Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK.

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

Respiratory symptoms, pulmonary function, and markers of inflammation among bar workers before and after a legislative ban on smoking in public places.

CONTEXT: Scotland prohibited smoking in confined public places on March 26, 2006. OBJECTIVE: To investigate the association of smoke-free legislation with symptoms, pulmonary function, and markers of inflammation of bar workers. DESIGN, SETTING, AND PARTICIPANTS: This prospective observational study was conducted in Tayside, Scotland from February-June 2006. One hundred five nonasthmatic and asthmatic nonsmoking bar workers were initially enrolled, of whom 77 completed the study per protocol. MAIN OUTCOME MEASURES: Respiratory and sensory symptoms, spirometry measurements, serum cotinine levels, peripheral inflammatory cell count, asthma quality-of-life scores, and exhaled nitric oxide levels were evaluated before and after introduction of the smoking ban. RESULTS: For the per-protocol analysis, the percentage of bar workers with respiratory and sensory symptoms decreased from 79.2% (n = 61) before the smoke-free policy to 53.2% (n = 41) (total change, -26%; 95% confidence interval [CI], -13.8% to -38.1%; P<.001) and 46.8% (n = 38) (-32.5%; 95% CI, -19.8% to -45.2%; P<.001) 1 and 2 months afterward. Forced expiratory volume in the first second increased from 96.6% predicted to 104.8% (change, 8.2%; 95% CI, 3.9% to 12.4%; P<.001) and then 101.7% (change, 5.1%; 95% CI, 2.1% to 8.0%; P = .002), and serum cotinine levels decreased from 5.15 ng/mL to 3.22 ng/mL (change, -1.93 ng/mL; 95% CI, -2.83 to -1.03 ng/mL; P<.001) and then 2.93 ng/mL (-2.22 ng/mL; 95% CI, -3.10 to -1.34 ng/mL; P<.001). The total white blood cell and neutrophil count was reduced from 7610 to 6980 cells/microL at 2 months (-630 cells/muL; 95% CI, -1010 to -260 cells/microL; P = .002) and from 4440 to 4030 cells/microL (-410 cells/microL; 95% CI, -740 to -90 cells/microL; P = .03), respectively. Asthmatic bar workers also had less airway inflammation, with a reduction in exhaled nitric oxide from 34.3 parts per billion (ppb) to 27.4 ppb 1 month after the ban (0.8-fold change; 95% CI, 0.67 to 0.96 ppb; P = .04), and Juniper quality-of-life scores increased from 80.2 to 87.5 points (7.3 points; 95% CI, 0.1 to 14.6 points; P = .049). CONCLUSIONS: Smoke-free legislation was associated with significant early improvements in symptoms, spirometry measurements, and systemic inflammation of bar workers. Asthmatic bar workers also had reduced airway inflammation and improved quality of life.

Epidermolysis bullosa simplex in Scotland caused by a spectrum of keratin mutations.

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder caused by mutations in keratins K5 (keratin 5) and K14 (keratin 14), with fragility of basal keratinocytes leading to epidermal cytolysis and blistering. Patients present with widely varying severity and are classified in three main subtypes: EBS Weber-Cockayne (EBS-WC), EBS Köbner (EBS-K), and EBS Dowling-Meara (EBS-DM), based on distribution and pattern of blisters. We could identify K5/K14 mutations in 20 out of the 43 families registered as affected by dominant EBS in Scotland; with previous studies this covers 70% of all Scottish EBS patients, making this the most comprehensively analyzed EBS population. Nine mutations are novel. All mutations lie within five previously identified rod domain hotspots and the severest blistering was associated with mutations in the helix boundary motifs. In some cases, the same mutation caused symptoms of EBS-WC and/or EBS-K, both within and between families, suggesting a contribution of additional factors to the phenotype. In some patients, no mutations were found in K5, K14, or K15, suggesting involvement of other genes. The results confirm that EBS is best considered as a single disorder with a spectrum of phenotypic variations, from severe EBS-DM at one extreme to mild EBS-WC at the other.