Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function

Serine proteases do not induce an increase in phosphorylation of myosin regulatory light chain.

<p>Phosphorylation of MLC was assessed using phospho-specific antibodies (Ser19/Thr18) and western blotting <b>A</b>. A representative blot of phosphorylated and total MLC. <b>B</b>. Densitometry was performed with normalization to actin and the ratios of phosphorylated to total MLC were determined. n = 3–4; ns, not significant compared to control as analyzed by ANOVA with Dunnett’s posthoc test. <b>C</b>. MLC phosphorylation was also determined by Phos-tag gels. SCBN cells were plated on transwells and treated for 15 minutes apically in Krebs with 135 BAU/mL trypsin (T) or 1.5 BAU/mL matriptase (MT). Lysate was collected and phosphorylation of MLC assessed by Phos-tag gel electrophoresis. Protein with zero, one, or two phosphorylations (0p, 1p, 2p) are labeled. The positive control is SCBN cells treated with 3 μM of the phosphatase inhibitor calyculin A (CA) apically for 30 minutes. A blot with n = 3 is shown in <b>C</b>, while densitometry is shown in <b>D</b> and is the ratio of the phosphorylated band over the total MLC (all three bands combined), n = 6. No significant differences are seen as assed by ANOVA within each phosphorylated group.</p

EGF induces an increase in TER in SCBN cells similar to serine proteases.

<p><b>A</b>. SCBN cells were examined for mRNA expression of EGFR, ErbB2, ErbB3, and ErbB4 using RT-PCR. <b>B</b>. SCBN cells were mounted in Ussing chambers and treated with 50 or 100 ng/mL EGF and change in TER determined, n = 3 *p<0.05 ** p<0.01 compared to control by ANOVA with Dunnett’s post-hoc test. <b>C</b>. In Ussing chambers, SBTI was added apically to the cells 10 minutes post EGF treatment and representative tracing is shown. <b>D</b>. No significant differences were found when SBTI was added to cells treated with EGF, n = 3.</p

Inhibition of CK2 completely prevents the serine protease induced increase in TER.

<p>SCBN cells were mounted in Ussing chambers and treated apically with 50 or 100 μM TBCA for 30 minutes prior to apical challenge with 45 BAU/mL trypsin. A representative tracing is shown in <b>A</b>. The peak change in TER post trypsin was determined and TBCA at 100 μM significantly reduces the change in TER in response to trypsin (<b>B</b>). * p<0.05 as assessed by ANOVA with Dunnett’s post hoc test compared to DMSO control. N = 5–8.</p

Src inhibition affects the initiation, but not sustained phase of the serine protease induced increase in TER.

<p>SCBN cells in Ussing chambers were treated with PP1 either 30 minutes before apical treatment with 45 BAU/mL trypsin or 10 minutes after trypsin challenge. Pretreatment with PP1 caused a reduction in the peak trypsin response as seen in the representative tracing in (<b>A</b>). The effect was dose dependent and quantified as the percent peak TER compared to the DMSO control in (<b>B</b>) (n = 3) *** p<0.001, ****<0.0001, $ p<0.05 vs 10 μM PP1 by ANOVA with Tukey’s post-hoc test. Treatment with PP1 after trypsin challenge resulted in no significant change in TER (n = 3). Representative tracing shown in <b>C</b>, and concentration response in <b>D</b>. SCBN cells grown on transwells were serum starved for 60 minutes with 1 μM PD153035 or DMSO and stimulated apically with 45 BAU/mL trypsin for 5 minutes. Cells were lysed and Src phosphorylation was determined via western blotting. Neither apical treatment with 45 BAU/mL trypsin, or treatment with PD153035 changed the phosphorylation of Src (<b>E</b>) (n = 3).</p

The serine protease induced increase in TER is partially dependent on EGFR.

<p>Confluent SCBN monolayers mounted in Ussing chambers were pre-treated apically with 0.1, 1, or 2 μM PD153035 or DMSO vehicle control for 20 minutes then stimulated apically with 50 ng/mL EGF. <b>A</b>. A representative tracing of n = 3. <b>B</b>. Percent change in TER was determined 15 minutes after addition of EGF. * p<0.05, ** p<0.01 compared to DMSO + EGF-treated control by ANOVA with Tukey’s post-hoc test. A concentration of 1 μM PD153035 was then used for subsequent experiments. <b>C</b>. SCBN cells were pretreated with 1 μM PD153035 for 20 minutes prior to the addition of 45 BAU/mL trypsin. A representative tracing is shown of n = 4. Percent peak TER <b>(E)</b> and the percent increase in TER 15 minutes post trypsin addition <b>(F)</b> were determined and PD153035 significantly reduced both parameters * p<0.05 as assessed by one sample T-test. <b>D</b>. PD153035 was added apically to cells after a new plateau had been reached, and a representative tracing shown of n = 6–7. <b>G</b>. The percent remaining trypsin-induced increase in TER was then determined. * p<0.05 as assessed by one sample T-test.</p

Summary of signaling pathways mediating the increased TER induced by apical trypsin in SCBN cells.

<p>Serine proteases (trypsin, matriptase) cleave an as yet unidentified surface molecule (or molecules) that trigger a rapid and sustained increase in TER (inset). The initiation phase <b>(1)</b> involves ERK1/2, PI3-K, CK2 and Src. The sustained phase <b>(2)</b> is partly dependent on EGFR tyrosine kinase activity which may be due to a Src-dependent EGFR transactivation. Our previous studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180259#pone.0180259.ref020" target="_blank">20</a>] demonstrated a downstream role for PKCζ-dependent phosphorylation of occludin. Indeed, occludin is the key tight junction protein responsible for the protease-mediated increase in TER [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180259#pone.0180259.ref022" target="_blank">22</a>].</p

The serine protease induced increase in TER is dependent on proteolytic activity.

<p>Confluent SCBN cells were mounted in Ussing chambers and treated apically with the serine protease inhibitors SBTI or aprotinin before or after apical treatment of 45 BAU/mL trypsin or 0.5 BAU/mL matriptase, respectively. <b>A</b>. Representative tracing of cells treated with 60 μg/mL SBTI 30 minutes prior to the addition of trypsin. <b>B</b>. Representative tracing of cells treated with trypsin followed by apical treatment with 60 μg/mL SBTI after 10 minutes. <b>C</b>. The percent sustained trypsin response was determined for cells treated with SBTI 10 minutes after trypsin treatment, n = 3–4. <b>D</b>. Cells were also treated 30 minutes post trypsin addition with SBTI, and a representative tracing of 60 μg/mL SBTI is shown. <b>E</b>. Percent sustained trypsin response was determined for cells treated 30 minutes post trypsin addition, n = 3–5. <b>F</b>. Representative tracing of cells treated apically with matriptase followed by 100 nM aprotinin after 10 min. <b>G</b>. Percent sustained matriptase (MT) response was determined for cells treated 10 minutes post matriptase addition was determined, n = 3. ** p<0.01, *** p<0.001 ****p<0.0001 compared to trypsin or matriptase treated only controls by ANOVA with Dunnett’s post-hoc test.</p

The serine protease mediated increase in TER is not dependent on MMPs.

<p>SCBN cells were mounted in Ussing chambers and treated with 1, 5, 10, 20, or 30 μM GM6001 for 30 minutes and stimulated with 45 BAU/mL trypsin. A representative tracing is shown in (<b>A</b>) and the percent change in TER 20 minutes post treatment with trypsin was determined from baseline taken as a percentage of the trypsin only control determined (<b>B</b>). No significant differences were observed comparing the groups to the untreated control, as determined by ANOVA. Cells were also pretreated with several concentrations of marimastat and TAPI-1 prior to apical trypsin treatment. Representative tracings are shown in (<b>C</b>) and (<b>E</b>) and summary data showing present change in TER are in (<b>D</b>) and (<b>F</b>). Like GM6001, no significant changes were observed in the response to trypsin with marimastat or TAPI-1.</p