A 14-kDa cathepsin L-derived carboxyl IGFBP-2 fragment is sequestered by cultured rat ileal crypt cells.
IGF-II gut drives mucosal growth during gestation. IGF binding protein-2 (IGFBP-2) 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.
Gordon
Phillip V
PV
Division of Neonatology, Department of Pediatrics, University of Virginia Health Sciences, Charlottesville, VA 22908, USA. pvg4n@virginia.edu
Paxton
Jessica B
JB
Kuemmerle
John F
JF
Fox
Nena S
NS
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D018938:Caco-2 Cells; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D007082:Ileum / Q000166:cytology* / Q000378:metabolism*; D007150:Immunohistochemistry; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism
D000906:Antibodies; D017077:Culture Media, Conditioned; D005938:Glucocorticoids; D018971:Insulin-Like Growth Factor Binding Protein 2; D010446:Peptide Fragments; D003907:Dexamethasone; D017526:Receptor, IGF Type 1; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat