We have previously hypothesized that IGF-I is a mediator of dexamethasone (DEX) effect in the newborn mouse ileum-a model designed to mimic the precocious mucosal maturation associated with spontaneous ileal perforations in extremely premature neonates. We have further investigated this hypothesis using in vivo and in vitro models of accelerated epithelial migration (a transient property, temporally associated with mucosal maturation). These experiments include a steroid-treatment model comparing IGF-I immunolocalization with bromodeoxyuridine (BrdU)-pulse-labeling, as a means of assessing epithelial cell migration, within the ileum of newborn mice that received either daily intraperitoneal injections of DEX (1 g/gm) or vehicle. Likewise, a transgenic newborn mouse model was used to compare the effect of IGF-I overexpression upon the clearance of BrdU-pulse-labeled epithelial cells traveling up the villus during the same time period. For our in vitro model, rat ileal epithelial cells (IEC-18) were cultured to confluence in serum-free media then treated with DEX, a stable IGF-I agonist, or nothing before being subjected to linear scarification. Serial photomicrographs of migrating cells were taken over time and the average speed was determined for each treatment condition. Our data demonstrate that IGF-I accelerates ileal epithelial cell migration in every model. However, DEX was only associated with accelerated epithelial cell migration in models where IGF-I (or a synthetic agonist) was highly abundant. In contrast, DEX by itself slowed migration speed in cell culture. These findings suggest that IGF-I may be a mediator of steroid effect during precocious maturation of the ileal mucosa. Normal mucosal maturation in rodents is triggered by a systemic cortisol surge that occurs during weaning (1). This process can be accelerated in suckling animals by exogenous administration of glucocorticoid in the weeks preceding weaning (2). However, administration of glucocorticoid in the first days of life results in a precocious maturation (characterized in the newborn mouse by mucosal growth concomitant with bowel wall thinning) (3). Further comparison between the precocious and the accelerated models of maturation in mouse pups reveals attenuated expression of maturation-related digestive enzymes in the younger animals (4). In addition to these developmental aberrancies, precocious maturation has also been linked to a neonatal disease state.The incidence of spontaneous ileal perforation in extremely low birth weight infants is approximately 5%, but the incidence is at least twice that when DEX is administered in the first days of life (5). Like the animal models, pathology from perforated ileal specimens demonstrates hypertrophied mucosa concomitant with a thinned bowel wall (6). Immunolocalization surveys of relevant growth factors during precocious maturation in the newborn mouse ileum have implicated two probable mediators of DEX effect (3,7,8). Whereas most growth factors are diminished by DEX treatment, IGF-I and tra...

Spontaneous intestinal perforations in extremely premature infants are associated with glucocorticoid-induced thinning of the ileal bowel wall. We have previously demonstrated that insulin-like growth factor-1 (IGF-1) is abundant within the submucosa of the newborn mouse ileum but is diminished by glucocorticoid exposure, concomitant with bowel wall thinning. These findings prompted us to hypothesize that IGF-I governs submucosal growth during neonatal gut development and that diminished IGF-I abundance results in submucosal thinning. Heterozygous IGF-I knockout, wild type and homozygous IGF-I over-expresser newborn mice were euthanized at 3 d of life. Additionally, wild type newborn mice received daily dexamethasone (DEX) (1g/gm/d) or vehicle control on days of life 1 and 2 and were also euthanized at 3 d of life. Their ileums were harvested, fixed and the resulting sections were processed in parallel for IGF-I immunohistochemistry and morphometric measurements of submucosal thickness and bowel diameter. Immunolocalization of IGF-I in each genotype was also compared with that seen in DEX-treated and control mice euthanized at the same time of life. IGF-I heterozygous knockouts had diminished submucosal IGF-I immunolocalization (similar to that seen in DEX-treated newborn mice) whereas homozygous IGF-I over-expressers exceeded that seen within wild type mice. IGF-I genotype and submucosal abundance was positively correlated with ileal submucosal thickness. DEX treatment of newborn mice diminished IGF-I and caused significant thinning of the submucosa compared with controls. Submucosal growth and thickness in the neonatal mouse ileum is governed by IGF-I and is diminished by dexamethasone treatment. Focal or spontaneous intestinal perforation in the extremely low birth weight infant is an emerging disease that was not commonly seen before the introduction of surfactant therapy. Today, now that the age of gestational viability has been significantly lowered, 4% to 5% of infants born weighing less than 1000 g will develop a sterile ileal perforation in the first week of life and twice that many will perforate if exposed to higher doses of glucocorticoids in the perinatal period (1). This disease causes significant morbidity and mortality in the neonate due to the subsequent development of peritonitis. Spontaneous perforations also represent a disturbing conundrum in neonatology, i.e. that effective therapies for prevalent developmental diseases (like respiratory distress syndrome) will likely result in the emergence of new and equally morbid diseases in the rescued survivors. In some cases, preexisting adjunct therapies can actually facilitate the genesis of such diseases.For example, several prominent clinical studies have demonstrated a relationship between perinatal dexamethasone treatment and spontaneous intestinal perforations (2-4). Histologic studies of perforated ileal tissue demonstrate a skewing of tissue development such that the mucosa is hypertrophied, the lumen is dilated and the bowel wall is thinned...

Glucocorticoids induce hypertrophy of the neonatal ileal mucosa but the molecular mechanisms behind this growth induction remain poorly understood. Ileal epithelial cells (IECs) are dependent upon IGF-II for proliferation both in vivo and in culture. The type-2 IGF receptor (IGFR-2) is a lysosomal transport protein that attenuates IGF-IIdriven growth and is highly abundant in the ileum. The cellular repressor of E1A-stimulated genes (CREG) is a secreted phosphoglycoprotein that affects cell fate via ligand binding with IGFR-2, although the mechanism by which it does so is unknown. We hypothesized that glucocorticoids might facilitate IGF-mediated hypertrophy through CREG-mediated degradation of IGFR-2. To test this hypothesis, confluent rat IECs (IEC-18) were cultured for 72 h with or without dexamethasone (DEX) and harvested for Western blot, immunocytochemistry, gene array and CREG immunoneutralization experiments.IGFR-2 and CREG immunohistochemistry were also performed in archived ileal specimens from control and DEX-exposed newborn mice and extremely premature infants to investigate in vivo and clinical relevance. DEX exposure was found to diminish IGFR-2 immunolocalization in cultured rat IECs, newborn mouse ileal mucosa and human neonatal ileal mucosa. Gene array data indicated that IGFR-2 expression was unchanged with DEX treatment, suggesting a mechanism of protein degradation. CREG immunolocalization and abundance was found to be increased by DEX and immunoneutralization of CREG resulted in the abolition of IGFR-2 degradation. We have concluded that CREG is a secreted mediator by which DEX induces degradation of IGFR-2 and speculate that this is a fundamental mechanism of mucosal growth induction.

has a high affinity for IGF-II and tightly regulates IGF-II availability during fetal and early neonatal growth. We have previously demonstrated that glucocorticoids alter IGF homeostasis in the neonatal ileum, but the mechanism(s) by which this occurs is poorly understood. We hypothesized that dexamethasone alters proteolytic regulation of IGFBP-2 in ileal crypt cells. To test this, ileal crypt [ileal epithelial (IEC)-18] cells were cultured in serum-free media and used to study IGFBP-2 catabolism by immunochemistry, gene array analysis, and pharmacological perturbation with dexamethasone. In addition, isolated human IGFBP-2, IGF-II, and cathepsins B, D, and L were utilized for in vitro protease assays. We found IGFBP-2 to be highly abundant in IEC-18 culture, and sequestration of carboxyl IGFBP-2 antigen was seen within vesicular bodies of some cells. Dexamethasone significantly decreased the number of these cells and decreased IGFBP-2 in the media. On gene array analysis, cathepsin L's message abundance was significantly increased by dexamethasone, and, by in vitro assay, cathepsin L created a 14-kDa carboxyl fragment that corresponded to the sole antigen detected in IEC-18 cell lysates as well as a 16.5-kDa fragment found in the media. The sequestered fragment size was formed preferentially when IGF-II was present, whereas the larger fragment size was formed preferentially when IGF-II was absent. Cathepsins B and D did not produce these fragments in vitro and were not detected in IEC-18 media. We conclude that dexamethasone alters IGFBP-2 catabolism through its effects on cathepsin L. dexamethasone; insulin-like growth factor; ileum; gut development FETAL GROWTH IS PREDOMINANTLY regulated by an autocrine/ paracrine growth factor known as IGF-II (9, 33). Syndromes of fetal overgrowth result from the disruption of IGF-II homeostasis, either by overexpression of IGF-II (13, 15) or by decreased IGF-II degradation (23, 41a). We postulate that altered catabolism of IGF binding proteins (IGFBPs) might be a potential regulatory mechanism as well. The fetal gut is particularly dependent on IGF-II and is disproportionately affected in these fetal overgrowth syndromes. Gut crypt cells utilize IGF-II to sustain proliferation in the postnatal periods (3). This arrangement holds true throughout life, as IGF-II is also a crucial growth factor for gastrointestinal cancers (10, 30, 31).IGFBP-2 has a high affinity for IGF-II and is coexpressed with IGF-II in the fetal gut (19,24,38,41). While IGFBP-2 inhibits IGF-II-mediated proliferation in cell culture models (21, 22), there is no intestinal phenotype in IGFBP-2 knockout mice (where overgrowth of the fetal gut might have been anticipated) (40), suggesting that its effects on IGF homeostasis are complex and balanced.IGFBP-2 has been found in a wide variety of vertebrates, including zebrafish, and is universally abundant in the developing gut (14). However, compared with mammalian IGFBP-2, zebrafish IGFBP-2 does not have the putative proteoglycanbinding site. This sequence is...

Background: IEC-18 cells are a non-transformed, immortal cell line derived from juvenile rat ileal crypt cells. They may have experimental advantages over tumor-derived gastrointestinal lineages, including preservation of phenotype, normal endocrine responses and retention of differentiation potential. However, their proclivity for spontaneous differentiation / transformation may be stereotypical and could represent a more profound experimental confounder than previously realized. We hypothesized that IEC-18 cells spontaneously diverge towards a uniform mixture of epigenetic fates, with corresponding phenotypes, rather than persist as a single progenitor lineage.