A Progressive Loss of phosphoSer138-Profilin Aligns with Symptomatic Course in the R6/2 Mouse Model of Huntington's Disease: Possible Sex-Dependent Signaling.

The R6/2 transgenic mouse model of Huntington's disease (HD) carries several copies of exon1 of the huntingtin gene that contains a highly pathogenic 120 CAG-repeat expansion. We used kinome analysis to screen for kinase activity patterns in neural tissues from wildtype (WT) and R6/2 mice at a pre-symptomatic (e.g., embryonic) and symptomatic (e.g., between 3 and 10 weeks postnatal) time points. We identified changes in several signaling cascades, for example, the Akt/FoxO3/CDK2, mTOR/ULK1, and RAF/MEK/CREB pathways. We also identified the Rho-Rac GTPase cascade that contributes to cytoskeleton organization through modulation of the actin-binding proteins, cofilin and profilin. Immunoblotting revealed higher levels of phosphoSer138-profilin in embryonic R6/2 mouse samples (cf. WT mice) that diminish progressively and significantly over the postnatal, symptomatic course of the disease. We detected sex- and genotype-dependent patterns in the phosphorylation of actin-regulators such a ROCK2, PAK, LIMK1, cofilin, and SSH1L, yet none of these aligned consistently with the changing levels of phosphoSer138-profilin. This could be reflecting an imbalance in the sequential influences these regulators are known to exert on actin signaling. The translational potential of these observations was inferred from preliminary observations of changes in LIMK-cofilin signaling and loss of neurite integrity in neural stem cells derived from an HD patient (versus a healthy control). Our observations suggest that a pre-symptomatic, neurodevelopmental onset of change in the phosphorylation of Ser138-profilin, potentially downstream of distinct signaling changes in male and female mice, could be contributing to cytoskeletal phenotypes in the R6/2 mouse model of HD pathology.

Responsive eLearning exercises to enhance student interaction with metabolic pathways.

Successful learning of biochemistry requires students to engage with the material. In the past this often involved students writing out pathways by hand, and more recently directing students to online resources such as videos, songs, and animated slide presentations. However, even these latter resources do not really provide students an opportunity to engage with the material in an active fashion. As part of an online introductory metabolism course that was developed at our university, we created a series of twelve online interactive activities using Adobe Captivate 9. These activities targeted glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogen metabolism, the citric acid cycle, and fatty acid oxidation. The interactive exercises consisted of two types. One involved dragging objects such as names of enzymes or allosteric modifiers to their correct drop locations such as a particular point in a metabolic pathway, a specific enzyme, and so forth. A second type involved clicking on objects, locations within a pathway, and so forth, in response to a particular question. In both types of exercises, students received feedback on their decisions in order to enhance learning. The student feedback received on these activities was very positive, and indicated that they found them to increase their confidence in the material and that they had learned the key principles of each pathway. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):223-229, 2018.

The Glutamine-Alanine Repeat Domain of TCERG1 is Required for the Inhibition of the Growth Arrest Activity of C/EBPα.

TCERG1 was characterized previously as a repressor of the transcription factor C/EBPα through a mechanism that involved relocalization of TCERG1 from nuclear speckles to pericentromeric regions. The inhibitory activity as well as the relocalization activity has been demonstrated to lie in the amino terminal half of the protein, which contains several discrete motifs including an imperfect glutamine-alanine (QA) repeat. In the present study, we showed that deletion of this domain completely abrogated the ability of TCERG1 to inhibit the growth arrest activity of C/EBPα. Moreover, the QA repeat deletion mutant of TCERG1 lost the ability to be relocalized from nuclear speckles to pericentromeric regions, and caused an increase in the average size of individual speckles. We also showed that deletion of the QA repeat abrogated the complex formation between TCERG1 and C/EBPα. Examination of mutants with varying numbers of QA repeats indicated that a minimal number of repeats are required for inhibitory activity as well as relocalization ability. These data contribute to our overall understanding of how TCERG1 can have gene-specific effects in addition to its more general roles in coordinating transcription elongation and splicing.

TCERG1 inhibits C/EBPα through a mechanism that does not involve sequestration of C/EBPα at pericentromeric heterochromatin.

Transcriptional elongation regulator 1 (TCERG1) is a nuclear protein that participates in multiple events that include regulating the elongation of RNA polymerase II and coordinating transcription and pre-mRNA processing. More recently, we showed that TCERG1 is also a specific inhibitor of the transcription factor CCAAT enhancer binding protein α (C/EBPα). Interestingly, the inhibition of C/EBPα by TCERG1 is associated with the relocalization of TCERG1 from the nuclear speckle compartment to the pericentromeric regions where C/EBPα resides. In the present study, we examined additional aspects of C/EBPα-induced redistribution of TCERG1. Using several mutants of C/EBPα, we showed that C/EBPα does not need to be transcriptionally competent or have anti-proliferative activity to induce TCERG1 relocalization. Moreover, our results show that C/EBPα does not need to be localized to the pericentromeric region in order to relocalize TCERG1. This conclusion was illustrated through the use of a V296A mutant of C/EBPα, which is incapable of binding to the pericentromeric regions of heterochromatin and thus takes on a dispersed appearance in the nucleus. This mutant retained the ability to redistribute TCERG1, however in this case the redistribution was from the nuclear speckle pattern to the dispersed phenotype of C/EBPα V296A. Moreover, we showed that TCERG1 was still able to inhibit the activity of the V296A mutant. While we previously hypothesized that TCERG1 might inhibit C/EBPα by keeping it sequestered at the pericentromeric regions, our new findings indicate that TCERG1 can inhibit C/EBPα activity regardless of the latter's location in the nucleus.

Nuclear redistribution of TCERG1 is required for its ability to inhibit the transcriptional and anti-proliferative activities of C/EBPalpha.

Transcription elongation regulator 1 (TCERG1) is an inhibitor of transcriptional elongation, and interacts with transcription and splicing factors, suggesting that it assists in coupling and coordinating these two processes. Recently we showed that TCERG1 possesses an additional activity, that being to repress the transactivation and anti-proliferative activities of the transcription factor CCAAT/Enhancer Binding Protein alpha (C/EBPalpha). In the present study, we provide evidence that TCERG1 functions as an inhibitor of C/EBPalpha rather than a transcriptional co-repressor. This conclusion was based on reporter gene experiments demonstrating that TCERG1 was able to reverse not only C/EBPalpha-mediated transactivation of promoter activity, but also C/EBPalpha-mediated transrepression of a promoter which is inhibited by C/EBPalpha. These observations, along with our previous findings that TCERG1 inhibits cellular proliferation conferred by C/EBPalpha, support the relabeling of TCERG1 as an inhibitor C/EBPalpha. Using mutants of TCERG1, we showed that the inhibitory activity lies in the amino terminal region. Because C/EBPalpha and TCERG1 have been shown to occupy different subnuclear compartments, we examined whether nuclear relocalization of either protein was involved in the inhibition of C/EBPalpha by TCERG1. Using confocal microscopy, we showed that TCERG1 localizes to nuclear speckles in the absence of C/EBPalpha. However, when co-expressed with C/EBPalpha, TCERG1 localizes to pericentromeric sites where C/EBPalpha resides. Nuclear redistribution of TCERG1 is required for its inhibitory activity, since mutants that did not display nuclear relocalization also lacked C/EBPalpha-inhibitory activity. We propose that TCERG1 inhibits C/EBPalpha activity by keeping it retained in inactive, pericentromeric heterochromatin.

Identification of a co-repressor that inhibits the transcriptional and growth-arrest activities of CCAAT/enhancer-binding protein alpha.

We used a yeast two-hybrid screening approach to identify novel interactors of CCAAT/enhancer-binding protein alpha (C/EBPalpha) that may offer insight into its mechanism of action and regulation. One clone obtained was that for CA150, a nuclear protein previously characterized as a transcriptional elongation factor. In this report, we show that CA150 is a widely expressed co-repressor of C/EBP proteins. Two-hybrid and co-immunoprecipitation analyses indicated that CA150 interacts with C/EBPalpha. Overexpression of CA150 inhibited the transactivation produced by C/EBPalpha and was also able to reverse the enhancing effect of the co-activator p300 on C/EBPbeta-mediated transactivation. Analysis of C/EBPalpha mutants indicated that CA150 interacts with C/EBPalpha primarily through a domain spanning amino acids 135-150. Chromatin immunoprecipitation assays showed that CA150 was present on a promoter that is repressed by C/EBPalpha but not present on a promoter that is activated by C/EBPalpha. Finally, we showed that in cells in which growth arrest had been induced by ectopic expression of C/EBPalpha, CA150 was able to release them from growth arrest. Interestingly, CA150 could not reverse the growth arrest produced by the minimal growth-arrest domain of C/EBPalpha (amino acids 175-217), suggesting that the effect of CA150 was directed at a region of C/EBPalpha outside of this minimal domain, consistent with our two-hybrid analysis. Taken together, these data indicate that CA150 is a co-repressor of C/EBP proteins and provides a possible mechanism for how C/EBPalpha can repress transcription of specific genes.

Troglitazone overcomes doxorubicin-resistance in resistant K562 leukemia cells.

Human myeloid leukemia cells become resistant to doxorubicin (DOX) treatment and this resistance is correlated with an increased glyoxalase 1 (GLO1) expression. Troglitazone (TRG) is an anti-diabetic thiazolidinedione drug previously used to treat insulin-resistance in Type 2 diabetes. We previously showed that TRG down regulates GLO1 gene expression in a number of cell types and reasoned that TRG might be a useful adjunct therapy to overcome DOX resistance. Here we show that TRG treatment overcomes the resistance to DOX in the DOX-resistant K562 human leukemia cells. Higher doses of TRG were found to alter histone H3:H2B ratios with a decreased ratio in DOX-sensitive and increased ratio in DOX-resistant lines. Furthermore, phosphorylated H3 was seen in DOX-resistant but not in DOX-sensitive cells. We conclude that the downstream effect of TRG in DOX-resistant cells may be interference with normal cell cycle events leading to genomic instability. Our data suggest that TRG may be a useful adjunct therapy in circumventing drug resistance in K562 leukemia cells.

Effect of overexpression and nuclear translocation of constitutively active PKB-alpha on cellular survival and proliferation in HepG2 cells.

Protein kinase B (Akt/PKB) is a key component in the PI 3-kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over-expression of PKB-alpha can prevent cell death following growth factor withdrawal, the long-term effects of stable over-expression of PKB-alpha on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB-alpha and compared its characteristics with HepG2 cells. Basal as well as insulin-stimulated levels of Ser(473) and Thr(308) phosphorylation in PKB-alpha transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB-alpha enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R(2) = 0.71) between cell proliferation and phosphorylated form of PKB-alpha at Thr(308) was observed along with higher levels of phosphorylated 3'-phosphoinositide-dependent kinase-1 (PDK-1). HepG2 cells with constitutive expression of active PKB-alpha also showed higher levels of phosphorylated p65 subunit of nuclear factor-kappaB (NFkappaB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB-alpha was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB-alpha renders HepG2 cells independent of serum based growth factors for survival and proliferation.

Troglitazone reduces heat shock protein 70 content in primary rat hepatocytes by a ubiquitin proteasome independent mechanism.

Troglitazone (TRG) is an antidiabetic agent that increases the insulin sensitivity of target tissues in non-insulin-dependent diabetes mellitus. Therapy with troglitazone has been associated with severe hepatic injury in a small percentage of patients and the mechanism of TRG-induced hepatotoxicity remains unclear. A family of highly conserved stress proteins identified as heat shock proteins (Hsps), are well-known to protect cells against a wide variety of toxic conditions such as extreme temperature changes, oxidative stress and toxic drugs. The stress-inducible Hsp 70 protein is one of the best-known endogenous factors protecting cells from injury under various stress conditions. Here we examined the effects of TRG on Hsp 70 mRNA and protein expression in primary cultures of rat hepatocytes. We also investigated the effects of TRG in an in vivo model by examining Hsp 70 protein levels in livers prepared from C57 mice fed a 0.2% dietary admixture of TRG. Levels of Hsp 70 mRNA increased in a concentration-dependent manner in rat hepatocytes treated for 8h with increasing concentrations of TRG. However, Hsp 70 protein levels decreased significantly in cells treated with increasing concentrations of TRG. C57 mice fed a 0.2% admixture of TRG for 10 days, also demonstrated decreased liver Hsp 70 protein levels. To investigate whether TRG decreased Hsp 70 protein levels by activating the ubiquitin-proteasome pathway, cells were pretreated with 10 microM lactacystin, a potent and specific inhibitor of this pathway. Lactacystin pretreatment failed to prevent TRG-induced decrease in Hsp 70 protein. The data suggests that TRG-induced effects may be mediated through another system of regulated proteolysis or may involve a post-transcriptional regulator mechanism. The mechanism of TRG-induced hepatotoxicity remains unclear, however, the effects induced by TRG on Hsp 70 may, in part, play a role.

Different transcription factor binding arrays modulate the cAMP responsivity of the phosphoenolpyruvate carboxykinase gene promoter.

The cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is mediated by a cAMP response unit, which includes three CCAAT/enhancer-binding protein (C/EBPs) sites, and a cAMP response element (CRE). Because both the CRE-binding protein and several C/EBP isoforms can to bind to the CRE with similar affinity, a variety of transcription factor bindings arrays in the cAMP response unit are possible that may affect the protein kinase A (PKA) responsivity of the promoter. To explore this issue, we have designed PEPCK promoter variants that have the native cis-elements within the cAMP response unit replaced with one or more LexA- and/or GAL4-binding sites. We also engineered the corresponding C/EBP and CRE-binding protein chimeras, which have their basic region leucine zipper domains replaced with LexA or GAL4 DNA-binding domains. Using this approach, we have reconstituted the PKA responsiveness of permissive PEPCK promoters in hepatoma cells and have characterized the PKA responsivity of the promoter under defined transcription factor occupancy patterns. Furthermore, analysis of deletion mutants of C/EBPalpha indicated that the domains that mediate its constitutive and PKA-inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. These results suggest that promoter context may influence which domains within a transcription factor are employed to mediate transactivation.

Conserved amino acids within CCAAT enhancer-binding proteins (C/EBP(alpha) and beta) regulate phosphoenolpyruvate carboxykinase (PEPCK) gene expression.

Thyroid hormone and cAMP stimulate transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). CCAAT enhancer-binding proteins (C/EBP(alpha) and beta) are involved in multiple aspects of the nutritional, developmental and hormonal regulation of PEPCK gene expression. Previously, we have identified a thyroid hormone response element in the PEPCK promoter and demonstrated that C/EBP proteins bound to the P3(I) site are participants in the induction of PEPCK gene expression by thyroid hormone and cAMP. Here, we identify several peptide regions within the transactivation domain of C/EBP(alpha) that enhance the ability of T(3) to stimulate gene transcription. We also demonstrate that several conserved amino acids in the transactivation domain of C/EBP(alpha) and C/EBPbeta are required for the stimulation of basal gene expression and identify amino acids within C/EBPbeta that participate in the cAMP induction of the PEPCK gene. Finally, we show that the CREB-binding protein (CBP) enhanced the induction of PEPCK gene transcription by thyroid hormone and that CBP is associated with the PEPCK gene in vivo. Our results indicate that both C/EBP proteins and CBP participate in the regulation of PEPCK gene transcription by thyroid hormone.

CCAAT/enhancer binding proteins: do they possess intrinsic cAMP-inducible activity?

CCAAT/enhancer binding proteins (C/EBPs) are transcription factors that are enriched in tissues which play a central role in energy metabolism, such as adipose and liver. Structure/function analyses of these proteins have identified several transactivation domains, some of which can physically interact with general transcription factors present in the preinitiation complex. C/EBPs are generally considered to be constitutively-acting factors, unlike other transcription factors whose activities can be regulated by covalent modification, binding of a specific ligand, etc. However, studies of the regulatory property of the phosphoenolpyruvate carboxykinase gene promoter have uncovered a role for C/EBPs in mediating cAMP responsiveness, and identified specific domains within the proteins, which mediate this effect. Interestingly, a number of other gene promoters that are activated in response to cAMP also contain binding sites for C/EBP, and these binding sites are often located within the region of the promoter that is responsible for mediating the acute responsiveness to cAMP. The evidence presented in this review provides compelling support for the hypothesis that C/EBPs have both constitutive and cAMP-inducible activities, and should be considered as a cAMP-responsive nuclear regulator.

Unique ability of troglitazone to up-regulate peroxisome proliferator-activated receptor-gamma expression in hepatocytes.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear receptor that is activated by the binding of an appropriate ligand. Several studies have demonstrated that certain ligands can also induce the expression of PPAR-gamma. In the present study, we examined the mechanism whereby this induction occurs by specifically addressing whether potentiation of the transactivation function of PPAR-gamma per se leads to induction of expression. We observed that thiazolidinediones, a group of insulin-sensitizing drugs, had differential effects, with troglitazone inducing protein levels of PPAR-gamma, while rosiglitazone, englitazone, and ciglitazone were without effect. Similarly, the prostaglandin metabolite 15-deoxy-Delta(12,14)-prostaglandin J(2) and the potent synthetic ligand GW1929 (N-(2-benzoyl phenyl)-L-tyrosine) also had no effect, as did ligands for other isoforms of PPAR. Since troglitazone has antioxidant properties, we also examined the effect of alpha-tocopherol and observed that it induced PPAR-gamma expression in a dose-dependent fashion. Finally, we found that mice fed troglitazone as a dietary admixture displayed an up-regulation of hepatic PPAR-gamma mRNA and protein, indicating that the mechanism of action is at the level of gene expression and not protein stability. These data indicate that 1) up-regulation of the transactivation function of PPAR-gamma does not alone account for the induction of expression of PPAR-gamma by troglitazone, and 2) an antioxidant-related mechanism may be involved.