Adrenergic signaling in human oral keratinocytes and wound repair

Abstract

Catecholamines are present in saliva, but their influence on oral epithelium is not understood. Because psychological stress increases salivary catecholamines and impairs oral mucosal wound healing, we sought to determine if epithelial adrenergic signaling could link these two findings. We found that cultured human oral keratinocytes (HOK) express the α(2B)- and β(2)-adrenergic receptors (ARs). Exposure of HOK to either epinephrine or the β-AR agonist, isoproterenol, reduced migratory speed and decreased in vitro scratch wound healing. Incubation with the β-AR antagonist timolol reversed the catecholamine-induced effects, indicating that the observed response is mediated by β-AR. Epinephrine treatment decreased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38; these decreases were also reversed with timolol. Cultured HOK express enzymes of the epinephrine synthetic pathway, and generate epinephrine. These findings demonstrate that stress-induced elevations of salivary catecholamines signal through MAPK pathways, and result in impaired oral keratinocyte migration required for healing.

Figure 1.

Expression of adrenergic receptors in HOK. (A) mRNA was isolated from HOK strains derived from three donors and hybridized to an Affymetrix HG-U133A microarray. The values in the table are signal intensities for hybridization with the microarray; values higher than 40 represent significant gene expression. Bold typeface indicates the 2 genes in the list that were significantly expressed. (B) Protein was isolated from HOK strains derived from four donors, and expression of the α_2B-AR and β₂-AR proteins was detected on a Western blot. Blots were stained with anti-α_2B-AR (Genex Bioscience, Hayward, CA, USA) at a 1:5000 dilution (2.3 µg/mL), followed by HRP-linked anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA) at a 1:1000 dilution (0.2 µg/mL), or with anti-β₂-AR (Abcam, Cambridge, MA, USA; clone Ab40834) at a 1:2500 dilution (0.2 µg/mL), followed by HRP-linked anti-goat secondary antibody (Abcam) at a 1:5000 dilution (0.4 µg/mL). α_2B-AR was detected at 49 kDa as expected based on its amino acid composition. Immuno-detected β₂-AR bands were evident at 49 kDa and 47 kDa, likely representing the palmitoylated and non-palmitoylated forms. Human epidermal keratinocytes were used as a positive control. 4, HOK from Donor 4; 5, HOK from Donor 5; 6, HOK from Donor 6; 7, HOK from Donor 7; 8, human epidermal keratinocytes from Donor 8.

Figure 2.

β-AR activation reduced HOK migration and in vitro wound closure. (A) Migratory speed of HOK that were plated on collagen-coated glass-bottomed culture dishes and treated with growth medium (Ctr), 1 µM isoproterenol (I), 20 µM timolol + 1 µM isoproterenol (T/I), or 20 µM timolol (T). Average speed in µm/min was calculated per treatment group, and we performed a one-way ANOVA followed by a two-sample, unequal variance, one-tailed Student’s t test to calculate significant differences between and among treatment groups. Panel A represents the mean values and standard errors of at least 7 experiments per treatment. The data represent 2 cell strains that were isolated from different donors. (B) Migratory speed of HOK that were treated with growth medium (Ctr), 1 µM epinephrine (E), 20 µM timolol + 1 µM epinephrine (T/E), or 20 µM timolol (T). Panel B represents the mean values and standard errors of at least 3 experiments per treatment. The data represent 6 cell strains that were isolated from different donors. *p ≤ 0.01 compared with Ctr; #p ≤ 0.05 compared with Ctr; **p ≤ 0.01 compared with I; and ***p ≤ 0.01 compared with E. (C) Scratch wounds were made in confluent cultures of HOK, previously treated with Mitomycin C, as described in MATERIALS & METHODS. Two scratches were made in each well, and two fields of view were photographed per scratch by means of an inverted Nikon Diaphot microscope. Images of the same field were captured at 0, 6, 12, and 24 hrs after the scratch was made. After wounding, the medium was replaced with growth medium (Ctr), 1 µM epinephrine (E), 20 µM timolol + 1 µM epinephrine (T/E), 20 µM timolol (T), or 20 µM yohimbine + 1 µM epinephrine (Y/E). The graph represents 2 HOK strains and displays the mean percentage healing calculated from 4 image areas on 2 scratches per treatment. *p ≤ 0.01 compared with Ctr; **p ≤ 0.05 compared with Ctr. (D) Actin and vinculin immunostaining revealed that cells that were treated with E had a non-migratory morphology. Cells were incubated overnight at 4°C with 33 µg/mL anti-vinculin (Sigma-Aldrich), followed by a two-hour incubation with 10 µg/mL AF594-goat-anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and 33nM AF488-phalloidin (Invitrogen). Cells treated with Ctr, T/E, or T all showed a similar migratory morphology. Bar = 10 microns.

Figure 3.

β-AR activation decreased ERK1/2 and p38 MAPK phosphorylation. Cells were grown in control medium until 60-80% confluent and treated for 60 min with growth medium (Ctr), 1 µM epinephrine (E), 20 µM timolol + 1 µM epinephrine (T/E), or 20 µM timolol (T). Immunoblotting with antibodies against P-ERK, ERK, P-p38 MAPK, and p38 MAPK (Cell Signaling Technology, Danvers, MA, USA) at a 1:1000 dilution (P-ERK, 0.1 µg/mL; ERK, 0.01 µg/mL; P-p38 MAPK, 35 ng/mL; p38 MAPK, 5 ng/mL) was followed by HRP-linked anti-rabbit secondary antibody (Cell Signaling Technology) at a 1:1000 dilution (0.2 µg/mL). Phosphorylated ERK and p38 MAPK were normalized to total ERK or p38 MAPK. The histogram represents the mean signal intensities and standard errors of at least 3 experiments per treatment. Panels A and B are data derived from 3 experiments. The data represent 2 cell strains that were isolated from different donors. *p ≤ 0.01 compared with Ctr.

Figure 4.

HOK can generate epinephrine. (A) Expression of enzymes tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT), involved in epinephrine synthesis. mRNA was isolated from HOK strains derived from Donors 1, 2, and 3 and hybridized to an Affymetrix HG-U133A microarray. The values in the table are signal intensities for hybridization with the microarray; values higher than 40 represent significant gene expression. (B) mRNA was isolated from HOK strains derived from Donors 4, 5, and 7, and Ct values for TH and PNMT were determined by real-time RT-PCR; values lower than 40 represent significant gene expression. (C) Epinephrine measured in HOK cellular extracts. Confluent cultures were extracted in 100 µL 0.1 M hydrochloric acid/100-mm culture dish, and 4 dishes were combined, sonicated, and used for triplicate measurements. Results are expressed as pg epinephrine per µg total protein in the sample. (D) Epinephrine measured in the culture medium conditioned by HOK. Results are expressed as the final concentration in the sample.