The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling.

IMPORTANCE Critical illness results in disability and reduced health-related quality of life (HRQOL), but the optimum timing and components of rehabilitation are uncertain. OBJECTIVE To evaluate the effect of increasing physical and nutritional rehabilitation plus information delivered during the post-intensive care unit (ICU) acute hospital stay by dedicated rehabilitation assistants on subsequent mobility, HRQOL, and prevalent disabilities. DESIGN, SETTING, AND PARTICIPANTS A parallel group, randomized clinical trial with blinded outcome assessment at 2 hospitals in Edinburgh, Scotland, of 240 patients discharged from the ICU between December 1, 2010, and January 31, 2013, who required at least 48 hours of mechanical ventilation. Analysis for the primary outcome and other 3-month outcomes was performed between June and August 2013; for the 6-and 12-month outcomes and the health economic evaluation, between March and April 2014. INTERVENTIONS During the post-ICU hospital stay, both groups received physiotherapy and dietetic, occupational, and speech/language therapy, but patients in the intervention group received rehabilitation that typically increased the frequency of mobility and exercise therapies 2-to 3-fold, increased dietetic assessment and treatment, used individualized goal setting, and provided greater illness-specific information. Intervention group therapy was coordinated and delivered by a dedicated rehabilitation practitioner. MAIN OUTCOMES AND MEASURES The Rivermead Mobility Index (RMI) (range 0-15) at 3 months; higher scores indicate greater mobility. Secondary outcomes included HRQOL, psychological outcomes, self-reported symptoms, patient experience, and cost-effectiveness during a 12-month follow-up (completed in February 2014). RESULTS Median RMI at randomization was 3 (interquartile range [IQR], 1-6) and at 3 months was 13 (IQR, 10-14) for the intervention and usual care groups (mean difference, −0.2 [95% CI, −1.3 to 0.9; P = .71]). The HRQOL scores were unchanged by the intervention (mean difference in the Physical Component Summary score, −0.1 [95% CI, −3.3 to 3.1; P = .96]; and in the Mental Component Summary score, 0.2 [95% CI, −3.4 to 3.8; P = .91]). No differences were found for self-reported symptoms of fatigue, pain, appetite, joint stiffness, or breathlessness. Levels of anxiety, depression, and posttraumatic stress were similar, as were hand grip strength and the timed Up & Go test. No differences were found at the 6-or 12-month follow-up for any outcome measures. However, patients in the intervention group reported greater satisfaction with physiotherapy, nutritional support, coordination of care, and information provision. CONCLUSIONS AND RELEVANCE Post-ICU hospital-based rehabilitation, including increased physical and nutritional therapy plus information provision, did not improve physical recovery or HRQOL, but improved patient satisfaction with many aspects of recovery.

Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP‐specific phosphodiesterase long isoforms. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. The PKA inhibitor, H89, ablated forskolin activation of wild‐type long PDE4 isoforms. Activated PKA caused the in vitro phosphorylation of recombinant wild‐type long PDE4 isoforms, but not those where the STR was mutated to alanine. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin‐challenged cells treated with H89. Neither was it evident in forskolin‐challenged cells expressing long isoforms where the STR had been mutated to alanine. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform‐specific N‐terminal region. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. Long PDE4 isoforms from all four sub‐families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed. British Journal of Pharmacology (2002) 136, 421–433; doi:

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42 MAPK ). These straddle the target residue, Ser 579 , for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH 2 -terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOHterminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.

IntroductionSevere sepsis is associated with high levels of morbidity and mortality, placing a high burden on healthcare resources. We aimed to study outcomes in the five years after severe sepsis.MethodsThis was a cohort study using data from a prospective audit in 26 adult ICUs in Scotland. Mortality was measured using clinical databases and quality of life using Short Form 36 (SF-36) at 3.5 and 5 years after severe sepsis.ResultsA total of 439 patients were recruited with a 58% mortality at 3.5 years and 61% mortality at 5 years. A total of 85 and 67 patients responded at 3.5 and 5 years follow-up, respectively. SF-36 physical component score (PCS) was low compared to population controls at 3.5 years (mean 41.8 (SD 11.8)) and at 5 years (mean 44.8 (SD 12.7)). SF-36 mental component score (MCS) was slightly lower than population controls at 3.5 years (mean 47.7 (SD 14.6)) and at 5 years after severe sepsis (mean 48.8 (SD 12.6)). The majority of patients were satisfied with their current quality of life (QOL) (80%) and all patients would be willing to be treated in an ICU again if they become critically ill despite many having unpleasant memories (19%) and recall (29%) of ICU events.ConclusionsPatients with severe sepsis have a high ongoing mortality after severe sepsis. They also have a significantly lower physical QOL compared to population norms but mental QOL scores were only slightly below population norms up to five years after severe sepsis. All survivors would be willing to be treated in an ICU again if critically ill. Mortality and QOL outcomes were broadly similar to other critically ill cohorts throughout the five years of follow-up.

1 Expressed in intact cells and in vitro, PDE4B and PDE4C isoenzymes of cyclic nucleotide phosphodiesterase (PDE), in common with PDE4D isoenzymes, are shown to provide substrates for C-terminal catalytic unit phosphorylation by the extracellular signal-regulated kinase Erk2 (p42 MAPK ). 2 In contrast, PDE4A isoenzymes do not provide substrates for C-terminal catalytic unit phosphorylation by Erk2. 3 Mutant PDE4 enzymes were generated to show that Erk2 phosphorylation occurs at a single, cognate serine residue located within the C-terminal portion of the PDE4 catalytic unit. 4 PDE4 long-form isoenzymes were markedly inhibited by Erk2 phosphorylation. 5 The short-form PDE4B2 isoenzyme was activated by Erk2 phosphorylation. 6 These functional changes in PDE activity were mimicked by mutation of the target serine for Erk2 phosphorylation to the negatively charged amino acid, aspartic acid. 7 Epidermal growth factor (EGF) challenge caused diametrically opposed changes in cyclic AMP levels in COS1 cells transfected to express the long PDE4B1 isoenzyme compared to cells expressing the short PDE4B2 isoenzyme. 8 We suggest that PDE4 enzymes may provide a pivotal point for integrating cyclic AMP and Erk signal transduction in cells with 4 genes encoding enzymes that are either insensitive to Erk2 action or may either be activated or inhibited. This indicates that PDE4 isoenzymes have distinct functional roles, giving credence to the notion that distinct therapeutic bene®ts may accrue using either PDE4 subfamily or isoenzyme-selective inhibitors.

Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.

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