Fibronectin-Integrin Signaling Is Required for L-Glutamine’s Protection against Gut Injury

<div><h3>Background</h3><p>Extracellular matrix (ECM) stabilization and fibronectin (FN)-Integrin signaling can mediate cellular protection. L-glutamine (GLN) is known to prevent apoptosis after injury. However, it is currently unknown if ECM stabilization and FN-Integrin osmosensing pathways are related to GLN’s cell protective mechanism in the intestine.</p> <h3>Methods</h3><p>IEC-6 cells were treated with GLN with or without FN siRNA, integrin inhibitor G<u>RGD</u>SP, control peptide G<u>RGE</u>SP or ERK1/2 inhibitors PD98059 and UO126 under basal and stressed conditions. Cell survival measured via MTS assay. Phosphorylated and/or total levels of cleaved caspase-3, cleaved PARP, Bax, Bcl-2, heat shock proteins (HSPs), ERK1/2 and transcription factor HSF-1 assessed via Western blotting. Cell size and F-actin morphology quantified by confocal fluorescence microscopy and intracellular GLN concentration by LC-MS/MS.</p> <h3>Results</h3><p>GLN’s prevention of FN degradation after hyperthermia attenuated apoptosis. Additionally, inhibition of FN-Integrin interaction by G<u>RGD</u>SP and ERK1/2 kinase inhibition by PD98059 inhibited GLN’s protective effect. G<u>RGD</u>SP attenuated GLN-mediated increases in ERK1/2 phosphorylation and HSF-1 levels. PD98059 and G<u>RGD</u>SP also decreased HSP levels after GLN treatment. Finally, G<u>RGD</u>SP attenuated GLN-mediated increases in cell area size and disrupted F-actin assembly, but had no effect on intracellular GLN concentrations.</p> <h3>Conclusion</h3><p>Taken together, this data suggests that prevention of FN degradation and the FN-Integrin signaling play a key role in GLN-mediated cellular protection. GLN’s signaling via the FN-Integrin pathway is associated with HSP induction via ERK1/2 and HSF-1 activation leading to reduced apoptosis after gut injury.</p> </div

ERK1/2 activation is involved in GLN’s protective mechanism and attenuates after FN-Integrin pathway inhibition.

<p>A) IEC-6 cells were treated with different concentrations of GLN (0, 2, 10, and 20 mM) with or without 1 h prior PD98059 treatment. Cell survival was measured via MTS assay. Results are shown as mean±SEM (n = 3). B) [T(P)²⁰²/Y(P)²⁰⁴]ERK1/2 and total ERK1/2 levels were determined by Western blot analysis after basal and stressed 43°C conditions without recovery. ERK1/2 activation is shown as mean fold change relative to total ERK1/2±SEM and ratioed to 0 mM GLN (n = 3).</p

GRGDSP’s effect on cell area size, F-actin morphology and intracellular GLN concentration.

<p>A) Cell area size is shown by fluorescence microscopy in IEC-6 cells under 0 mM GLN conditions and 10 mM GLN treatment without inhibitor G<u>RGD</u>SP (Images A, C, E, G) or after 1 h prior treatment with G<u>RGD</u>SP (Images C, D, F, H) in basal and stressed conditions (43°C). F-actin is shown in green and nuclei in blue. All images taken 15 min following exposure of cells to GLN in non-HS cells and 15 min post-HS in stressed cells. All images were aquired using the same exposure times, and were renormalized the same for optical comparison (n = 4). Scalebars = 33 µm. B) Cell area size data are shown in fold increase under non-HS (37°C) and HS (43°C) conditions (n = 3–4) C) IEC-6 cells were treated as descibed in A). GLN levels in cell extracts were quantified using LC-MS/MS and are shown as fold change± SEM ratioed to 0 mM GLN group (n = 6–7). D) Fluorescence microscopy was used for the morphological analysis of the distribution of F-actin. Microfilaments were visualized with Alexa Flour 488 phalloidin antibody. Representative results are shown from 4 independent fluorescence microscopy experiments for each condition. Scalebars: 45 µm.</p

Proposed working model.

<p>GLN is protective in the intestine by preventing FN degradation after thermal injury, as well as by activating the protective FN-Integrin signaling pathway. GLN phosphorylates ERK1/2 via the FN-Integrin pathway leading to HSF-1 activation, which enhances HSP expression to prevent apoptosis.</p

GRGDSP and PD98059 affect GLN-mediated increases in HSP70 expression.

<p>A) IEC-6 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050185#pone-0050185-g002" target="_blank">Fig. 2B</a>. HSP70 expression was determined by Western blot analysis. In addition, ß-actin was monitored to normalize total blotted protein. Data are shown as mean fold change relative to HS 0 mM GLN±SEM (n = 5). B) Western blot of HSP70 and ß-actin are shown after PD98059 treatment. Results are ratioed to HS 0 mM GLN groups and represent means±SEM (n = 3).</p

GLN is protective by preventing FN degradation after HS.

<p>A) FN levels from IEC-6 cells, with or without GLN treatment under unstressed or stressed conditions, were determined by Western blot with ß-actin as loading control after 0 h recovery. Densitometric analysis of FN expression as mean fold change relative to 0 mM GLN cells±SEM (n = 4). B) Western blot and densitometric analysis of FN after 3 h recovery is shown (n = 4). C) FN expression of transfected IEC-6 cells with no siRNA, NC siRNA, or FN siRNA (n = 3) are presented. D) Caspase-3 and cleaved caspase-3 expression was determined via Western blot in basal and HS conditions after IEC-6 cells were transfected with no siRNA, NC siRNA, or FN siRNA (n = 3). E) Densitometric analysis of cleaved caspase-3 levels is shown as mean fold change relative to HS 0 mM GLN cells ±SEM (n = 3).</p

GLN’s intestinal protection is inhibited by GRGDSP.

<p>A) IEC-6 cells were treated for 1 h with either media, G<u>RGD</u>SP or G<u>RGE</u>SP before the cells were treated with 0, 2, 10 or 20 mM GLN. Cell survival, following lethal HS (44°C), was measured via MTS assay. All groups were normalized to their non-HS controls to account for differences in cell growth. Assays were carried out in triplicate, experiments were performed 4 times and shown as mean±SEM. B) IEC-6 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050185#pone-0050185-g002" target="_blank">Fig. 2A</a>, but they underwent non-lethal HS (43°C). After 3 hours recovery at 37°C, Bax and Bcl-2 levels were measured by Western blot. Bax was ratioed to anti-apoptotic marker Bcl-2 and shown in fold change±SEM (n = 4). C) Representative Western blot of PARP and cleaved PARP and densitometric analysis of cleaved PARP ratioed to HS 0 mM GLN are displayed. Cleaved PARP levels are presented as fold change±SEM (n = 4).</p