Browsing by Subject "cAMP"

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    AB affects apoE transcriptionally through the activation of B-AR, cAMP and AP-2
    (2009-09) Rossello, Ximena Serenella
    Two key players in the development of Alzheimer’s disease (AD) are amyloid beta protein (Aβ) and apolipoprotein E (apoE). We and others have reported that Aβ elevates apoE protein levels in astrocytes, which in turn could alter lipid trafficking and cell function. The mechanism for the Aβ-induced increase in apoE levels is not clearly understood. We propose that Aβ affects apoE transcriptionally through the activation of the beta-adrenergic receptor (βAR), cAMP and the activator protein 2 (AP-2). To test this hypothesis it was first determined if the stimulation of apoE protein levels by Aβ was triggered by an upregulation of apoE mRNA, in contrast to changes in secretion or degradation. The results show a time-dependent increase in apoE mRNA expression levels with peak expression reached after 1 hour of Aβ treatment. βAR antagonists were used to evaluate the involvement of the βAR. The antagonists significantly inhibited the Aβ-induced stimulation of apoE mRNA and protein levels. In order to further understand the mechanism behind these results we assessed cAMP role in the proposed Aβ-apoE pathway. This second messenger has been associated with AD and has been shown to elevate apoE message and secretion levels. The data shows an Aβ-dependent elevation in cAMP levels as well as an increase in apoE levels after dBcAMP treatment, confirming the activation of a cAMP-dependent pathway. In addition, I provide evidence that confirms the participation of the transcription factor AP-2, specifically that of AP-2β. AP-2 is known to be unregulated by cAMP and to bind to the apoE promoter. I report an increase in AP-2β translocation to the nucleus after both cAMP and Aβ treatment and confirm its participation in the activation of the apoE promoter. In conclusion, my work reveals a novel pathway for Aβ stimulation of apoE abundance in astrocytes involving βAR and the transcription factor AP-2β. These findings not only help clarify the relationship between Aβ and apoE but also help understand AD progression and possibly show a mechanism that could aid in the fight against this fast growing disease.
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    Membrane and nuclear progesterone receptors activate tumor suppressive signaling mechanisms in cell line models of human ovarian cancer
    (2010-07) Charles, Nathan Jon
    Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Mortality rates for ovarian cancer have been unchanged for more than 70 years even though surgical and chemotherapeutic strategies have become considerably more sophisticated. While a lack of clinical success is largely due to a poor etiologic understanding, recent observations suggest that the ovarian steroid hormone progesterone may be an endogenous ovarian cancer tumor suppressor. Therefore, the goal of our studies was to define progesterone receptor action in ovarian cancer and identify the signaling mechanisms responsible for its tumor suppressive function. Membrane progesterone receptors (mPRs) represent a newly defined class of ~40 kDa GPCR-like progesterone receptors belonging to the adipoQ receptor ( PAQR ) gene family. Never studied in cancerous cells of ovarian surface epithelial origin, we identified positive expression of each isoform (mPRα/PAQR7, mPRβ/PAQR8, and mPRγ/PAQR5) in a panel of ovarian cancer cell lines. Contrary to breast cancer cells, progesterone stimulation of these receptors in ovarian cancer cells increased intracellular cAMP levels and cAMP response element (CRE) transcriptional activity, but required high, pregnancy equivalent levels of progesterone as well as β 1,2 -adrenergic receptor co-stimulation. High-dose progesterone exposure also increased phosphorylation of the stress-activated JNK1/2 and p38 mitogen activated protein kinases (MAPKs). In particular, mPR-mediated p38 activation was responsible for increasing BAX mRNA expression; a pro-apoptotic Bcl family member. These results demonstrate that functionally active mPRs are capable of activating signaling pathways associated with tumor suppression in ovarian cancer cells. Clinical observations have shown that nuclear progesterone receptor (PR) expression is downregulated as ovarian tumors become progressively more malignant. Overexpression of PR in the ES-2 ovarian cancer cell line inhibited cellular proliferation and increased cell survival. As a result, prolonged ligand stimulation caused PR-expressing cells to undergo cellular senescence and exit the cell-cycle into G 0 . Senescent cells expressed significantly higher mRNA levels of the cell-cycle inhibitor p21. When the inhibition of PR-induced p21 expression was removed by stably downregulating STAT3 expression, PR-mediated senescence occurred more quickly and p21 induction was enhanced as cells arrested in the G1 phase of the cell-cycle. However, when increased p21 expression was prevented, ligand-stimulated PR-expressing cells also exhibited a heightened senescence response. These findings, along with the observation that ligand-stimulated PR-positive primary human ovarian cancer cells also become senescent, support our conclusion that PR-mediated cellular senescence is an endogenous ovarian cancer tumor suppressive mechanism. Taken together, these results demonstrate that progesterone receptors possess tumor suppressing characteristics in ovarian cancer cells, and warrant further investigation into the use of progesterone as an ovarian cancer chemopreventive and chemotherapeutic agent.