure to the xenoestrogen, diethylstilbestrol (DES), during development of the uterus increased tumor incidence, multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Uterine leiomyomas are ovarian-hormone dependent tumors that develop from the uterine myometrium. DES exposure was shown to developmentally program the myometrium, causing increased expression of estrogen-responsive genes prior to the onset of tumors. Loss of function of the normal Tsc-2 allele remained the rate-limiting event for tumorigenesis; however, tumors that developed in exposed animals displayed an enhanced proliferative response to ovarian steroid hormones relative to tumors that developed in unexposed animals. Furthermore, the studies presented herein identify developmental periods during which target tissues are maximally susceptible to developmental programming. These data suggest that exposure to environmental factors during critical periods of development can permanently alter normal physiological tissue responses and thus lead to increased disease risk in genetically susceptible individuals. ^

The effect of genotype-by-environment interaction on longitudinal triglyceride profiles in the Bogalusa Heart Study

Triglyceride levels are a component of plasma lipids that are thought to be an important risk factor for coronary heart disease and are influenced by genetic and environmental factors, such as single nucleotide polymorphisms (SNPs), alcohol intake, and smoking. This study used longitudinal data from the Bogalusa Heart Study, a biracial community-based survey of cardiovascular disease risk factors. A sample of 1191 individuals, 4 to 38 years of age, was measured multiple times from 1973 to 2000. The study sample consisted of 730 white and 461 African American participants. Individual growth models were developed in order to assess gene-environment interactions affecting plasma triglycerides over time. After testing for inclusion of significant covariates and interactions, final models, each accounting for the effects of a different SNP, were assessed for fit and normality. After adjustment for all other covariates and interactions, LIPC -514C/T was found to interact with age3, age2, and age and a non-significant interaction of CETP -971G/A genotype with smoking status was found (p = 0.0812). Ever-smokers had higher triglyceride levels than never smokers, but persons heterozygous at this locus, about half of both races, had higher triglyceride levels after smoking cessation compared to current smokers. Since tobacco products increase free fatty acids circulating in the bloodstream, smoking cessation programs have the potential to ultimately reduce triglyceride levels for many persons. However, due to the effect of smoking cessation on the triglyceride levels of CETP -971G/A heterozygotes, the need for smoking prevention programs is also demonstrated. Both smoking cessation and prevention programs would have a great public health impact on minimizing triglyceride levels and ultimately reducing heart disease. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Signaling pathways in the transcriptional and post-translational regulation of the human glutathione S -transferase P1 gene

The human glutathione S-transferase P1 (GSTP1) protein is an endogenous inhibitor of c-jun N-terminal kinases (JNKs) and an important phase II detoxification enzyme. ^ Recent identification of a cAMP response element (CRE) in the 5 ′-region of the human GSTP1 gene and several putative phosphorylation sites for the Ser/Thr protein kinases, including, cAMP-dependent protein kinases (PKAs), protein kinases C (PKCs), and JNKs in the GSTP1 protein raised the possibility that signaling pathways may play an important role in the transcriptional and post-translational regulation of GSTP1 gene. This study examined (a) whether the signaling pathway mediated by CAMP, via the GSTP1 CRE, is involved in the transcriptional regulation of the GSTP1 gene, (b) whether signaling pathways mediated by the Ser/Thr protein kinases (PKAs, PKCs, and JNKs) induce post-translational modification, viz. phosphorylation of the GSTP1 protein, and (c) whether such phosphorylation of the GSTP1 protein alters its functions in metabolism and in JNK signaling. ^ The first major finding in this study is the establishment of the human GSTP1 gene as a novel CAMP responsive gene in which transcription is activated via an interaction between PKA activated CRE binding protein-1 (CREB-1) and the CRE in the 5′-regulatory region. ^ The second major finding in this study is the observation that the GSTP1 protein undergoes phosphorylation and functionally activated by second messenger-activated protein kinases, PKA and PKC, in tumor cells with activated signaling pathways. Following phosphorylation by PKA or PKC, the catalytic activity of the GSTP1 protein was significantly enhanced, as indicated by a decrease in its Km (2- to 3.6-fold) and an increase in Kcat/ Km (1.6- to 2.5-fold) for glutathione. Given the frequent over-expression of GSTP1 and the aberrant PKA/PKC signaling cascade observed in tumors, these findings suggest that phosphorylation of GSTP1 may contribute to the malignant progression and drug-resistant phenotype of these tumors. ^ The third major finding in this study is that the GSTP1 protein, an inhibitor of JNKs, undergoes significant phosphorylation in tumor cells with activated JNK signaling pathway and in those under oxidative stress. Following phosphorylation by JNK, the ability of GSTP1 to inhibit JNK downstream function, i.e. c-jun phosphorylation, was significantly enhanced, suggesting a feedback mechanism of regulation of JNK-mediated cellular signaling. (Abstract shortened by UMI.) ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^