LTA represents a class of amphiphilic molecules anchored to the outer face of the cytoplasmic membrane in gram-positive bacteria and is commonly released during cell growth, especially under antibiotic therapy
[1, 2]. It can cause cytokine induction in mononuclear phagocytes
. In previous studies, LTA concentrations of 0.2~50 μg/ml were detected and stimulated activity of polymononuclear leucocyte functions and release of TNF-α in peripheral blood mononuclear cells
[23, 24]. Meanwhile, LTA levels at the infectious site can reach a high level of 26,694 ng/mL
. The concentration of LTA used in this study was < 50 μg/ml. Therefore, our results show that LTA at clinically relevant concentrations can activate alveolar type II epithelial cells by stimulating production of surfactants.
During bacterial infection, endotoxins, including LTA and LPS, increase capillary permeability and enhance expressions of cellular adhesion molecules, proinflammatory cytokines, and chemokines
[1, 15]. These endotoxins can lead to most of the clinical manifestations of bacterial infection and are associated with ALI
[4, 5]. In addition, LTA can trigger lung inflammation and causes neutrophil influx into the lungs
[15, 26]. This study showed that in response to LTA stimulation, levels of SP-A mRNA and protein in alveolar A549 cells were time-dependently augmented. SP-A contributes to the pulmonary host defense
[10, 16, 27]. A previous study reported that when sp
a gene expression was knocked-out, susceptibility of the lungs to pathogenic infection was simultaneously raised
. Our previous study also showed that LPS-mediated toll-like receptor (TLR) 2 signaling in human alveolar epithelial cells might increase SP-A biosynthesis and subsequently lead to an inflammatory response in the lungs
. As a result, SP-A could be an effective biomarker for detecting pulmonary infection by gram-negative or -positive bacteria.
This study showed that LTA increased the expression of NF-κB and its translocation from the cytoplasm to nuclei. NF-κB is a typical transcription factor in response to stimulation by LTA
. LTA can bind CD14 and then stimulates TLR activation
[16, 29]. After LTA associates with TLR2, NF-κB can be activated by protein kinases and is then translocated to nuclei from the cytoplasm
. NF-κB regulates certain gene expressions to control cell proliferation, differentiation, and death
[30, 31]. A previous study showed that LTA induced cyclooxygenase-2 expression in epithelial cells via IκB degradation and successive p65 NF-κB translocation
. LTA could induce SP-A mRNA expression in A549 cells. Our bioinformatic search revealed that NF-κB-DNA-binding motifs were found in the promoter regions of the sp
a gene. Suppressing NF-κB activation using BAY 11–7082 simultaneously inhibited LTA-induced SP-A mRNA expression. Thus, LTA transcriptionally induces SP-A expression through inducing NF-κB expression and translocation.
Our present results revealed that the phosphorylation of ERK1/2 was associated with NF-κB activation. Sequentially, ERK1/2-activated IκBα kinase can phosphorylate IκB at two conserved serine residues in the N-terminus, triggering the degradation of this inhibitor and allowing for the rapid translocation of NF-κB into nuclei
[16, 20]. Accordingly, LTA-induced activation of A549 cells is mainly due to the improvement in ERK1/2 phosphorylation. Roles of ERK1 and ERK2 in LTA-induced SP-A expression were not determined in this study but will be validated using RNA interference in our next study. There is growing evidence that the ERK signaling pathway, which contributes to regulating inflammatory events
. Therefore, LTA regulates SP-A expression in alveolar type II epithelial cells in the course of eliciting ERK1/2 phosphorylation and subsequent activation of the transcription factor, NF-κB.
ERK activation is mediated by at least three different pathways: a Raf/MEK-dependent pathway, a PI3K/Raf-independent pathway that strongly activates MEK, and a third undetermined pathway that directly activates ERK proteins
. This study showed that LTA time-dependently increased levels of phosphorylated MEK1. Thus, one of the possible reasons explaining why LTA stimulates ERK1/2 activation is the increase in MEK1 phosphorylation. MAPK-regulating signals place this family of protein kinases in an apparently linear signaling cascade downstream of growth factor receptors, adaptor proteins, guanine-nucleotide exchange factors, Ras, Raf, and MEK
. The present study demonstrates that LTA can induce SP-A expression via MEK-dependent activation of the protein kinase ERK1/2-signaling pathway.