Cigarette smoke and its component acrolein augment IL-8/CXCL8 mRNA stability via p38 MAPK/MK2 signaling in human pulmonary cells.

Interleukin-8 (IL-8/CXCL8) is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined the acute effect of aqueous cigarette smoke extract (CSE) on IL-8 expression in primary human pulmonary cells, in particular in normal human bronchial smooth muscle cells (HBSMCs). IL-8 mRNA levels increased upon CSE exposure in a concentration- and time-dependent manner, and such an effect was accompanied by IL-8 secretion. CSE-evoked elevation of IL-8 mRNA was mimicked by its component acrolein. Both CSE and acrolein induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, accompanied by the phosphorylation of MAPK-activated kinase 2 (MK2), a known downstream substrate of the p38 MAPK, both in HBSMCs and in human airway epithelial cells. Furthermore, pharmacological inhibition of p38 MAPK or MK2 strongly accelerated the decay of IL-8 mRNA levels upon stimulation with CSE or acrolein and subsequent blockade of mRNA neosynthesis with actinomycin D in pulmonary structural cells (HBSMCs and airways epithelial cells) as well as in human alveolar macrophages. Conversely, pharmacological inhibition of ERK1/2 signaling inhibited CSE-induced steady-state levels of IL-8 mRNA without affecting mRNA stability, thus suggesting inhibition at the transcriptional level. In sum, p38 MAPK/MK2 signaling is an important posttranscriptional mechanism underlying upregulation of IL-8 mRNA levels elicited by CSE and acrolein. Given the pivotal role of IL-8 in neutrophil chemotaxis and activation, our results shed light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.

ENPP1 affects insulin action and secretion: evidences from in vitro studies.

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.

In skeletal muscle advanced glycation end products (AGEs) inhibit insulin action and induce the formation of multimolecular complexes including the receptor for AGEs.

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.