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

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

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

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

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

A pan-JAK inhibitor prevents the decrease in AQP1 expression following treatment with IFNγ.

<p>(A) Immunoblots showing expression of phosphorylated and total STAT1, phosphorylated and total STAT3, AQP1 and actin in CMT93 cells pretreated with a pan-JAK inhibitor (20 μM for 2 hr) or DMSO vehicle followed by treatment with or without IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of phospho-STAT1 normalized to total (t) STAT1 expression. (C,D) Densitometry graph of phospho-STAT3α and phospho-STAT3β each normalized to total (t) STAT3 expression. (E) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO + IFNγ; ^$$$p < 0.001 vs. DMSO alone and pan-JAK Inhibitor alone; ^#p < 0.05 vs. DMSO alone. Blots are representative of 4 separate experiments.</p

Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation - Table 3

<p>Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation - Table 3 </p