Insulin-like growth factor binding protein-5 expression by human trabecular meshwork.
OBJECTIVE
The authors found transcript expression for insulin-like growth factor binding protein-5 (IGFBP-5) while screening for uniquely expressed trabecular meshwork (TM) mRNAs. Because the insulin-like growth factor (IGF) autocrine-paracrine system may provide an important signaling mechanism between TM cells and the outflow pathway, the expression of IGFBP-5 and IGF-I receptor in the TM was characterized.


METHODS
Poly(A+) RNA was isolated from cell cultures of human TM, ciliary body, retinal pigment epithelium, and skin fibroblasts and subjected to reverse transcription-polymerase chain reaction (RT-PCR) differential display analysis. A unique 980-bp band present in the TM was cloned and sequenced. Additional PCR and Northern analyses were used to define trabecular IGFBP-5 expression. Western immunoblots and confocal immunohistochemistry were used to evaluate the protein expression patterns of IGFBP-5 and the IGF-I receptor. IGF-I and IGF-II were added to trabecular cells in culture, and matrix metalloproteinase production was evaluated.


RESULTS
A unique differential display band was identified in the TM. Sequencing of this band identified it as the 3'-untranslated region of IGFBP-5. RT-PCR, using a variety of specific primers for IGFBP-5, Northern analysis, Western immunoblots, and immunohistochemical analysis, confirmed that IGFBP-5 was expressed in the TM. However, IGFBP-5 was also present at low levels in the ciliary body and skin fibroblasts by Northern and Western analysis, in contrast with the differential display findings. In addition, the IGF-I receptor was expressed by the TM and showed cell-surface staining by immunohistochemistry. Trabecular IGFBP-5 was distributed throughout the meshwork in the extracellular matrix and the cells with more staining in the juxtacanalicular region than in the uveal meshwork. IGF-I, but not IGF-II, modestly increased trabecular stromelysin and gelatinase B but not collagenase, gelatinase A, or tissue inhibitor of metalloproteinases 1 or 2.


CONCLUSIONS
IGFBP-5 and IGF-I receptor were expressed at significant levels by TM cells and may serve an important role in trabecular function.
Wirtz
M K
MK
Casey Eye Institute, Oregon Health Sciences University, Portland 97201, USA.
Xu
H
H
Rust
K
K
Alexander
J P
JP
Acott
T S
TS
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D005109:Extracellular Matrix / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000378:metabolism; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger; D012313:RNA; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; D008666:Metalloendopeptidases