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Limitations
There are limitations to our work. First, observational studies are subject to inherent limitations in the study design leading to unmeasured differences in the study population and unmeasured confounders despite all possible adjustments. PPIs use may be a surrogate of comorbidities and thus, the observed association may have been affected by selective overuse of PPIs in high risk groups. For example, the potential interaction between PPIs and Clopidogrel found in observational studies was refuted in randomized controlled trials [52]. Second, the use of PPIs was based on electronic and prescription records, rather than by actual use by the patient. Third, there is presence of publication bias, and substantial amount of heterogeneity in the included studies. There are many patient level parameters which may have led to substantial heterogeneity. Nevertheless, investigating these variables is only possible with individual patient data meta-analysis. Fourth, all statistical methods used to assess for publication bias or residual confounding are subject to certain assumptions and have inherent limitations. For example, funnel plot asymmetry can be due to between studies heterogeneity rather than publication bias [31]. Given these limitations, focus on hand hygiene as one of the cornerstones of prevention of nosocomial transmission of C. difficile is warranted. Several studies have documented the reduction of rates of hospital acquired infection by improvement in the compliance with hand washing by healthcare workers between episodes of contact with patients [53].

Conclusions
In this rigorously conducted systemic review and meta-analysis, we found very low quality evidence in support of an association between PPI use and risk of CDI. This association was weakened by the presence of significant heterogeneity and publication bias. Our findings are re-assuring that PPIs use in the general population does not pose a significant CDI risk. On the other hand, our findings warrant judicious and evidence-based use of PPI in patients at high risk for CDI.

Abstract
Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade.
Citation: Cirit M, Grant KG, Haugh JM (2012) Systemic Perturbation of the ERK Signaling Pathway by the Proteasome Inhibitor, MG132. PLoS ONE 7(11): e50975. doi:10.1371/journal.pone.0050975 Editor: Laszlo Buday, Hungarian Academy of Sciences, Hungary Received September 17, 2012; Accepted October 30, 2012; Published November 30, 2012 Copyright: ?2012 Cirit et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the National Institutes of Health (GM088987). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction
Signal transduction pathways and networks direct cell responses largely through post-translational modifications, e.g., phosphorylation/dephosphorylation of their protein components. But the rates of these modifications depend in turn on the intracellular concentrations of enzymes and other regulatory proteins; thus, mechanisms governing protein synthesis and degradation are equally central to the regulation of cell signaling. The ubiquitin-proteasome pathway is an essential quality control mechanism directing degradation of mislocated, misfolded, and damaged proteins, and, by tempering the expression levels of specific signaling proteins, it also exerts a level of control over cell physiology [1]. Poly-ubiquitinated proteins, targeted by E3 ubiquitin ligases, can be recognized and degraded by the 26S proteasome, a multi-subunit, multi-catalytic protease machine [2]. Proteasome inhibitors have shown great promise as cancer therapeutics because they impact a variety of mechanisms affecting tumor cell proliferation and survival; proteasome inhibition interferes with cell cycle progression, upregulates tumor suppressors such as p53, and diminishes activation of pro-proliferation pathways such as those controlled by NFkB and extracellular signal-regulated kinases (ERKs) [3,4]. The mitogen-activated protein kinases (MAPKs) ERK2/ MAPK1 and ERK1/MAPK3 (hereafter referred to collectively as ERK1/2) are activated by phosphorylation in a canonical Raf R MEK R ERK kinase cascade in response to most growth factors and cytokines, and ERK1/2 phosphorylate more than 150 cytosolic and nuclear substrates [5,6]. Thus, they are master controllers of cell proliferation, differentiation, and migration. ERK signaling is inappropriately activated in a wide array of
human cancers, which can be caused by an activating mutation in one of the upstream signaling proteins or through overexpression of growth factors or growth factor receptors [7,8]. The dual specificity phosphatases (DUSPs) have been linked to dephosphorylation of ERK1/2 and other MAPKs [9], and in many contexts, DUSP expression levels are known to be regulated through the ubiquitin-proteasome degradation pathway [10?5]. Accordingly, cells treated with MG132 or other proteasome inhibitors exhibit higher expression of MKP3/DUSP6, an ERK1/ 2-specific DUSP, accompanied by lower levels of ERK phosphorylation stimulated by growth factors [12?4]. Considering that a host of intracellular proteins are affected by proteasome inhibition, coupled with evidence that knockdown of MKP3 expression enhances growth factor-stimulated ERK phosphorylation in some contexts [13] but not in others [16], led us to question whether or not the diminution of ERK signaling in MG132-treated cells could be attributed solely to upregulation of MKP3 and other DUSPs. In this short paper, we confirm that MG132 treatment reduces phosphorylation of ERK in fibroblasts stimulated with platelet-derived growth factor (PDGF) or basic fibroblast growth factor (FGF) and show that this is caused by two parallel effects. For a given level of MEK activation, ERK phosphorylation is reduced, consistent with the proposed upregulation of ERK phosphatase activity, but maximal MEK activation is also diminished.

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