Matrix GLA protein function in human trabecular meshwork cells: inhibition of BMP2-induced calcification process.
OBJECTIVE
The matrix GLA (MGP) gene has been found to be among the 10 most highly expressed genes in the human trabecular meshwork (TM), and its expression is affected by conditions associated with glaucoma. Because MGP protein has been shown to play a key role in inhibiting calcification in cartilage and arterial vessels, MGP's function in human TM was investigated.


METHODS
Perfused TM tissue and primary human TM (HTM) cells originated from donors of nonglaucomatous eyes. MGP mRNA was assayed by relative quantitative and real-time PCR. AdhMGP recombinant adenovirus was generated by bacterial transposition. Western blot analyses were cross-reacted with MGP N-terminal- and conformational-specific antibodies. MGP/BMP2 colocalization was analyzed by confocal microscopy. gamma-Carboxylation activity was measured by incorporation of 14CO2 into FLEEL synthetic peptide. Alkaline phosphatase (ALP) activity was used as a marker of osteogenic differentiation and a calcification precursor. Calcification was assessed by measuring direct calcium (o-cresolphthalein). Normalization was conducted with a telomerase probe (genomic DNA).


RESULTS
HTM cells contained high levels of gamma-carboxylase activity and were able to convert MGP to its active conformation. Overexpression of MGP in HTM cells reduced ALP activity in a model of BMP2-induced osteogenesis. MGP colocalized intracellularly with BMP2. HTM cells aged in culture exhibited increased calcium content, increased ALP, decreased normalized MGP expression and lower gamma-carboxylase activity.


CONCLUSIONS
MGP protein is active and functions as an inhibitor of BMP2-induced ALP activity in the HTM cells. The human TM may undergo a calcification process with age. Inhibition of the calcification mechanism mediated by MGP could be used to regulate resistance and elevated IOP.
Xue
Wei
W
Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7041, USA.
Wallin
Reidar
R
Olmsted-Davis
Elizabeth A
EA
Borrás
Teresa
T
D000256:Adenoviridae / Q000235:genetics; D000293:Adolescent; D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002114:Calcinosis / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000502:physiology*; D019735:Carbon-Carbon Ligases / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006801:Humans; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D000097568:Matrix Gla Protein
D000469:Alkaline Phosphatase; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D002118:Calcium; D002135:Calcium-Binding Proteins; D019735:Carbon-Carbon Ligases; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000097568:Matrix Gla Protein; C525718:BMP2 protein, human; C020660:glutamyl carboxylase