Paul A. Townsend

Molecular regulation of cardiac hypertrophy.

Heart failure is one of the leading causes of mortality in the western world and encompasses a wide spectrum of cardiac pathologies. When the heart experiences extended periods of elevated workload, it undergoes hypertrophic enlargement in response to the increased demand. Cardiovascular disease, such as that caused by myocardial infarction, obesity or drug abuse promotes cardiac myocyte hypertrophy and subsequent heart failure. A number of signalling modulators in the vasculature milieu are known to regulate heart mass including those that influence gene expression, apoptosis, cytokine release and growth factor signalling. Recent evidence using genetic and cellular models of cardiac hypertrophy suggests that pathological hypertrophy can be prevented or reversed and has promoted an enormous drive in drug discovery research aiming to identify novel and specific regulators of hypertrophy. In this review we describe the molecular characteristics of cardiac hypertrophy such as the aberrant re-expression of the fetal gene program. We discuss the various molecular pathways responsible for the co-ordinated control of the hypertrophic program including: natriuretic peptides, the adrenergic system, adhesion and cytoskeletal proteins, IL-6 cytokine family, MEK-ERK1/2 signalling, histone acetylation, calcium-mediated modulation and the exciting recent discovery of the role of microRNAs in controlling cardiac hypertrophy. Characterisation of the signalling pathways leading to cardiac hypertrophy has led to a wealth of knowledge about this condition both physiological and pathological. The challenge will be translating this knowledge into potential pharmacological therapies for the treatment of cardiac pathologies.

STAT-1 Interacts with p53 to Enhance DNA Damage-induced Apoptosis

The STAT-1 transcription factor has been implicated as a tumor suppressor by virtue of its ability to inhibit cell growth and promoting apoptosis. However, the mechanisms by which STAT-1 mediates these effects remain unclear. Using human and mouse STAT-1-deficient cells, we show here that STAT-1 is required for optimal DNA damage-induced apoptosis. The basal level of the p53 inhibitor Mdm2 is increased in STAT-1(-/-) cells, suggesting that STAT-1 is a negative regulator of Mdm2 expression. Correspondingly, both basal p53 levels, and those induced by DNA damage were lower in STAT-1(-/-) cells. In agreement with this lower p53 response to DNA damage in cells lacking STAT-1, the induction of p53 responsive genes, such as Bax, Noxa, and Fas, was reduced in STAT-1-deficient cells. Conversely, STAT-1 overexpression enhances transcription of these genes, an effect that is abolished if the p53 response element in their promoters is mutated. Moreover, STAT-1 interacts directly with p53, an association, which is enhanced following DNA damage. Therefore, in addition to negatively regulating Mdm2, STAT-1 also acts as a coactivator for p53. Hence STAT-1 is another member of a growing family of protein partners able to modulate the p53-activated apoptotic pathway.

Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis

We have previously demonstrated that STAT‐1 plays a critical role in promoting apoptotic cell death in cardiac myocytes following ischemia/reperfusion (I/R) injury. Epigallocatechin‐3‐gallate (EGCG), the major constituent of green tea, has recently been reported to inhibit STAT‐1 activity in noncardiac cells. In the present study, we have assessed the protective effects of EGCG and green tea extract (GTE) infusion on both cultures of cardiac myocytes and the isolated rat heart. EGCG reduced STAT‐1 phosphorylation and protected cardiac myocytes against I/R‐induced apoptotic cell death. Moreover, EGCG reduced the expression of a known STAT‐1 pro‐apoptotic target gene, Fas receptor. More interestingly, oral administration of GTE as well as EGCG infusion limited the extent of infarct size and attenuated the magnitude of myocyte apoptosis in the isolated rat heart exposed to I/R injury. This reduction cell death was associated with improved hemodynamic recovery and ventricular function in the ischemic/reperfused rat heart. This is the first report to show that consumption of green tea is able to mediate cardioprotection and enhance cardiac function during I/R injury. Because GTE‐mediated cardioprotection is achieved, at least in part, through inhibition of STAT‐1 activity, we may postulate that a similar action can be implemented in the clinical setting to minimize STAT‐1 activation levels in patients with acute coronary artery disease (CAD).

BAG-1: a multifunctional regulator of cell growth and survival

BAG-1 is multifunctional protein which interacts with a wide range of cellular targets to regulate growth control pathways important for normal and malignant cells, including apoptosis, signaling, proliferation, transcription and cell motility. Of particular relevance to tumour cells, BAG-1 interacts with the anti-apoptotic BCL-2 protein, various nuclear hormone receptors and the 70 kDa heat shock proteins, Hsc70 and Hsp70. Interaction with chaperones may account for many of the pleiotropic effects associated with BAG-1 overexpression. Recent studies have shown that BAG-1 expression is frequently altered in malignant cells, and BAG-1 expression may have clinical value as a prognostic/predictive marker. This review summarises current understanding of molecular mechanisms of BAG-1 expression and function.

A Cyclic Peptide Inhibitor of HIF‑1 Heterodimerization That Inhibits Hypoxia Signaling in Cancer Cells

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1β protein–protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein–protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

K-ATP channel gene expression is induced by urocortin and mediates its cardioprotective effect

Background—Urocortin is a novel cardioprotective agent that can protect cardiac myocytes from the damaging effects of ischemia/reperfusion both in culture and in the intact heart and is effective when given at reperfusion. Methods and Results—We have analyzed global changes in gene expression in cardiac myocytes after urocortin treatment using gene chip technology. We report that urocortin specifically induces enhanced expression of the Kir 6.1 cardiac potassium channel subunit. On the basis of this finding, we showed that the cardioprotective effect of urocortin both in isolated cardiac cells and in the intact heart is specifically blocked by both generalized and mitochondrial-specific KATP channel blockers, whereas the cardioprotective effect of cardiotrophin-1 is unaffected. Conversely, inhibiting the Kir 6.1 channel subunit greatly enhances cardiac cell death after ischemia. Conclusions—This is, to our knowledge, the first report of the altered expression of a KATP channel subunit induced by a cardioprotective agent and demonstrates that KATP channel opening is essential for the effect of this novel cardioprotective agent.

PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73

ABSTRACT p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73α, -β, and -γ bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73α, although not the C-terminally truncated isoforms p73β and -γ, and this requires the RING finger domain of PIAS-1. The ΔNp73α isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G1 and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G2 arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice

BackgroundSurfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency.MethodsSP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test.Main ResultsAfter 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment.ConclusionTreatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

STAT-1 facilitates the ATM activated checkpoint pathway following DNA damage.

STAT-1 plays a role in mediating stress responses to various stimuli and has also been implied to be a tumour suppressor. Here, we report that STAT-1-deficient cells have defects both in intra-S-phase and G2-M checkpoints in response to DNA damage. Interestingly, STAT-1-deficient cells showed reduced Chk2 phosphorylation on threonine 68 (Chk2-T68) following DNA damage, suggesting that STAT-1 might function in the ATM-Chk2 pathway. Moreover, the defects in Chk2-T68 phosphorylation in STAT-1-deficient cells also correlated with reduced degradation of Cdc25A compared with STAT-1-expressing cells after DNA damage. We also show that STAT-1 is required for ATM-dependent phosphorylation of NBS1 and p53 but not for BRCA1 or H2AX phosphorylation following DNA damage. Expression levels of BRCT mediator/adaptor proteins MDC1 and 53BP1, which are required for ATM-mediated pathways, are reduced in cells lacking STAT-1. Enforced expression of MDC1 into STAT-1-deficient cells restored ATM-mediated phosphorylation of downstream substrates. These results imply that STAT-1 plays a crucial role in the DNA-damage-response by regulating the expression of 53BP1 and MDC1, factors known to be important for mediating ATM-dependent checkpoint pathways.