Cyclic adenosine 3', 5'-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs). Here we identify a cAMP-responsive signalling complex maintained by the muscle-specific A-kinase anchoring protein (mAKAP) that includes PKA, PDE4D3 and Epac1. These intermolecular interactions facilitate the dissemination of distinct cAMP signals through each effector protein. Anchored PKA stimulates PDE4D3 to reduce local cAMP concentrations, whereas an mAKAP-associated ERK5 kinase module suppresses PDE4D3. PDE4D3 also functions as an adaptor protein that recruits Epac1, an exchange factor for the small GTPase Rap1, to enable cAMP-dependent attenuation of ERK5. Pharmacological and molecular manipulations of the mAKAP complex show that anchored ERK5 can induce cardiomyocyte hypertrophy. Thus, two coupled cAMP-dependent feedback loops are coordinated within the context of the mAKAP complex, suggesting that local control of cAMP signalling by AKAP proteins is more intricate than previously appreciated.

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 upregulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably Cutaneous melanoma is a form of skin cancer characterized by aggressive metastatic growth and poor prognosis. 1 The incidence of melanoma continues to increase in many parts of the world.2 The median survival time of patients with metastatic melanoma is 6 months, and the 5-year survival rate is less than 5%.3 Genetic factors and exposure to ultraviolet radiation are risk factors for melanoma pathogenesis.4

Hypoxia in solid tumors is associated with the development of chemoresistance. Although many studies have focused on the effect of hypoxia on drug-induced apoptosis, the effect of nonapoptotic pathways on hypoxia-induced drug resistance has not been previously investigated. Here, we determined the effects of hypoxia on multiple forms of drug-induced death in human MDA-MB-231 breast carcinoma cells. Clonogenic assays showed that preexposure to hypoxia leads to resistance to various classes of chemotherapeutic agents, including anthracyclines (daunorubicin and doxorubicin), epipodophyllotoxins (etoposide), and anthracenediones (mitoxantrone). Results revealed a high degree of heterogeneity in nuclear and cytoplasmic alterations in response to acute drug exposure; however, the majority of exposed cells displayed morphologic and biochemical changes consistent with drug-induced senescence. Hypoxia decreased only the proportion of cells in the senescent population, whereas the small proportion of cells exhibiting features of apoptosis or mitotic catastrophe were unaffected. Similar results were obtained with human HCT116 colon carcinoma cells, indicating that the protective effect of hypoxia on drug-induced senescence is not unique to MDA-MB-231 cells. Treatment of MDA-MB-231 cells with small interfering RNA targeting the A-subunit of hypoxiainducible factor-1 (HIF-1), a key regulator of cellular adaptations to hypoxia, prevented hypoxia-induced resistance. HIF-1A small interfering RNA also selectively abolished the hypoxia-induced changes in the senescent population, indicating that the increased survival was due to protection against drug-induced senescence. These results support a requirement for HIF-1 in the adaptations leading to drug resistance and reveal that decreased druginduced senescence is also an important contributor to the development of hypoxia-induced resistance.

Maladaptive cardiac hypertrophy can progress to congestive heart failure, a leading cause of morbidity and mortality in the United States. A better understanding of the intracellular signal transduction network that controls myocyte cell growth may suggest new therapeutic directions. mAKAP is a scaffold protein that has recently been shown to coordinate signal transduction enzymes important for cytokine-induced cardiac hypertrophy. We now extend this observation and show mAKAP is important for adrenergic-mediated hypertrophy. One function of the mAKAP complex is to facilitate cAMPdependent protein kinase A-catalyzed phosphorylation of the ryanodine receptor Ca 2+ -release channel. Experiments utilizing inhibition of the ryanodine receptor, RNA interference of mAKAP expression and replacement of endogenous mAKAP with a mutant form that does not bind to protein kinase A demonstrate that the mAKAP complex contributes to pro-hypertrophic signaling. Further, we show that calcineurin A␤ ␤ associates with mAKAP and that the formation of the mAKAP complex is required for the full activation of the pro-hypertrophic transcription factor NFATc. These data reveal a novel function of the mAKAP complex involving the integration of cAMP and Ca 2+ signals that promote myocyte hypertrophy. Journal of Cell Science 5638 from the nuclear envelope, we showed that mAKAP was required for the induction of myocyte hypertrophy by this cytokine-regulated signaling pathway (Dodge-Kafka et al., 2005). We now report that mAKAP is important for the induction of myocyte hypertrophy by adrenergic receptor signaling. The ␤-adrenergic receptor stimulates adenylate cyclase and increases intracellular cAMP levels and, subsequently, PKA activity. mAKAP-bound PKA can phosphorylate associated RyR2 (Kapiloff, 2002;Marx et al., 2000;Ruehr et al., 2003), potentiating channel activity (Hain et al., 1995;. It is generally understood that RyR2 mediates Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum during excitation-contraction coupling (Fill and Copello, 2002). We have found, however, a small pool of RyR2 associated with mAKAP at the nuclear envelope ). mAKAP-associated RyR2 might regulate the release of Ca 2+ from adjacent, peri-nuclear sarcoplasmic reticulum or the release of putative stores within the nuclear envelope (Abrenica and Gilchrist, 2000). We now present evidence that mAKAP is integral to the induction of ␤-adrenergic-stimulated hypertrophy by participating in the CaN-NFATc signaling pathway. We show further that mAKAP is also relevant to ␣-adrenergic signaling. In contrast to the ␤-adrenergic receptor, the ␣-adrenergic receptor activates phospholipase C and phosphatidyl inositol pathways, resulting in increased intracellular Ca 2+ levels and activation of MAP kinases (including MEK5 and ERK5) and CaN (Dorn and Force, 2005;Nicol et al., 2001). Given the ability of mAKAP to anchor ERK5, PKA, RyR2, and, as shown below, CaN, to the nuclear envelope, we propose that the mAKAP scaffold may serve as an integrator for hypertrophic signa...

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MicroRNAs play important roles in gene regulation

Dysfunction of key miRNA pathways regulating basic cellular processes is a common driver of many cancers. However, the biological roles and/or clinical applications of such pathways in Merkel cell carcinoma (MCC), a rare but lethal cutaneous neuroendocrine (NE) malignancy, have yet to be determined. Previous work has established that miR-375 is highly expressed in MCC tumors, but its biological role in MCC remains unknown. Herein, we show that elevated miR-375 expression is a specific feature of well-differentiated MCC cell lines that express NE markers. In contrast, miR-375 is strikingly down-regulated in highly aggressive, undifferentiated MCC cell lines. Enforced miR-375 expression in these cells induced NE differentiation, and opposed cancer cell viability, migration, invasion, and survival, pointing to tumor-suppressive roles for miR-375. Mechanistically, miR-375-driven phenotypes were caused by the direct post-transcriptional repression of multiple Notch pathway proteins (Notch2 and RBPJ) linked to cancer and regulation of cell fate. Thus, we detail a novel molecular axis linking tumor-suppressive miR-375 and Notch with NE differentiation and cancer cell behavior in MCC. Our findings identify miR-375 as a putative regulator of NE differentiation, provide insight into the cell of origin of MCC, and suggest that miR-375 silencing may promote aggressive cancer cell behavior through Notch disinhibition.

Objectives: Providing diabetes self-management education (DSME) in an evidence-based format that is accessible and tailored to the population needs is crucial for individuals living with diabetes mellitus. Our qualitative study explores the experiences of older adults living with diabetes while residing in a rural setting. Methods: Adults aged 65 or older and residing in a rural area of Ontario completed a photovoice activity and semi-structured interviews to illustrate their experience of living with diabetes and accessing DSME. Results: Fourteen participants (11 males; mean age = 74 years) completed the photovoice activity and interview. Four main themes were identified pertaining to learning about diabetes education, the depth and breadth of learning, applying knowledge to daily life, and engaging older adults in DSME. Discussion: Diabetes self-management education should account for older adults’ preferences in learning about diabetes and self-management to promote access to evidence-based information, bolster knowledge and self-management efficacy, and improve disease control.