Mutations in BRAF, a component of extracellular signalregulated kinases 1 and 2 (ERK) cascade, are frequent in melanoma. It is important to understand how BRAF mutations contribute to malignant traits including anchorage-and growth factor-independence. We have previously shown that efficient activation of ERK in normal human epidermal melanocytes (NHEM) requires both adhesion to the extracellular matrix and growth factors. Mutant V599E BRAF is sufficient to promote ERK activation independent of adhesion and growth factors. Here, we analysed regulation of G1 cell cycle events in NHEM and human melanoma cells. We show that S phase entry in NHEM requires both adhesion and growth factor signaling through the MEK-ERK pathway. This control correlates with induction of cyclin D1 and downregulation of p27 Kip1 , two key G1 cell cycle events. In melanoma cells expressing V599E BRAF, cyclin D1 was constitutively expressed independent of adhesion but dependent upon MEK activation and nuclear accumulation of ERK. Reduction of cyclin D1 levels by RNA interference inhibited S phase entry in melanoma cells. Importantly, expression of V599E BRAF in NHEM was sufficient to promote cyclin D1 promoter activity in the absence of adhesion. Additionally, p27Kip1 levels were downregulated in V599E BRAF-expressing melanoma cells and active BRAF was sufficient to downregulate p27 Kip1 in serum-starved NHEM. Thus, adhesion-growth factor cooperation, leading to efficient activation of ERK, regulates cyclin D1 and p27Kip1 levels in human melanocytes and mutant BRAF overrides adhesion-growth factor control of these two G1 cell cycle proteins in melanomas. These findings provide important insight into how BRAF mutations contribute to aberrant human melanocyte proliferation.

While it is known that the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemic injury in the heart and brain, its role in kidney injury is unclear. Here we determined the effects on ischemia-reperfusion injury of the 20-HETE analogues, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid (5,14−20-HEDE), and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14−20-HEDGE), and of the inhibitor of 20-HETE synthesis N-hydroxy-N-(4-butyl-2 methylphenyl) formamidine (HET0016). Using Sprague-Dawley rats we found that while treatment with the inhibitor exacerbated renal injury, infusion of both 5,14−20-HEDE and 5,14−20-HEDGE significantly attenuated injury when compared to vehicle or inhibitor-treated rats. Medullary blood flow, measured by laser-Doppler flowmetry, decreased to half of the baseline one hour after reperfusion in the control rats, but 5,14−20-HEDGE completely prevented this. Treatment of control animals with 5,14−20-HEDGE increased urine output and sodium excretion without altering their mean arterial pressure or glomerular filtration rate. Our results suggest that 20-HETE analogues protect the kidney from ischemia-reperfusion injury by inhibiting renal tubular sodium transport and preventing the post-ischemic fall in medullary blood flow. Analogues of 20-HETE may be useful in the treatment of acute ischemic kidney injury.

Pathological conditions such as hypertension and hyperglycemia as well as abrasions following balloon angioplasty all lead to endothelial dysfunction that impacts disease morbidity. These conditions are associated with the elaboration of a variety of cytokines and increases in p38 activity in endothelial cells. However, the relationship between enhanced p38 activity and endothelial cell function remains poorly understood. To investigate the effect of enhanced p38 MAPK activity on endothelial cell function, we expressed an activated mutant of MEK6 (MEK6E), an upstream regulator of p38. Expression of MEK6E activated p38 and resulted in phosphorylation of its downstream substrate, heat shock protein 27 (Hsp27). Activation of p38 was not sufficient to induce apoptosis; however, it did induce p38-dependent cell cycle arrest. MEK6E expression was sufficient to inhibit ERK phosphorylation triggered by growth factors and integrin engagement. MAPK phosphatase-1 (MKP-1) expression was increased upon p38 activation, and expression of a "substrate-trapping" MKP-1 was sufficient to restore ERK activity. Activation of p38 was sufficient to induce cell migration, which was accompanied by alterations in actin architecture characterized by enhanced lamellipodia. Co-expression of a mutant form of Hsp27, lacking all three phosphorylation sites, reversed MEK6E-induced cell migration and altered the cytoskeletal changes induced by p38 activation. Collectively, these results suggest that cellular decisions regarding migration and proliferation are influenced by p38 activity and that prolonged activation of p38 may result in an anti-angiogenic phenotype that contributes to endothelial dysfunction.

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Aims/hypothesis Diabetic patients are at increased risk of cardiomyopathy, acute myocardial infarction and loss of cardiac progenitor cells (CPCs), but the aetiology is poorly understood. We hypothesised a significant role for mannose-binding lectin (MBL) in cardiomyopathies associated with hyperglycaemia. Methods The role of MBL in myocardial ischaemia and reperfusion (MI/R) injury was investigated in wild-type (WT) and MBL-null mice following 2 weeks of streptozotocin-induced hyperglycaemia. Results Hyperglycaemic WT mice presented with significantly decreased left ventricular ejection fractions and increased serum troponin I levels and myocardial inflammation compared with non-diabetic WT mice following MI/R. Hyperglycaemic MBL-null mice or insulin-treated diabetic WT mice were significantly protected from MI/R injury compared with diabetic WT mice. In an additional study using diabetic WT mice, echocardiographic measurements demonstrated signs of dilative cardiomyopathy, whereas heart:body weight ratios suggested hypertrophic cardiac remodelling after 2 weeks of hyperglycaemia. Immunohistochemical analysis of CPCs showed significantly lower numbers in diabetic WT hearts compared with non-diabetic hearts. Insulin-treated diabetic WT or untreated diabetic MBL-null mice were protected from dilative cardiomyopathy, hypertrophic remodelling and loss of CPCs. Conclusions/interpretationThese data demonstrate that MBL may play a critical role in diabetic MI/R injury. Further, the absence of MBL appears to inhibit hypertrophic remodelling and hyperglycaemia-induced loss of CPCs after just 2 weeks of hyperglycaemia in mice.