Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 223-251). Also available online through Digital Dissertations.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22150号 / 医博第4541号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 稲垣 暢也, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na +/H+ exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-α (PFT-α), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 μM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-α followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-α also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-α interferes with multiple steps of Dox-induced apoptosis.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: Cancer is an emerging health problem in South Africa, with breast cancer being one of the leading cancers affecting women globally. Therefore, there is a need to find novel targets to improve the therapeutic options for these patients. A recently proposed target is the mitogen-activated protein kinase phosphatase-1 (MKP-1). Studies have suggested that mitogen-activated protein kinase phosphatases are involved in the development of cancer and play an important role in the response of cancer cells to chemotherapy. Additionally, numerous studies have indicated that there is increased expression of MKP-1 in breast cancers where its over-expression is proposed to be a significant mediator in chemo-resistance. We propose that inhibition of MKP-1 will increase the cytotoxic effect of doxorubicin in breast cancer cells, thus making the cells more responsive to treatment leading to increased cell death through autophagy and apoptosis. Methods: In MDA-MB231 cells, MKP-1 was inhibited using sanguinarine or MKP-1 siRNA and this was compared to a known inducer of MKP-1, dexamethasone. MDA-MB231 cells were treated with doxorubicin alone or in combination with MKP-1 inhibitors or an inducer. Following treatment, cell death was determined by trypan blue and a caspase glo assay as well as with western blotting. Autophagy was determined by western blotting and flow cytometry. LC3 and p62 were used as markers of autophagy and caspase 3 and PARP as apoptosis markers. Likewise, the level of MKP-1 expression under conditions of MKP-1 induction, inhibition or silencing was evaluated by means of western blotting. C57BL6 tumour bearing mice was used to analyse apoptosis and autophagy in vivo under conditions of MKP-1 inhibition, using sanguinarine, together with doxorubicin treatment. Western blotting was used to determine levels of caspase 3, LC3, p62 and MKP-1 expression. Results and discussion: A concentration and time curve indicated that 5 μM doxorubicin reduced cell viability in the MDA-MB231 cells significantly after 24 hours of treatment. MKP-1 expression was significantly reduced with sanguinarine and MKP-1 siRNA. Furthermore, our results indicate a significant increase in apoptosis in MDA-MB231 cells when treated with doxorubicin, under conditions of MKP-1 inhibition or MKP-1 silencing. Also, an increase in autophagic activity was observed following treatment with doxorubicin in combination with sanguinarine. Whole excised tumours of C57BL6 mice also showed an increase in apoptosis and autophagy following treatment with sanguinarine in combination with doxorubicin. This indicates that the inhibition of MKP-1 with sanguinarine sensitized the MDA-MB231 cells and E0771 cell tumours to doxorubicin-induced-apoptosis through a mechanism involving autophagy. Conclusion: This is an encouraging finding that could hopefully be used in future studies to overcome doxorubicin-resistance in breast cancer cells overexpressing MKP-1. Targeting MKP-1 can have potential therapeutic benefits for breast cancer patients by making chemotherapy more effective. Sanguinarine thus has potential to be developed as a clinically relevant inhibitor of MKP-1 which could provide a novel avenue for therapeutic intervention in combination with chemotherapy in breast cancer patients. / AFRIKAANSE OPSOMMING: Inleiding: Kanker is 'n vinnig groeiende gesondheidsprobleem in Suid-Afrika, met borskanker as een van die vernaamste kankers wat vroue wêreldwyd raak. Daar is dus 'n behoefte aan nuwe terapeutiese opsies vir hierdie pasiënte en mitogeen-geaktiveerde proteïenkinase fosfatase-1 (MKP-1) is onlangs voorgestel as ‘n moontlike teiken. Verskeie studies toon dat mitogeen-geaktiveerde proteïenkinase fosfatases betrokke is by die ontwikkeling van kanker en ook belangrike rolspelers is in die reaksie van kanker op chemoterapie. Daarbenewens toon talle studies dat daar verhoogde MKP-1 uitdrukking in borskanker is, asook dat dit ‘n belangrike bemiddelaar is vir die weerstand wat borskanker teen chemoterapie bied. Ons het dus voorgestel dat die inhibisie van MKP-1 die sitotoksiese effek van doxorubicin op borskanker selle sal verhoog; sodoende sal die kanker selle beter reageer op behandeling en dit sal dus lei tot verhoogde seldood deur autofagie en apoptose. Metodes: MKP-1 is geïnhibeer met behulp van sanguinarine of MKP-1 siRNA in MDA-MB231 selle en dit is vergelyk met 'n bekende MKP-1 induseerder, dexamethasone. MDA-MB231 selle is met doxorubicin alleen behandel of in kombinasie met MKP-1 inhibeerders of ‘n induseerder. Seldood is bepaal deur middel van ‘n trypan blou en kaspase toetsingsmetode, asook met die westelike kladtegniek. Autofagie is bepaal deur westelike kladtegniek en vloeisitometrie. LC3 en p62 is gebruik as merkers van autofagie en kaspase 3 en PARP is as apoptose merkers gebruik. MKP-1 uitdrukking is geëvalueer deur middel van westelike kladtegniek. C57BL6 muise met kankeragtige gewasse is gebruik om apoptose en autofagie in vivo te ondersoek. MKP-1 is geïnhibeer met sanguinarine en die muise is behandel met ‘n kombinasie van sanguinarine en doxorubicin. Kaspase 3, LC3, p62 en MKP-1 uitdrukking is bepaal deur middel van die westelike kladtegniek. Resultate en bespreking: ‘n Konsentrasie en tyd kurwe het aangedui dat 5 μM doxorubicin die MDA-MB231 selle se lewensvatbaarheid aansienlik verminder het na 24 uur. MKP-1 uitdrukking is ook aansienlik verminder met sanguinarine en MKP-1 siRNA. Verder dui die resultate op 'n beduidende toename in apoptose in MDA-MB231 selle na behandeling met doxorubicin onder toestande van MKP-1 inhibisie. 'n Toename in autofagiese aktiwiteit is waargeneem na behandeling met doxorubicin en sanguinarine. Die kankeragtige gewasse van die C57BL6 muise toon ook 'n toename in apoptose en autofagie na behandeling met sanguinarine en doxorubicin. Hierdie resultate dui daarop dat die inhibisie van MKP-1 met sanguinarine die MDA-MB231 selle en E0771 sel gewasse gesensitiseer het tot doxorubicin-geïnduseerde apoptose deur middel van ‘n meganisme wat autofagie insluit. Gevolgtrekking: Hierdie bevinding kan hopelik in toekomstige studies gebruik word om doxorubicin weerstand te oorkom in borskanker selle waar MKP-1 verhoog is. Deur MKP-1 te teiken, kan dit lei tot potensiële terapeutiese voordele vir borskanker pasiënte en sodoende kan dit chemoterapie meer effektief maak. Sanguinarine het dus die potensiaal om ontwikkel te word as ‘n klinies relevante inhibeerder van MKP-1 wat sodoende kan dien as terapeutiese intervensie in kombinasie met chemoterapie vir borskanker pasiënte.

Fibroblast growth factor (FGF) signalling plays many fundamentally important roles during the development of the mammalian embryo. However, its effects on pluripotent stem cells derived from mouse and human embryos appear to be markedly different. FGF2 is routinely added to culture medium for propagating undifferentiated human (hES) cells, whereas in mouse (mES) cell cultures FGFs have been described as regulators of their differentiated progeny. To assess the effect of FGF signalling on undifferentiated mES cells, the effects of FGF2 and 4 were analysed in the presence of saturating and sub-saturating levels of the inhibitor of differentiation, leukaemia inhibitory factor (LIF). Mouse ES cell self-renewal was quantified by measuring the expression of the stem cell specific reporter Oct4-LacZ in biochemical and fluorometric assays. Treatment with FGF reduced the expression of the OCT4-LacZ reporter, even under saturating concentrations of LIF and this was mirrored by decreased levels of OCT4 protein. Furthermore, treatment with FGF leads to upregulation of the ectodermal differentiation marker Pax6. These results suggest that FGF signalling has a direct impact on undifferentiated mES cells, and actively promotes their differentiation. To asses the effect of FGF signalling on hES cells without the influence of undefined factors, a feeder and serum free system was developed. Cells growing in this conditions for >20 passages maintained expression of surface (SSEA3 and TRA1-60 and 81) and internal (OCT4) markers specific for undifferentiated hES cells. Expression of these markers was dependant on the continuous presence of FGF2. Indeed, withdrawal of FGF2 resulted in a rapid decrease of in hES cell growth and of the emergence of cell flattened morphology and of the surface marker SSEA1, changes typically associated with differentiation. Two important signals activated by FGF in hES cells are the ERK/MAPK and PI3K pathways. To assess their functional relevance, hES cell cultures were treated with the drugs UO126 and LY294002, inhibitors of the MAPK and PI3K pathways respectively. Drug mediated suppression of the phosphorylation of these pathways, correlated with a reduction in cell growth, flattening of the colonies and reduction in SSEA4 expression. Use of SB431542, specific inhibitor of TGFβ/activin type I receptor kinase (Alk5) also resulted in the flattening of the colonies and the appearance of dispersed cells. Therefore, inhibition of MAPK and PI3K appears to impair growth and self-renewal in hES cells and this may be happening in conjunction with TGFβ/Activin pathway. Taken together, these results suggest that FGF signalling has opposite effects in mouse and human ES cells: inducing differentiation in mES and sustaining self-renewal in hES.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors. The beta-arrestins, adaptor proteins involved in GPCR desensitization, may also act as scaffolds for signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. The MAPK family, which includes the extracellular-signal regulated kinases (ERK) 1 and 2, promotes cellular differentiation and proliferation. Herein, the activation of ERK1/2 upon stimulation of the GPCR bradykinin type 2 receptor (B2R) with bradykinin was examined. Various B2R mutants with modified C-termini were employed to examine the temporal kinetics of ERK1/2. One of these receptor mutants displayed a loss of beta-arrestin binding as well as greatly enhanced ERK1/2 activation, compared to the wild-type receptor, when a cluster of serine/threonine residues important for B2R internalization was mutated. The other receptor mutants exhibited a correlation between their affinity for beta-arrestin and the intensity of ERK1/2 activation. Data from a mouse embryonic fibroblast cell line null for beta-arrestin suggested that beta-arrestin is involved in late-phase ERK1/2 activation by the B2R. These data point to the involvement of beta-arrestin in the activation of the ERK1/2 MAPKs through the B2R.