Anti-microbial peptides are essential for the intestinal innate immunity that protects the intestinal epithelia from attacks by foreign pathogens. Human β-defensin (HBD) is one of the pivotal anti-microbial peptides that are expressed in the colonic epithelia. This study investigated the effect and the signaling mechanism of inducible β-defensin HBD2 by an essential amino acid, isoleucine (Ile) in colonic epithelial cells. Here we examined the expression level of HBD2 on induction of Ile in epithelial cells, and checked this pathway. HBD2 mRNA was induced by co-incubation with IL-1 α and Ile in Caco2 cells, but not by Ile alone. An inhibitor of either ERK or Gi, a subunit of G-proteins, reduced the induction of HBD2 mRNA by Ile. The treatment with Ile also increased the intracellular calcium ion concentration, thus suggesting that the GPCR and ERK signaling pathway mediate the effects of Ile. These results indicate that an essential amino acid, Ile, enhances the expression of an inducible β-defensin, namely HBD2, by IL-1 α through the activation of GPCRs and ERK signaling pathway. The administration of Ile may therefore represent a possible option to safely treat intestinal inflammation.
The innate immunity system plays a significant role in inactivating pathogenic microbes which invade the host, particularly in the intestinal tract and skin, in which anti-microbial peptides are thought to be essential from attacks by bacteria, fungi and viruses [
Human colonic epithelial Caco2 cells purchased from the ATCC (American Type Culture Collection, Manassas, VA) were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% (vol/ vol) fetal bovine serum (FBS), 2 mM L-glutamine, 25 U/mL penicillin, 25 μg/mL streptomycin (all from Invitrogen/GIBCO, Grand Island, NY) and MEM non-essential amino acid solution (SIGMA, Hertfordshire, UK) in a humidified atmosphere of 5% CO2. The cells were plated on 6 well plates or 35 mm dishes and then were allowed to differentiate for 10 - 14 days before the experiments were performed.
After the Caco2 cells were grown in serum-free media for 24 hours, various concentrations of Ile (0, 5, 10, 20, 50, 100, 200 and 500 ng/ml) and/or IL-1α (0, 1, 5, 10, 20, 50 ng/ml) were added to the cells in 6 well plates. For the analysis of their inhibitory effects, an ERK pathway inhibitor (PD98059; Cell signaling, Boston, USA) or a GPCRs inhibitor (Pertussis toxin (PTX); SIGMA) was added 2 hours or 16 hours, respectively, before the harvest of the cells. RNA was harvested from the Caco2 cells using Ultrespec TM (BIOTECX, Houston, TX, USA) and then was reverse-transcribed using the Oligo (dT) 20 primer (Invitrogen, Carlsbad, CA, USA) and Superscript II RT (Invitrogen). The HBD2 mRNA was amplified on a Light Cycler system (Roche) using specific primers (sense, 5-ggtataggcgatcctgttacctgc-3), antisense, 5-tcatggctttttgcagcattttgttc-3) in triplicate. The averaged mRNA expression of HBD2 was normalized to GAPDH expression (LightCycler-primer set Human GAPDH (search LC, Heidelberg, Germany)).
The cells were washed 3 times with PBS and treated with Cell Lysis Buffer (Cell Signaling) with a Protease Inhibitor Cocktail Kit (PIERCE, Rockford, USA). Each lysate was centrifuged and the HBD2 protein in the supernatant was measured by a Human BD-2 ELISA Development Kit (PeproTech, Hamburg, Germany).
After the incubation of Caco2 cells with inducers such as Ile for various times, the cells were treated with Cell Lysis Buffer (Cell Signaling) with a Protease Inhibitor Cocktail Kit (PIERCE, Rockford, USA) as described above. Forty μg of each sample was resolved by NuPAGE Bis-Tris polyacrylamid gel (12%) (Invitrogen) and immediately transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) using 1X transfer buffer (25 mM Tris pH 8.8, 192 mM glycine with 15% [vol/vol] methanol). The PVDF membranes were incubated in PBS with 0.05% (vol/vol) Tween 20 (T-PBS) containing 5% (wt/ vol) milk for 1 hour at room temperature to block nonspecific binding. The blots were incubated overnight at 4˚C with an anti-rabbit p44/p42 phosphoor total-ERK antibody (Cell Signaling) as the primary antibody. The blots were washed five times for 10 minutes each in T-PBS at room temperature, incubated for 60 minutes in HRP-conjugated rabbit anti-mouse IgG (Jackson Immunoresearch, West Grove, PA) in T-PBS, washed four times in TPBS, once in PBS, and developed using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Massachusetts, USA).
Caco2 cells were incubated with 5 μM Fluo4 AM (Invitrogen) for 40 minutes, and then the cell images were obtained with a confocal laser capture microscope. The images were analyzed with an image-analytical software program (ZEN, 2007) to calculate the intensity of the fluorescence emitted from the cells.
All measured values are presented as the means ± standard deviation, and the statistical examinations were performed using Student’s t-test. A p-value < 0.05 was considered to be statistically significant.
The HBD mRNA expression was examined by real-time PCR. HBD2 mRNA was not detected after the treatment with any concentration of Ile alone. IL-1α induced HBD2 mRNA expression (
the effect of a co-incubation with IL-1α and Ile on the expression of HBD mRNA was investigated. As shown in
Because it has been proposed that the activation of the
ERK signaling pathway mediated the increase in the expression of HBD2 [
While most amino acids are absorbed by specific transporters expressed at the cell membrane, some of them are known to act as ligands for GPCRs. For example, glutamic acid is absorbed into the epithelia, and is also bound to a taste receptor which is one of the GPCRs. Many types of GPCRs, such as taste receptor and GPR35, are expressed in the intestinal epithelia [18-20]. In addi-
tion, the functions of some GPCRs are associated with the expression of anti-microbial peptides in neutrophils and gingival cells [21,22]. Furthermore, an α subunit of the GPCRs, Gi, is known to be involved in the activation of the ERK signaling pathway [23-29], suggesting a possibility that GPCRs are involved in the induction of HBD2 mRNA by Ile in the intestinal epithelia. To examine the influence of the GPCRs on the induction of HBD2 mRNA, Caco2 cells were incubated with an inhibitor of GPCRs, Pertussis toxin, for 16 hours before the treatment with IL-1α and Ile. As shown in
The present study demonstrated that an essential amino
acid, Ile, augmented the expression of HBD2 induced by IL-1α through the activation of the ERK signaling pathway, while Ile alone did not change the HBD2 expression. Furthermore, this Ile effect was reduced by an inhibitor of GPCRs, Pertussis toxin, illustrating that GPCRs were also involved in the induction of HBD2 by the combination of IL-1α and Ile. While it has been suggested that Ile induces the transcription of the promoter of the HBD2 gene [
IL-1α activates the ERK signaling pathway, which is known to be important for HBD2 expression [
Our data are the first to show that the induction of HDB2 by Ile was mediated by GPCRs, which are seventransmembrane domain receptors known to bind many ligands, such as chemical mediators and amino acids. GPCRs comprise a large protein family of transmembrane receptors that sense molecules outside the cell [
It has been proposed that the defensins expression decrease in patients with CD [5,14]. Therefore, the induction of HBD2 by the administration of Ile is a possible option to treat CD. We also confirmed that oral intake of Ile caused changes in the microflora in CD patients, possibly through the induction of HBD2 expression (data not shown). It was also reported that dexamethasone, a major drug used for the treatment of CD, induced HBD2 in the presence of pro-inflammatory cytokines [
This study was supported by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.K., #15390223 and #19390194, T.I., #20590733 and M.F., #23590931) and by the Intractable Disease, the Health and Labour Sciences Research Grants from Ministry of Health, Labor and Welfare in Japan.