Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function.

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (alpha-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male alpha-MHC-Spry1 mice compared to control mice. Ventricular function of alpha-MHC-Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.

Mixed-Phenotype Acute Leukemia: Diagnostic Criteria and Pitfalls.

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous category in the World Health Organization classification that comprises acute leukemias with discrete admixed populations of myeloid and lymphoid blasts ("bilineal") or with extensive coexpression of lymphoid and myeloid markers in a single blast population ("biphenotypic"). Flow cytometric findings suggestive of MPAL are often met with consternation by pathologists and oncologists alike, owing to unfamiliarity with the disease and uncertainty about how MPAL fits into established paradigms for treatment of acute leukemia. The purpose of this review is to explain the diagnostic criteria for MPAL, summarize its biological and clinical features, and address common diagnostic pitfalls of these unusual leukemias.

Identification and regulation of Sprouty1, a negative inhibitor of the ERK cascade, in the human heart.

We screened a compendium of gene profiles from 19 paired human heart samples harvested at the time of implant and explant of a left ventricular assist device (LVAD) for novel genes regulating the Ras/MEK/ERK cascade. From this analysis we identified Sprouty1, an evolutionally conserved gene that acts as an intrinsic inhibitor of the Ras/MEK/ERK pathway. Sprouty1 mRNA and protein were significantly upregulated in the heart in response to mechanical unloading with a LVAD. The upregulation of Sprouty1 in the heart following mechanical unloading was accompanied by a significant decrease in phosphorylated ERK1/2. Gain of function experiments demonstrated that upregulation of Sprouty1 in isolated cardiac myocytes led to a significant decrease and altered kinetics of ERK1/2 phosphorylation. Immunohistochemistry of human hearts revealed that Sprouty1 was also expressed in the microvasculature. Upregulation of Sprouty1 in endothelial cells led to a significant decrease in VEGF-induced endothelial cell proliferation. To our knowledge, these findings are the first to define Sprouty expression in the heart and suggest that Sprouty1 may serve as an intrinsic mediator governing ventricular remodeling through a coordinated coupling of both myocyte and vascular alterations in response to mechanical load.

Progesterone receptors induce FOXO1-dependent senescence in ovarian cancer cells.

Loss of nuclear progesterone receptors (PR) and low circulating progesterone levels are associated with increased ovarian cancer (OC) risk. However, PR are abundantly expressed in a significant percentage of serous and endometrioid ovarian tumors; patients with PR+ tumors typically experience longer progression-free survival relative to those with PR-null tumors. The molecular mechanisms of these protective effects are poorly understood. To study PR action in OC in the absence of added estrogen (i.e., needed to induce robust PR expression), we created ES-2 OC cells stably expressing vector control or GFP-tagged PR-B (GFP-PR). Progestin (R5020) stimulation of ES-2 cells stably expressing GFP-PR induced cellular senescence characterized by altered cellular morphology, prolonged survival, senescence-associated ß-galactosidase activity, G1 cell cycle arrest and upregulation of the cell cycle inhibitor, p21, as well as the Forkhead-box transcription factor, FOXO1; these results repeated in unmodified ER+/PR+ PEO4 OC cells. PR-B and FOXO1 were detected within the same PRE-containing regions of the p21 upstream promoter. Knockdown of p21 resulted in molecular compensation via FOXO1-dependent upregulation of numerous FOXO1 target genes (p15, p16, p27) and an increased rate of senescence. Inhibition of FOXO1 (with AS1842856) or stable FOXO1 knockdown inhibited progestin-induced p21 expression and blocked progestin-induced senescence. Overall, these findings support a role for PR as a tumor suppressor in OC cells, which exhibits inhibitory effects by inducing FOXO1-dependent cellular senescence. Clinical "priming" of the PR-FOXO1-p21 signaling pathway using PR agonists may provide a useful strategy to induce irreversible cell cycle arrest and thereby sensitize OC cells to existing chemotherapies as part of combination "two-step" therapies.

Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications.

The recent discovery of a novel, membrane localized progestin receptor (mPR) unrelated to the classical progesterone receptor (PR) in fishes and its subsequent identification in mammals suggests a potential mediator of non-traditional progestin actions, particularly in tissues where PR is absent. While early studies on mPR focused on final oocyte maturation in fishes, more current studies have examined mPRs in multiple mammalian systems in both reproductive and non-reproductive tissues as well as in diseased tissues. Here we review the current data on mPR in mammalian systems including male and female reproductive tracts, liver, neuroendocrine tissues, the immune system and breast and ovarian cancer. We also provide new data demonstrating mPR expression in the RAW 264.7 immune cell line and bone marrow-derived macrophages as well as mPR expression and downstream gene regulation in ovarian cancer cells.

Expression of membrane progesterone receptors (mPR/PAQR) in ovarian cancer cells: implications for progesterone-induced signaling events.

The high mortality rates associated with ovarian cancer are largely due to a lack of highly effective treatment options for advanced stage disease; a time when initial diagnosis most commonly occurs. Recent evidence suggests that the steroid hormone, progesterone, may possess anti-tumorigenic properties. With the discovery of a new class of membrane-bound progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) gene family in the ovary, there are undefined mechanisms by which progesterone may inhibit tumor progression. Therefore, our goal was to define potential mPR-dependent signaling mechanisms operative in ovarian cancer cells. We detected abundant mPRα (PAQR7), mPRß (PAQR8), and mPRγ (PAQR5), but not classical nuclear PR (A or B isoforms) mRNA expression and mPRα protein expression in a panel of commonly used ovarian cancer cell lines. In contrast to mPR action in breast cancer cells, progesterone alone failed to induce changes in cyclic adenosine monophosphate (cAMP) levels in ovarian cancer cells. However, progesterone enhanced cAMP production by ß(1,2)-adrenergic receptors and increased isoproterenol-induced transcription from a cAMP response element (CRE)-driven reporter gene. Independently of ß-adrenergic signaling, we additionally observed activation of both JNK1/2 and p38 MAPK in response to progesterone alone. This finding was supported by the results of a screen for potential mPR gene targets. Progesterone induced a significant increase in transcription of the pro-apoptotic marker BAX, whose activity and expression has been linked to JNK1/2 and p38 signaling. Inhibitors of JNK, but not p38, blocked progesterone-induced BAX expression. Taken together, these observations implicate at least two distinct signaling pathways that may be utilized by mPRs in ovarian cancer cells that exhibit regulatory genomic changes. These studies on mPR signaling in ovarian cancer lay the foundation for future work aimed at understanding how progesterone exerts its anti-tumorigenic effects in the ovary and suggest that pharmacologic activation of mPRs, abundantly expressed in ovarian cancers, may provide a new treatment option for patients with advanced stage disease.

Chronic treatment with clenbuterol modulates endothelial progenitor cells and circulating factors in a murine model of cardiomyopathy.

The purpose of this study was to determine the effects of chronic treatment with the beta 2 adrenergic receptor agonist clenbuterol on endothelial progenitor cells (EPC) in a well-characterized model of heart failure, the muscle LIM protein knockout (MLP(-/-)) mouse. MLP(-/-) mice were treated daily with clenbuterol (2 mg/kg) or saline subcutaneously for 6 weeks. Clenbuterol led to a 30% increase in CD31(+) cells in the bone marrow of MLP(-/-) heart failure mice (p < 0.004). Clenbuterol did not improve ejection fraction. Clenbuterol treatment in MLP(-/-) mice was associated with significant changes in the following circulating factors: tissue inhibitor of metalloproteinase-type 1, leukemia inhibitory factor 1, C-reactive protein, apolipoprotein A1, fibroblast growth factor 2, serum glutamic oxaloacetic transaminase, macrophage-derived chemokine, and monocyte chemoattractant protein-3. Clenbuterol treatment in the MLP(-/-) model of heart failure did not rescue heart function, yet did increase CD31(+) cells in the bone marrow. This is the first evidence that a beta 2 agonist increases EPC proliferation in the bone marrow in a preclinical model of heart failure.

Molecular signature of recovery following combination left ventricular assist device (LVAD) support and pharmacologic therapy.

AIMS: A novel combination therapy consisting of a left ventricular assist device (LVAD) combined with pharmacologic therapy including the selective beta(2)-agonist, clenbuterol, has shown promise in restoring ventricular function in patients with heart failure. The aim of this study was to identify common genes and signalling pathways whose expression was associated with reversal of heart failure and restoration of ventricular function. METHODS AND RESULTS: Microarray analysis was performed on six paired human heart samples harvested at the time of LVAD implant and at the time of LVAD explant for recovery of ventricular function (post). Follow-up data shows that the improvements in ventricular function have been maintained for an average of 3.8 years post-explant. Analysis of the gene expression data revealed: (i) a significant association of integrin pathway signalling with recovery and (ii) the identification of several novel targets including, EPAC2, in the well-described cAMP pathway whose expression was down-regulated with recovery, and was associated with improvements in cardiac contractility, metabolism, and function. CONCLUSION: This data set represents the first description of signalling pathways associated with the functional recovery of end-stage human heart failure and the identification of new targets in the human heart that are modified by this combination therapy.