Activation Of P38 MAPK In Cells Research Articles

Desmoglein 3 Inhibits Keratinocyte Migration by Suppressing p38MAPK Activity

The desmosomal transmembrane adhesion molecule desmoglein 3 (Dsg3) is required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 loss of function, either by siRNA silencing or by peptides interfering with Dsg3 binding, increases the activity of p38MAPK. In this study, we aim to investigate the physiological role of Dsg3‐dependent p38MAPK suppression. Dsg3‐deficient mice display repetitive skin erosions most of which rapidly heal spontaneously. In lysates from wound biopsies but also from perilesional areas, p38MAPK activation was detectable compared to control animals. Similarly, in skin biopsies of pemphigus patients which suffer from impaired cell cohesion due to autoantibodies targeting Dsg3, p38MAPK activity was increased in areas of epidermal blistering. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK‐dependent manner. Indeed, silencing of Dsg3 resulted in increased p38MAPK activity which was paralleled by enhanced migration speed. In line with this, scratch wounded keratinocyte monolayers exhibited p38MAPK activation in cells lining the wound edge. Importantly, migration was efficiently blocked by inhibition of p38MAPK both in control and Dsg3 knockdown keratinocytes. These data indicate that Dsg3 via p38MAPK inhibition prevents a switch from an adhesive to a migratory keratinocyte phenotype. Upon wounding, impaired Dsg3 adhesion may thus favor wound closure by facilitating p38MAPK‐dependent migration.

A Subpopulation of Blasts with Attenuated p38MAPK Response Is Seen in Virtually All AML Patients and AML Cell Lines and Is Defined By Cells with Augmented Lipid-Associated Anti-Oxidant Defense

The serine/ threonine kinase, p38MAPK is activated by phosphorylation in response to a variety of cellular stresses including oxidative stress. Prolonged p38MAPK activation drives cell-cycle arrest and apoptosis; and in HSC activation of p38MAPK leads to a loss of reconstituting capacity (Ito et al, Nat.Med. 2006;12:446-451). In cancer, p38MAPK responses are often attenuated and cancer models suggest that this is a necessary adaptation for transformation (Dolado et al, Cancer Cell 2007;11:191-205). Previously we have shown that 60% of acute myeloid leukemia (AML) patients constitutively generate significantly more extracellular reactive oxygen species (ROS) than normal hematopoietic CD34+ cells (Hole et al, Blood 2013;122:3322-3330). Despite this, AML blasts showed low or absent p38MAPK phosphorylation; even in patients generating high levels of ROS. Here we examine p38MAPK activation at the single cell level in primary AML blasts using flow cytometry.We challenged AML blasts with a dose of hydrogen peroxide (H2O2) sufficient to completely activate p38MAPK in normal CD34+ cells (1 mM for 30 min), where the threshold for activation was defined as the 95th percentile of basal p38MAPK activation in unstimulated cells. Attenuated responses to H2O2 were seen in 14/15 (93%) of patients; where 16-95% of the total blast population failed to activate p38MAPK. These non-responding cells are hereafter termed “Δpp38MAPK cells” and were absent in normal CD34+ cells (p < 0.01; Figure 1). Examination of a panel of 6 AML cell lines showed that each of the lines contained Δpp38MAPK cells at different frequencies: MV4-11 (10%); HL60 (10%); KG-1 (15%), U937 (30%), NB4 (50%), THP-1 (65%). Further analysis showed that Δpp38MAPK cells were not distinguished by cell cycle phase, immunophenotype or reduced viability in either cell lines or AML blasts. These data suggest that nearly all AML patients harbor a population of blasts which have developed resistance to p38MAPK activation. We reasoned that failure to respond could arise either through defective p38MAPK signaling or because of enhanced anti-oxidative protection in a subpopulation of cells. To investigate the latter, we labelled cells with the lipophilic oxidation probe, C11 -BODIPY or the cytosolic oxidant probe, CM-DCFDA and monitored the oxidative response to H2O2 at the single cell level in the AML cell lines: KG-1, MV4-11 and THP-1. In each case C11 -BODIPY oxidation exactly matched the heterogeneous profile of p38MAPK activation in these cells, whereas CM-DCFDA showed only homogeneous responses to H2O2 induction. These data show that Δpp38MAPK cells are defined by an enhanced membrane-associated anti-oxidant capacity and we are currently analyzing this resistant subpopulation to identify the molecules responsible.To examine whether p38MAPK responsiveness influenced responsiveness to pro-oxidant drugs, we selected KG-1 and THP-1 cells (as representative examples of strong and weak p38MAPK responses respectively) and tested their sensitivity to the pro-oxidant drugs, phenethyl isothiocyanate (PEITC) and buthionine sulfoximine (BSO). We found that the IC50 was higher for THP-1 for both PEITC (KG-1 = 0.6µM; THP-1 = 7.5µM) and BSO (KG-1 = 50µM; THP-1 = 70µM), indicating that the p38MAPK responsiveness limits the effectiveness of pro-oxidant drugs. We next examined whether promoting p38MAPK activation could augment the effects of these pro-oxidants. We used the p38MAPK activator 2-benzylidene-3-(cyclohexylamino)-1-indanone (BCI), which promotes activation of p38MAPK via inhibition of a p38MAPK phosphatase, DUSP1. This compound weakly promoted phosphorylation of p38MAPK in THP-1 cells and consistent with this, had no effect on the efficacy of these compounds in these cells. However, BCI potently activated p38MAPK in KG-1 cells and showed synergy with BSO in this context (CI = 0.3; Figure 2) indicating that where BCI is effective in activating p38MAPK it can promote the effectiveness of pro-oxidant drugs.In summary, we show for the first time that AML patients almost universally display attenuated p38MAPK responses in all or part of the blast population and we suggest that this trait may be selected for to maintain self-renewing potential under the pro-oxidative conditions found in the leukemic marrow. Further we show that by manipulating p38MAPK activity, we can augment the potency of the pro-oxidant compound BSO. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells

BackgroundAortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. MethodsTLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity. ResultsInterstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity. ConclusionsExposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.

A Specific Mechanomodulatory Role for p38 MAPK in Embryonic Joint Articular Surface Cell MEK-ERK Pathway Regulation

Mechanisms regulating cell behavior and extracellular matrix composition in response to mechanical stimuli remain unresolved. Our previous studies have established that the MEK-ERK cascade plays a specific role in the mechano-dependent joint formation process by promoting the assembly of pericellular matrices reliant upon hyaluronan (HA) for their integrity. Here we demonstrate: (i) novel cross-talk between p38 MAPK and MEK-ERK signaling pathways that is specific for mechanical stimuli and (ii) a role for p38 MAPK in facilitating HA production by cells derived from the articular surface of embryonic chick tibiotarsal joints. We find that p38 MAPK blockade restricts pericellular assembly of HA-rich matrices and reduces basal as well as mechanical strain-induced release of HA. p38 MAPK blockers potentiated early strain-induced increases but restricted sustained increases in MEK/ERK phosphorylation at later times; c-Fos hyperphosphorylation at threonine 325 was found to parallel this p38 MAPK-mediated modulation of ERK activation. In contrast, p38 MAPK inhibitors had no detectable effect on the ERK activation induced by fibroblast growth factor 2 or pervanadate, a phosphatase inhibitor, and MEK inhibitors did not influence p38 MAPK phosphorylation, confirming both the specificity and unidirectionality of p38 MAPK-ERK cross-talk. Immunochemical and immunoblotting studies revealed constitutive p38 MAPK activation in cells at, or derived from, developing articular joint surfaces. Unlike the MEK-ERK pathway, however, p38 MAPK was not further stimulated by mechanical stimulation in vitro. Thus, p38 MAPK specifically facilitates ERK activation and downstream signaling in response to mechanical stimuli. These results suggest that constitutively active p38 MAPK serves an essential, permissive role in mechanically induced changes in ERK activation and in the accumulation of HA-rich extracellular matrices that serve a key role in joint development.