Department of Pharmacology & Toxicology

Many laboratory animal studies of migraine have employed electrophysiological techniques to assess neuronal sensitization, but few have examined behaviors using International Headache Society criteria, which are based on behavioral changes including duration and intensity of pain and avoidance of routine activity. Fewer still have attempted to correlate the appearance of these diagnostic symptoms with changes in the activity of pain-related neurotransmitters and neuromodulators, such as calcitonin gene related peptide (CGRP). A vasoactive neuropeptide, CGRP might contribute to the vasodilatory component of migraine, and to the pain associated with this condition as it is present in nociceptors, including those in the trigeminal ganglion that innervate cerebral vasculature. Previous work has shown that CGRP levels are increased in animal models of inflammatory pain and in the external jugular vein of humans during migraine attacks. The CGRP receptor is comprised of three proteins: a G-protein coupled receptor called calcitonin-like receptor (CLR), a receptor activity-modifying protein (RAMP1), and a coupling protein, receptor component protein (RCP). Thus, the availability and sensitivity of this receptor is subject to regulation at numerous levels. The objectives of this study were to develop a preclinical behavioral model of chronic migraine, to test sensory and motor behaviors relevant to International Headache Society diagnostic criteria, to examine whether there are sex differences in these behaviors, and to assess whether alterations in the expression pattern of genes encoding CGRP and its receptor components are associated with sex differences or changes in pain-related behaviors. Male and female Sprague-Dawley rats were implanted with a dural cannula placed over the occipital cortex. Groups of rats were treated with 10 or 20 microliter volumes of an inflammatory soup containing 1 mM each of histamine, serotonin, and bradykinin, as well as 0.1 mM prostaglandin E2 (pH 5.5). A control group received sterile phosphate-buffered saline (pH 7.4) alone. Baseline behavioral testing was conducted on all eight groups of animals prior to surgery and seven days later. The inflammatory soup or control solution was administered supradurally 3 times/week for a total of eight applications. Locomotor activity was assessed using force plate actimetry during and following application of the inflammatory soup or vehicle. Total RNA was isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Real-time polymerase chain reaction was used to quantify the expression of amplified constructs using gene specific primers for CGRP, RAMP1, CLR, and RCP. The results reveal pronounced sex differences in behavior following application of the inflammatory soup. Female rats displayed dose-dependent migraine-related behaviors and a longer duration of these effect in measurements of distance traveled, bouts of low mobility, and spatial confinement. Both males and females experienced allodynia following exposure to the inflammatory mixture. Moreover, females displayed a higher baseline gene expression of CGRP and lower baseline gene expression of RAMP1, CLR, and RCP in the medulla than male animals. In addition to these baseline differences, gene expression of CLR and RCP was induced in the medulla of female rats but not in males. No sex difference in CGRP gene expression was noted in the trigeminal ganglia, although females do have a lower baseline expression of CGRP receptors, RAMP1, CLR, and RCP than males. It was also found that in the trigeminal ganglia RAMP1, CLR and RCP are inducible, especially in males, and that at least a portion of these responses are the result of volume effects associated with application to the dura of the inflammatory soup or vehicle. These findings indicate significant sex dependent changes in rat locomotor activity and CGRP-related gene expression in the brainstem and trigeminal ganglia associated with the application to the dura of an inflammatory soup. As the behavioral endpoints utilized in this study correspond to clinical signs considered by the International Headache Society as diagnostic for migraine, these data confirm that CGRP and its receptors are involved in migraine pathophysiology and reveal for the first time that alterations in the response to this peptide may be related to the increased prevalence of migraine in females. Such findings could be of value in devising new therapeutic strategies for the treatment of the debilitating condition.

Liver-enriched nuclear receptors (NRs) collectively function as metabolic and toxicological `sensors' that mediate liver-specific gene-activation in mammals. NR-mediated gene-environment interaction regulates important steps in the hepatic uptake, metabolism and excretion of glucose, fatty acids, lipoproteins, cholesterol, bile acids, and xenobiotics. While it is well-recognized that ligand-binding is the primary mechanism behind activation of NRs, recent research is revealing that multiple signal transduction pathways modulate NR-function in liver. The interface between specific signal transduction pathways and NRs helps to determine their overall responsiveness to various environmental and physiological stimuli. The pregnane x receptor (PXR, NR1I2) was identified in 1998 as a member of the NR superfamily of ligand-activated transcription factors. PXR is activated by a broad range of lipophilic compounds in a species-specific manner. The primary function ascribed to PXR is the homeostatic control of steroids, bile acids, and xenobiotics. This function is mediated through PXR's ability to coordinately activate gene expression and regulate the subsequent activity of phase I and phase II metabolic enzymes, as well as several membrane transporter proteins. While PXR likely evolved primarily to protect the liver from toxic assault, its activation also represents the molecular basis for an important class of drug-drug, herb-drug, and food-drug interactions. While ligand binding is the primary mode of PXR activation, several signal transduction pathways interface with the PXR protein to determine its overall responsiveness to environmental stimuli. Multiple signaling pathways modulate the activity of PXR, likely through direct alteration of the phosphorylation status of the receptor and its protein cofactors. Therefore, specific combinations of ligand binding and cell signaling pathways affect PXR-mediated gene activation and determine the overall biological response. This dissertation contributes to the molecular understanding of the regulation of PXR by novel agonists, cAMP-dependent protein kinase (PKA) signaling, and phosphorylation. The results presented here were primarily obtained from mouse and tissue culture systems. This dissertation identifies Tian Xian, a traditional Chinese herbal anti-cancer remedy, as a novel PXR activator. This evidence suggests that Tian Xian should be used cautiously by cancer patients taking chemotherapy due to its potential to increase the metabolism of co-administered medications. In addition, data presented here show that activation of PKA signaling modulates PXR activity in a species-specific manner. It is further revealed that PXR exists as phospho-protein in vivo and that the activation of PKA signaling modulates the phospho-threonine status of PXR. Finally, the potential phosphorylation sites within the PXR protein are identified. These phosphorylation sites are characterized, using a phosphomimetic and phospho-deficient site-directed mutagenesis based approach, based on their ability to modulate PXR activity. Taken together, the work presented in this dissertation contributes to understanding the interface between ligands, signal transduction pathways and PXR activity, which is critical for the development of safe and effective therapeutic strategies.

Disruption of normal apoptosis can contribute to the onset of cancer. Additionally, many cancer drugs are effective for their ability to initiate the apoptotic process. Often this involves the activation of the mitochondria-mediated pathway. The existing paradigm of mitochondria-mediated apoptosis, which can be activated by DNA damage, indicates this pathway proceeds in a linear fashion. Mitochondria outer membrane permeabilization (MOMP) and the release of intermembrane space proteins (e.g., cytochrome c) are early events during mitochondria-mediated apoptotic signaling. In addition, Apaf-1 is a critical component of the mitochondrial pathway and is generally thought to reside downstream of MOMP. My dissertation investigates the molecular requirements essential for MOMP during stress-induced and receptor-mediated apoptosis. Distinct clones of Jurkat T-lymphocytes were used in which the mitochondria-mediated pathway had been inhibited at three different steps. The first aim investigated the molecular requirements necessary for Bak activation. Apaf-1-deficient cells and cells overexpressing full-length XIAP or the BIR1/BIR2 domains of XIAP were refractory mitochondrial apoptotic events. These data suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. In so-called types II cells, the mitochondria-mediated pathway is required for cell death upon stimulation of the receptor-mediated pathway. In the second aim, I investigated the molecular requirements necessary for Fas-mediated apoptosis. Interestingly, Apaf-1-deficient type II Jurkat cells were sensitive to anti-Fas. Inhibiting downstream caspases decreased all anti-Fas-induced apoptotic changes. Combined, my findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis does not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. In the third aim, mitochondrial apoptotic events were examined after prolonged treatment with etoposide ( 6 h). Total cellular cytochrome c and Smac were decreased after 24 h of incubation. Interestingly, inhibition of the 26S proteasome by co-treatment of Apaf-1-deficient cells with bortezomib or MG132 led to the robust retention of total cellular cytochrome c and Smac. Combined, the major findings suggest that proteasomal degradation is largely responsible for the loss of intracellular cytochrome c and Smac in Apaf-1-deficient cells incubated with etoposide over extended time periods.

Liver-enriched nuclear receptors (NRs) collectively function as metabolic and toxicological `sensors' that mediate liver-specific gene-activation in mammals. NR-mediated gene-environment interaction regulates important steps in the hepatic uptake, metabolism and excretion of glucose, fatty acids, lipoproteins, cholesterol, bile acids, and xenobiotics. While it is well-recognized that ligand-binding is the primary mechanism behind activation of NRs, recent research is revealing that multiple signal transduction pathways modulate NR-function in liver. The interface between specific signal transduction pathways and NRs helps to determine their overall responsiveness to various environmental and physiological stimuli. The pregnane x receptor (PXR, NR1I2) was identified in 1998 as a member of the NR superfamily of ligand-activated transcription factors. PXR is activated by a broad range of lipophilic compounds in a species-specific manner. The primary function ascribed to PXR is the homeostatic control of steroids, bile acids, and xenobiotics. This function is mediated through PXR's ability to coordinately activate gene expression and regulate the subsequent activity of phase I and phase II metabolic enzymes, as well as several membrane transporter proteins. While PXR likely evolved primarily to protect the liver from toxic assault, its activation also represents the molecular basis for an important class of drug-drug, herb-drug, and food-drug interactions. While ligand binding is the primary mode of PXR activation, several signal transduction pathways interface with the PXR protein to determine its overall responsiveness to environmental stimuli. Multiple signaling pathways modulate the activity of PXR, likely through direct alteration of the phosphorylation status of the receptor and its protein cofactors. Therefore, specific combinations of ligand binding and cell signaling pathways affect PXR-mediated gene activation and determine the overall biological response. This dissertation contributes to the molecular understanding of the regulation of PXR by novel agonists, cAMP-dependent protein kinase (PKA) signaling, and phosphorylation. The results presented here were primarily obtained from mouse and tissue culture systems. This dissertation identifies Tian Xian, a traditional Chinese herbal anti-cancer remedy, as a novel PXR activator. This evidence suggests that Tian Xian should be used cautiously by cancer patients taking chemotherapy due to its potential to increase the metabolism of co-administered medications. In addition, data presented here show that activation of PKA signaling modulates PXR activity in a species-specific manner. It is further revealed that PXR exists as phospho-protein in vivo and that the activation of PKA signaling modulates the phospho-threonine status of PXR. Finally, the potential phosphorylation sites within the PXR protein are identified. These phosphorylation sites are characterized, using a phosphomimetic and phospho-deficient site-directed mutagenesis based approach, based on their ability to modulate PXR activity. Taken together, the work presented in this dissertation contributes to understanding the interface between ligands, signal transduction pathways and PXR activity, which is critical for the development of safe and effective therapeutic strategies.

Disruption of normal apoptosis can contribute to the onset of cancer. Additionally, many cancer drugs are effective for their ability to initiate the apoptotic process. Often this involves the activation of the mitochondria-mediated pathway. The existing paradigm of mitochondria-mediated apoptosis, which can be activated by DNA damage, indicates this pathway proceeds in a linear fashion. Mitochondria outer membrane permeabilization (MOMP) and the release of intermembrane space proteins (e.g., cytochrome c) are early events during mitochondria-mediated apoptotic signaling. In addition, Apaf-1 is a critical component of the mitochondrial pathway and is generally thought to reside downstream of MOMP. My dissertation investigates the molecular requirements essential for MOMP during stress-induced and receptor-mediated apoptosis. Distinct clones of Jurkat T-lymphocytes were used in which the mitochondria-mediated pathway had been inhibited at three different steps. The first aim investigated the molecular requirements necessary for Bak activation. Apaf-1-deficient cells and cells overexpressing full-length XIAP or the BIR1/BIR2 domains of XIAP were refractory mitochondrial apoptotic events. These data suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. In so-called types II cells, the mitochondria-mediated pathway is required for cell death upon stimulation of the receptor-mediated pathway. In the second aim, I investigated the molecular requirements necessary for Fas-mediated apoptosis. Interestingly, Apaf-1-deficient type II Jurkat cells were sensitive to anti-Fas. Inhibiting downstream caspases decreased all anti-Fas-induced apoptotic changes. Combined, my findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis does not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. In the third aim, mitochondrial apoptotic events were examined after prolonged treatment with etoposide ( 6 h). Total cellular cytochrome c and Smac were decreased after 24 h of incubation. Interestingly, inhibition of the 26S proteasome by co-treatment of Apaf-1-deficient cells with bortezomib or MG132 led to the robust retention of total cellular cytochrome c and Smac. Combined, the major findings suggest that proteasomal degradation is largely responsible for the loss of intracellular cytochrome c and Smac in Apaf-1-deficient cells incubated with etoposide over extended time periods.

Disruption of normal apoptosis can contribute to the onset of cancer. Additionally, many cancer drugs are effective for their ability to initiate the apoptotic process. Often this involves the activation of the mitochondria-mediated pathway. The existing paradigm of mitochondria-mediated apoptosis, which can be activated by DNA damage, indicates this pathway proceeds in a linear fashion. Mitochondria outer membrane permeabilization (MOMP) and the release of intermembrane space proteins (e.g., cytochrome c) are early events during mitochondria-mediated apoptotic signaling. In addition, Apaf-1 is a critical component of the mitochondrial pathway and is generally thought to reside downstream of MOMP. My dissertation investigates the molecular requirements essential for MOMP during stress-induced and receptor-mediated apoptosis. Distinct clones of Jurkat T-lymphocytes were used in which the mitochondria-mediated pathway had been inhibited at three different steps. The first aim investigated the molecular requirements necessary for Bak activation. Apaf-1-deficient cells and cells overexpressing full-length XIAP or the BIR1/BIR2 domains of XIAP were refractory mitochondrial apoptotic events. These data suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. In so-called types II cells, the mitochondria-mediated pathway is required for cell death upon stimulation of the receptor-mediated pathway. In the second aim, I investigated the molecular requirements necessary for Fas-mediated apoptosis. Interestingly, Apaf-1-deficient type II Jurkat cells were sensitive to anti-Fas. Inhibiting downstream caspases decreased all anti-Fas-induced apoptotic changes. Combined, my findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis does not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. In the third aim, mitochondrial apoptotic events were examined after prolonged treatment with etoposide ( 6 h). Total cellular cytochrome c and Smac were decreased after 24 h of incubation. Interestingly, inhibition of the 26S proteasome by co-treatment of Apaf-1-deficient cells with bortezomib or MG132 led to the robust retention of total cellular cytochrome c and Smac. Combined, the major findings suggest that proteasomal degradation is largely responsible for the loss of intracellular cytochrome c and Smac in Apaf-1-deficient cells incubated with etoposide over extended time periods.

The therapeutic benefits of atypical antipsychotics are proposed to be mediated by antagonism and subsequent desensitization of 5-HT2A receptor signaling; however, the mechanisms underlying this desensitization response are not yet understood. We hypothesize that the desensitization of 5-HT2A receptors induced by atypical antipsychotics is dependent on activation of the JAK-STAT pathway. To test this hypothesis, we used a cell line, A1A1v cells, that natively expresses 5-HT2A receptor signaling system, and further confirmed the findings in rats. In A1A1v cells, we confirmed that treatment with both olanzapine and clozapine desensitizes 5-HT2A receptor signaling. Furthermore, olanzapine treatment also increased RGS7 mRNA and protein levels which were dependent on activation of JAK-STAT pathway. Similar results were found with MDL100907, a specific 5HT2A receptor antagonist; RGS7 protein and mRNA levels were increased along with activation of the JAK-STAT pathway, suggesting that antagonism of 5-HT2A receptors is sufficient to induce these changes. In addition, we also found an increase in STAT3 binding to the putative RGS7 promoter region with olanzapine treatment suggesting that the increase in RGS7 expression could be directly mediated by the JAK-STAT pathway. An increase in RGS protein could mediate desensitization of 5-HT2A receptor signaling by its GAP activity. Lastly, inhibition of the JAK-STAT pathway significantly attenuated olanzapine-induced desensitization of 5-HT2A receptor signaling in A1A1v cells. Similar findings were also observed in rats treated with olanzapine for 7 days. We found a decrease in 5-HT2A receptor-stimulated PLC activity in the frontal cortex which was dependent on activation of JAK-STAT pathway. Consistent with the cell culture data, the olanzapine-induced increase in RGS7 proteins and mRNA levels were dependent on activation of the JAK-STAT pathway. Olanzapine treatment significantly reduced plasma levels of oxytocin, adrenocorticotrophic hormone (ACTH), and corticosterone. Surprisingly, 5-HT2A receptor-stimulated oxytocin and corticosterone levels were also decreased in a dose-dependent manner by the JAK inhibitor whereas ACTH levels were not altered. Further studies are needed to investigate the role of the JAK-STAT pathway in the regulation of hormone levels. Taken together, these results from experiments in cells in culture and in rats suggest that increases in RGS7 expression via increased activation of the JAK-STAT pathway are necessary for antipsychotic-induced desensitization of 5-HT2A receptor signaling.