Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma.
OBJECTIVE
The primary causative factor of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP) due to increased aqueous humor (AH) outflow resistance, which is associated with morphologic and biochemical changes in the trabecular meshwork (TM). Patients with glaucoma have elevated levels of transforming growth factor (TGF)-beta2 in their AH, and TGF-beta has been shown to increase TM extracellular matrix (ECM) production. The bone morphogenetic protein (BMP) signaling pathway modifies TGF-beta signaling in several different tissues, and a prior study demonstrated that TM cells and tissues express members of the BMP gene family. The purpose of this study was to determine whether BMPs can alter TGF-beta2 signaling in the TM and whether there are defects in BMP signaling in glaucoma.


METHODS
ELISA, Western immunoblot analysis, and immunohistochemistry were used to evaluate the expression of BMP proteins in TM cells and tissues. ELISA was used to determine the effects of TGF-beta2 and BMPs on TM fibronectin (FN) secretion. Gene expression was determined by gene microarrays and quantitative (q)PCR. Perfusion-cultured human anterior segments were used to study the effects of altered BMP signaling on IOP.


RESULTS
The human TM synthesized and secreted BMP-4 as well as expressed BMP receptor subtypes BMPRI and BMPRII. TM cells responded to exogenous BMP-4 by phosphorylating Smad signaling proteins. Cultured human TM cells treated with TGF-beta2 significantly increased FN levels, and BMP-4 blocked this FN induction. The expression of BMP family genes in normal and glaucomatous TM cells was profiled and significant elevation of mRNA and protein levels of the BMP antagonist gremlin were found in glaucomatous TM cells. In addition, Gremlin was present in human aqueous humor and in the perfusate medium of perfusion-cultured human eyes. Gremlin blocked the negative effect of BMP-4 on TGF-beta-induction of FN. Recombinant Gremlin added to the medium of ex vivo perfusion-cultured human eye anterior segments caused the glaucoma phenotype of elevated IOP.


CONCLUSIONS
These results are consistent with the hypothesis that, in POAG, elevated expression of Gremlin by TM cells inhibits BMP-4 antagonism of TGF-beta2 and leads to increased ECM deposition and elevated IOP.
Wordinger
Robert J
RJ
Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas, USA.
Fleenor
Debra L
DL
Hellberg
Peggy E
PE
Pang
Iok-Hou
IH
Tovar
Tara O
TO
Zode
Gulab S
GS
Fuller
John A
JA
Clark
Abbot F
AF
D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000378:metabolism; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism; D005902:Glaucoma, Open-Angle / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007429:Intraocular Pressure / Q000187:drug effects; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000378:metabolism / Q000494:pharmacology*
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D005353:Fibronectins; C486399:GREM1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D051785:Smad Proteins; D053781:Transforming Growth Factor beta2; D052005:Bone Morphogenetic Protein Receptors, Type I; D052006:Bone Morphogenetic Protein Receptors, Type II