Bone morphogenetic protein-2: a potential regulator in scleral remodeling.
OBJECTIVE
Bone morphogenetic protein 2 (BMP-2) is a member of the main subgroup of bone morphogenetic proteins within the transforming growth factor-beta superfamily. BMP-2 is involved in numerous cellular functions including development, cell proliferation, apoptosis, and extracellular matrix synthesis. We examined BMP-2 expression in human scleral fibroblasts (HSF) and assessed the effects of recombinant human BMP-2 (rhBMP-2) on HSF proliferation, matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-2 (TIMP-2).


METHODS
We used confocal fluorescence microscopy (CFM) to study BMP-2 distribution in HSF cells and frozen human scleral sections. The influence of rhBMP-2 on cell proliferation at different concentrations (0 ng/ml, 1 ng/ml, 10 ng/ml, and 100 ng/ml) was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effects of rhBMP-2 on the cell cycle were investigated with flow cytometric analysis. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine MMP-2 and TIMP-2 mRNAs and secreted proteins in HSF that were incubated with rhBMP-2.


RESULTS
BMP-2 protein expression from human sclera was confirmed by CFM. Cell proliferation was significantly increased with 100 ng/ml rhBMP-2 in a time-dependent manner (p<0.05). The HSF cell cycle moved to the S and S+G(2)M phases after rhBMP-2 stimulation at 100 ng/ml compared to normal cells (p<0.05). TIMP-2 mRNA levels were significantly increased in HSF incubated for 24 h with 100 ng/ml rhBMP-2 (p<0.01). A 48 h incubation with 10 ng/ml or 100 ng/ml rhBMP-2 resulted in significantly increased TIMP-2 mRNA and protein expression and significantly decreased MMP-2 mRNA expression (p<0.01) while MMP-2 protein expression significantly decreased at 100 ng/ml rhBMP-2 (p<0.01).


CONCLUSIONS
Human sclera fibroblasts expressed BMP-2, which promoted cell proliferation, and elicited changes in MMP-2 and TIMP-2, might influence extracellular matrix synthesis.
Hu
Jianmin
J
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, P R China.
Cui
Dongmei
D
Yang
Xiao
X
Wang
Shaowei
S
Hu
Shoulong
S
Li
Chuanxu
C
Zeng
Junwen
J
D000293:Adolescent; D000328:Adult; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D009928:Organ Specificity / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012590:Sclera / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2