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Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins¹

JIANHU ZHANG^*,, WEI LI^,, MATTHEW A. SANDERS^*,, BAUER E. SUMPIO^,, ASIT PANJA and MARC D. BASSON^*,,2

^* Departments of Surgery, Wayne State University;
John D. Dingell VA Medical Center,
Yale University,
Tianjin Medical University Cancer Hospital, China;
West Haven VA Hospital, and
Department of Medicine, State University of New York, Syracuse, New York, USA

²Correspondence: Surgical Service (11Surg), John D. Dingell VA Medical Center, 4646 John R. St., Detroit, MI 48201-1932, USA. E-mail: Marc.Basson{at}med.va.gov

SPECIFIC AIM

Complex patterns of mechanical deformation may stimulate intestinal epithelial proliferation. Since the matrix modulates static intestinal epithelial biology, we examined whether matrix proteins could influence the intestinal epithelial response to cyclic strain in vitro (using human Caco-2 and nontransformed human intestinal primary epithelial cells, HIPEC, as models); finding that fibronectin inhibited the effects of cyclic strain, we sought the mechanism of this effect.

PRINCIPAL FINDINGS

1. Cyclic strain on basement membrane collagen I and collagen IV stimulates proliferation and activates FAK and MAPK similarly, but strain yields different results on fibronectin or laminin substrates
10% strain at 10 cycles/min was similarly mitogenic on collagen I and collagen IV. Stimulation of proliferation on laminin was less, but significant. However, strain inhibited proliferation on fibronectin-coated membranes. Strain stimulated phosphorylation of focal adhesion kinase (FAK), ERK 1, and ERK 2 in cells cultured on collagen I, collagen IV, and laminin substrates, although phosphorylation on laminin appeared weaker. No phosphorylation occurred on fibronectin. The MEK inhibitor PD98059 completely blocked strain-induced proliferation on collagen I but did not affect static proliferation on collagen I or fibronectin or strain-stimulated proliferation on fibronectin. In contrast, EGF stimulated proliferation and ERK phosphorylation equivalently on collagen I or fibronectin.

2. Fibronectin inhibits the effects of strain
Plasma fibronectin (300 µg/mL, plasma level) inhibited the mitogenic effects of strain in cells on collagen I (Fig. 1 A)as well as FAK and ERK phosphorylation by strain (Fig. 1B ), but did not affect proliferation or ERK phosphorylation induced by EGF. Precoating with saturating concentrations of collagen I and fibronectin together prevented the mitogenic effects of strain.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of plasma fibronectin on strain-induced Caco-2 proliferation and FAK and ERK1,2 phosphorylation. All cells were cultured on collagen I (Col I) substrates. For proliferation studies, A) equimolar (0.68 µM) plasma fibronectin (pFN), collagen IV (Col IV), or laminin (LN) was added to the medium under static conditions (open bars) or during strain (shaded bars). Cells were cultured for 24 h after an initial time 0 measurement in parallel plates before trypsinization and counting. Cell number increased in response to strain when no matrix proteins were added to the medium () or in the presence of added collagen IV or laminin, but not in the presence of added plasma fibronectin (*P<0.01, n=6, 1 of 3 similar). In parallel signal studies, B) cells were cultured in the absence (-pFN) or presence (+pFN) of 0.68 µM plasma fibronectin for 3 h, then subjected to strain for 0, 10, or 60 min. The cells were lysed and subjected to Western blot with antibodies specific for FAK-397 phosphorylation or ERK 1,2 phosphorylation. Membranes were reprobed for total FAK or ERK as controls for protein loading. FAK397 and ERK phosphorylation increased in response to strain in the absence, but not in the presence of added plasma fibronectin (1 of 3 similar).

3. Strain is mitogenic for nontransformed human enterocytes (HIPEC); plasma fibronectin inhibits this

4. Ligation of the 5 or v integrin subunit, which may mediate adhesion to fibronectin, blocks the effects of strain on Caco-2 proliferation and FAK and ERK phosphorylation
5 and v integrin subunits were expressed in homogenates of normal human colonic mucosa from three patients, in HIPEC from three patients, and in Caco-2 cells. These cells express the 2 (involved in collagen binding) and 6 (involved in laminin binding) integrin subunits. Antibody to either the 5 or v integrin subunit blocked both the mitogenic effects of cyclic strain (Fig. 2 ,top) and phosphorylation of FAK and ERK by cyclic strain (Fig. 2 , bottom). However, neither antibody to the 2 subunit nor antibody to 6 prevented the stimulation of proliferation or phosphorylation of ERK in response to strain.

View larger version (47K): [in this window] [in a new window]   Figure 2. Effect of functional antibodies to integrin subunits on strain-associated FAK and ERK1,2 activation and Caco-2 proliferation. Caco-2 cells were cultured on collagen I. Either no anti-integrin antibody () or functional (blocking) antibodies to the 2, 5, v, or 6 integrin subunits were added to the medium. Top panel: Cells were cultured under static conditions (open bars) or conditions of cyclic strain (shaded bars) for 24 h in the absence or presence of anti-integrin subunit antibodies before counting. Plates counted before addition of the antibodies and initiation of strain provided time 0 measurements (*P<0.05, n=6, 1 of 3 similar). For signal studies, cells were harvested 10 min after the initiation of strain for FAK studies and 30 min after the initiation of strain for ERK studies. Lysates were and subjected to Western blot with antibodies specific for phosphorylated FAK-397 or phosphorylated ERK 1 and ERK 2, then stripped and reprobed for total FAK and ERK protein to control for protein loading (1 of 4 similar for each). Strain stimulated FAK and ERK phosphorylation and increased cell number under control conditions and in the presence of antibody to the 2 or 6 integrin subunits, but not in the presence of antibody to the 5 or v subunits.

CONCLUSIONS AND SIGNIFICANCE

Physical force effects have been studied in diverse cell types, with some similarities and some differences in findings. Little information exists about matrix modulation of strain effects. Indeed, the effects of strain varied with the matrix to which the cells were exposed. Either fibronectin or ligation of fibronectin binding integrin receptors inhibited the mitogenic and signal effects of strain. The effect seemed specific since fibronectin did not inhibit the effects of EGF. These results represent the first report that nontransformed primary human intestinal epithelial cells respond to strain and fibronectin similar to the Caco-2 cell model.

Some signaling induced by strain probably occurs at the focal adhesion complex, a cluster of transmembrane integrins, cytoplasmic signal molecules, and cytoskeletal anchors where FAK is activated. Such FAK activation may represent modulation of integrin-associated signal transduction by strain or "inside-out" mechanotransduction associated with cytoskeletal movement.

That ERK activation is required for the mitogenic effects of strain on collagen but does not affect basal Caco-2 proliferation on collagen nor basal or strain-treated proliferation on fibronectin illustrates an interesting specificity of cell types since ERK activation is not required for strain to stimulate proliferation in endothelial cells or inhibit it in osteoblasts. In contrast, ERK mediates other effects of strain in smooth muscle cells.

Not only are the effects of strain on intestinal epithelial cells matrix dependent, but fibronectin may regulate the effects of other matrix proteins since adding fibronectin blocks these effects.

Our results do not imply that fibronectin inhibits intestinal epithelial proliferation in static cells. The proliferation of primary intestinal epithelial cells or Caco-2 cells varies with the matrix substrate on which the cells are plated under static conditions, and unstimulated static Caco-2 cell proliferation on fibronectin exceeds proliferation on tissue culture plastic. Moreover, fibronectin does not inhibit the mitogenic effects of EGF. Instead, fibronectin appears to specifically inhibit the mitogenic effects of a physical force, cyclic deformation. In contrast, fibronectin binding by FET colon cancer cells via 5ß1 integrin suppresses proliferation by inhibiting ERK and cyclin-dependent kinases. Thus, altered tissue or plasma fibronectin levels in development or disease might regulate the effects of force on the gut mucosa.

We hypothesized that fibronectin modulation of strain effects might be mediated by fibronectin-binding integrins. Fibronectin binds to 5ß1, 4ß1, vß3, vß6, and IIß integrins, but Caco-2_BBE cells do not express 4 and IIß3 exists in platelets. Although controversy exists in the literature, we found immunoreactivity to both the 5 and the v integrin subunit in Caco-2 cells and primary human intestinal epithelial cells. Indeed, antibody to either the 5 or the v integrin subunit blocked both mitogenic effects and ERK activation by cyclic strain; neither anti-2 nor anti-6 had such effects.

Functional anti-integrin antibodies have been reported to affect static and strain-induced cell proliferation and phenotype by interfering with cell-matrix interactions. Since the cells we treated with functional antibodies to the or v subunits were maintained on collagen and since these antibodies had the same effect as fibronectin itself, it seems unlikely that the antibodies disrupted cell-fibronectin interactions. However, it has been suggested that IIbß3 integrin–matrix engagement can cluster the engaged integrin into a focal adhesion complex and alter the functions of 5ß1 and 2ß1 integrins. A similar mechanism may be responsible for integrin cross-talk in the model used in this study. Possible alternate explanations include disorganization of the cytoskeleton or signal complexes induced by ligation of "irrelevant" fibronectin binding integrins during culture on collagen, alterations in cell expression of other integrin heterodimers induced by fibronectin-receptor ligation, or induction of an as yet unidentified overriding negative signal, which then blocks the signal pathway induced by strain.

The intestinal mucosa is subject to complex physical forces in vivo engendered by villus motility and contraction, contact with luminal contents and opposing mucosa, and strain and pressure from peristalsis. Epithelial remodeling in response to nutrients, lactation, or small bowel resection may result in mucosal deformation. Intestinal epithelial proliferation and phenotype alter during starvation or ileus when patterns of mucosal strain are altered. Repetitive deformation and luminal pressure stimulate mucosal tyrosine kinase activity in anesthetized rats, and Caco-2 cell tyrosine kinase activity is similarly activated by repetitive deformation in vitro. Tyrosine kinase inhibitors block the effects of strain on Caco-2 proliferation and differentiation. Although diverse tyrosine kinases may be involved in strain signaling, the integrin-associated tyrosine kinase FAK is required for ERK activation by strain in Caco-2 cells and ERK activation is required for the effects of strain on proliferation and differentiation.

Collagen I and III dominate the interstitial matrix of the bowel, but gut basement membrane is primarily collagen IV and laminin. The basement membrane and the interstitial matrix contain tissue fibronectin in lesser amounts. Fibronectin is abundant in plasma in an alternately spliced form present in tissue matrix. Proliferating intestinal epithelial cells in crypts are exposed to tissue fibronectin in the basement membrane and plasma fibronectin in the matrix and extracellular fluid. Higher levels of fibronectin in the crypt matrix may control strain-stimulated intestinal epithelial proliferation. Tissue fibronectin is lost from the mucosa in development at the time of villus tip outgrowth. Neonates exhibit substantially higher mucosal proliferation than adults and lower plasma fibronectin levels. Plasma fibronectin tends to be decreased in acute inflammatory bowel disease when mucosal proliferation is high. Clearly an observation of correlation does not necessarily imply causality in complex pathophysiologic states. Other diseases occur in which fibronectin levels and mucosal proliferation are actually parallel and an inhibition of the mitogenic effect of strain by fibronectin may be overshadowed by other mitogenic stimuli. For instance, in hyperthyroidism, thyroid hormone directly drives both mucosal proliferation and plasma fibronectin levels. Nevertheless, these results raise the possibility that fibronectin inhibition of the mitogenic effects of strain may contribute to normal intestinal mucosal development and maintenance of the intestinal proliferative crypt compartment, as well as to the hyperproliferative state in inflammatory bowel disease.

There are manifest differences between regular strain on a uniform membrane in culture and the irregular repetitive mucosal strains that occur in vivo in response to peristalsis, villous motility, mucosal interaction with luminal contents, and other biological processes. Furthermore, the effects of forces in vivo on the gut mucosa must represent the sum of diverse endocrine, paracrine, neural, and metabolic influences engendered by force effects on other cell types in addition to direct effects of strain on intestinal epithelial cells. Nevertheless, these data raise the possibility that physiologic or pathophysiologic mucosal strain patterns may modulate intestinal epithelial proliferation in a manner regulated by plasma and tissue fibronectin levels.

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0663fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0663fje

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