Breast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target. / text

As intestinal epithelial cells mature, they continuously transition from proliferation to differentiation to apoptosis under the influence of cell signalling pathways including c-Jun N-terminal kinase (JNK). Glutathione S-transferases (GSTs) are cytoprotective detoxification enzymes, some of which, including GSTA1, also sequester and inhibit JNK through complex formation. Thus, GSTA1 may be a key sensor of cellular state and regulator of responses to cell stress stimuli. The focus of this research study was to investigate the functional importance of GSTA1 in two contexts: 1) modulating complex integrity with JNK and activation of JNK by oxidative stress, 2) controlling cellular transitioning between proliferation, differentiation and apoptosis. In the first study, the impact of GSTA1 levels on dissociation of GSTA1-JNK complexes and JNK activation in response to cellular stress was investigated in the human colonic adenocarinoma Caco-2 cells. The pro-oxidant menadione caused GSTA1-JNK complex dissociation in preconfluent Caco-2 cells, whereas postconfluent cells were relatively resistant to this effect. Preconfluent cells were more sensitive than postconfluent cells to menadione-induced cytotoxicity. Additionally, menadione-induced JNK activation was transient since removal of the stimulus resulted in re-association of GSTA1 with JNK and significantly reduced cytotoxicity. Over-expression and knockdown of GSTA1 affected the degree of GSTA1-JNK complex association without altering the JNK activation. However, enhanced GSH levels by N-acetyl cysteine blocked menadione-induced complex dissociation and JNK activation in Caco-2 cells. The results suggest that the mechanism of menadione-mediated JNK activation involves the production of reactive oxygen species, likely superoxide anion, and that the level of intracellular GSH plays an important role in preventing menadione-induced GSTA1-JNK complex dissociation and subsequent JNK activation. The functional importance of GSTA1 in controlling cellular proliferation, differentiation and apoptosis was investigated. Sodium butyrate (NaB) is a short-chain fatty acid, physiologically present in the human large intestine and modulates transitioning of cell states in colon cancer cell lines. GSTA1 levels increased in association with differentiation markers in postconfluent Caco-2 cells. Forced expression of GSTA1 significantly reduced cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increased cells in S-phase and associated cell proliferation. NaB (1 mM) reduced Caco-2 cell proliferation, increased differentiation and up-regulated GSTA1 activity. In contrast, higher dose of NaB (10 mM) caused toxicity in preconfluent cells via apoptosis through caspase-3 activation in association with reduced GSTA1 activity. GSTA1 down-regulation by siRNA did not alter NaB-induced differentiation or the sensitivity of Caco-2 cells to NaB-induced apoptosis. Furthermore, NaB (10 mM) caused GSTA1-JNK complex dissociation but did not affect JNK activation. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.

Axon degeneration is a selective elimination of axons, which plays a crucial role during development, injury, and maintenance of neuronal connections. The p75 neurotrophin receptor (NTR) is responsible for maintaining the specificity of neuronal connectivity in parts of the adult brain by inducing the degeneration of aberrantly growing axons into myelinated tracts. The objective of this study is to identify and characterize the signaling pathways used by p75NTR to mediate axon degeneration on myelin. Since p75NTR signals via JNK/Bax/caspase pathway to cause apoptosis, I asked whether this pathway might also be involved in axon degeneration. I observed that inhibition of JNK or Bax significantly decreased myelin-induced axonal degeneration, while depolarization of axons with potassium chloride prevented axonal degeneration on myelin. Together, these results suggest that p75NTR-dependent, myelin-mediated axon degeneration occurs via JNK/BAX signaling, and that neural activity is important for the prevention of myelin-induced axonal degeneration.

Axon degeneration is a selective elimination of axons, which plays a crucial role during development, injury, and maintenance of neuronal connections. The p75 neurotrophin receptor (NTR) is responsible for maintaining the specificity of neuronal connectivity in parts of the adult brain by inducing the degeneration of aberrantly growing axons into myelinated tracts. The objective of this study is to identify and characterize the signaling pathways used by p75NTR to mediate axon degeneration on myelin. Since p75NTR signals via JNK/Bax/caspase pathway to cause apoptosis, I asked whether this pathway might also be involved in axon degeneration. I observed that inhibition of JNK or Bax significantly decreased myelin-induced axonal degeneration, while depolarization of axons with potassium chloride prevented axonal degeneration on myelin. Together, these results suggest that p75NTR-dependent, myelin-mediated axon degeneration occurs via JNK/BAX signaling, and that neural activity is important for the prevention of myelin-induced axonal degeneration.

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

Thesis (Ph.D.)--Georgetown University, 2009. / Includes bibliographical references.

Salamanders have an extraordinary ability to regenerate appendages after loss or amputation, irrespective of age. My dissertation research explored the possibility that regenerative ability is associated with the evolution of novel, salamander-specific genes. I utilized transcriptional and genomic databases for the axolotl to discover previously unidentified genes, to the exclusion of other vertebrate taxa. Among the genes identified were multiple mmps (Matrix metalloproteases) and a jnk1/mapk8 (c-jun-N-terminal kinase) paralog. MMPs function in extracellular matrix remodeling (ECM) and tissue histolysis, processes that are essential for successful regeneration. Jjnk1/mapk8 plays a pivotal role in regulating transcription in response to cellular stress stimuli, including ROS (reactive oxygen species). Discovery of these novel genes motivated further bioinformatic studies of mmps and wet-lab experiments to characterize JNK and ROS signaling. The paralogy of the newly discovered mmps and orthology of 15 additional mmps was established by analyses of predicted, protein secondary structures and gene phylogeny. A microarray-analysis identified target genes downstream of JNK signaling that are predicted to function in cell proliferation, cellular stress response, and ROS production. These inferences were validated by additional experiments that showed a requirement for NOX (NADPH oxidase) activity, and thus presumably ROS production for successful tail regeneration. In summary, my dissertation identified novel, salamander-specific genes. The functions of these genes suggest that regenerative ability is associated with a diverse extracellular matrix remodeling and/or tissue histolysis response, and also stress-associated signaling pathways. The bioinformatic findings and functional assays that were developed to quantify ROS, cell proliferation, and mitosis will greatly empower the axolotl embryo model for tail regeneration research.

Palmitate (PA), one of the most prevalent saturated fatty acids, causes myocardial dysfunction. However, the mechanisms by which PA induces cell apoptosis and autophagy remain to be elucidated. We showed that autophagy was induced in an mTORC1-dependent way and played a protective role against PA-induced apoptosis, which was verified by pretreatment with 3-methyladenine (3MA) and rapamycin. However, p62 began to accumulate after 18 h treatment with PA, suggesting prolonged exposure to PA lead to an impairment of autophagic flux. PA enhanced ROS production as well as activated p38-mitogen-activated protein kinase (p38 MAPK) and c-jun NH2 terminal kinases (JNKs). The antioxidant N-Acety-L-Cysteine (NAC) was found to attenuate the JNK and p38 MAPK activation with a concomitant reduction of PA-induced autophagy and apoptosis. Furthermore, both JNK and p38 MAPK inhibitors were shown to directly abrogate caspase 7 cleavage as well as the conversion of LC3BI to LC3BII. Thus, we demonstrate that PA stimulates autophagy and apoptosis via ROS-dependent JNK and p38 MAPK pathways.

Matrix metalloproteinases (MMPs), a family of extracellular endopeptidases, are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Interleukin-1β (IL-1β), increased in the heart post-myocardial infarction (post-MI), plays a protective role in the pathophysiology of left ventricular (LV) remodeling following MI. Here we studied expression of various angiogenic genes affected by IL-1β in cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of MMP-2. cDNA array analysis of 96 angiogenesis-related genes indicated that IL-1β modulates the expression of numerous genes, notably increasing the expression of MMP-2, not MMP-9. RT-PCR and Western blot analyses confirmed increased expression of MMP-2 in response to IL-1β. Gelatin in-gel zymography and Biotrak activity assay demonstrated that IL-1β increases MMP-2 activity in the conditioned media. IL-1β activated ERK1/2, JNKs, and protein kinase C (PKC), specifically PKCα/β1, and inhibition of these cascades partially inhibited IL-1β-stimulated increases in MMP-2. Inhibition of PKCα/β1 failed to inhibit ERK1/2. However, concurrent inhibition of PKCα/β1 and ERK1/2 almost completely inhibited IL-1β-mediated increases in MMP-2 expression. Inhibition of p38 kinase and nuclear factor-κB (NF-κB) had no effect. Pretreatment with superoxide dismutase (SOD) mimetic, MnTMPyP, increased MMP-2 protein levels, whereas pretreatment with SOD and catalase mimetic, EUK134, partially inhibited IL-1β-stimulated increases in MMP-2 protein levels. Exogenous H2O2 significantly increased MMP-2 protein levels, whereas superoxide generation by xanthine/xanthine oxidase had no effect. This in vitro study suggests that IL-1β modulates expression and activity of MMP-2 in CMECs.

Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK–JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF−/− mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF−/− littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK–JNK–CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.