Bone morphogenic proteins 2 and 4 and their receptors in the adult human cornea.
OBJECTIVE
To examine the expression of transforming growth factor family members bone morphogenic proteins 2 and 4 (BMP2, BMP4), their receptor mRNAs, and BMP2 and BMP4 proteins in the cells of the human cornea. The effects of BMP2 and BMP4 on corneal fibroblast proliferation and apoptosis were also examined.


METHODS
Reverse transcription-polymerase chain reaction, immunoprecipitation, and western blot analysis were used to examine mRNA and protein expression in cultured human corneal cells. Immunocytochemistry was applied to examine protein localization in fresh frozen human cornea cells. Stimulation and inhibition of nuclear factor-kappaB (NF-kappaB) activation was evaluated by gel shift assay. Apoptosis was examined using trypan blue exclusion, laddering of DNA, CPP32 assay, and transmission electron microscopy. Proliferation was monitored by counting cells.


RESULTS
BMP2 and BMP4 mRNAs and proteins were expressed in cultured human corneal epithelial cells, keratocytes, and corneal endothelial cells. BMP2 and BMP4 were detected in each major corneal cell type in fresh frozen human cornea. BMP receptor IA, IB, and II mRNAs were also detected in cultured human corneal epithelial cells, keratocytes, and endothelial cells. BMP2 and BMP4 stimulated activation of NF-kappaB. Actinomycin D and SN50 peptide, but not SN50M control peptide, inhibited NF-kappaB activation in response to BMP2 or BMP4. BMP2 and BMP4 stimulated apoptosis of corneal fibroblast cells when NF-kappaB activation was inhibited with the nonselective inhibitor actinomycin D or selective inhibitor SN50. The nonsteroidal anti-inflammatory agents ketorolac tromethamine and diclofenac sodium augmented the effect of BMP2 on corneal fibroblast apoptosis. BMP2 and BMP4 both stimulated proliferation of corneal fibroblast cells in the absence of inhibitors of NF-kappaB activation.


CONCLUSIONS
BMP2, BMP4, and their receptors are expressed in the cells of the adult human cornea. The functions regulated by these cytokines may include keratocyte proliferation and apoptosis.
Mohan
R R
RR
Eye Institute and the Department of Cell Biology, The Cleveland Clinic Foundation, Ohio, USA.
Kim
W J
WJ
Mohan
R R
RR
Chen
L
L
Wilson
S E
SE
D000328:Adult; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D052004:Bone Morphogenetic Protein Receptors; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication / Q000187:drug effects; D004357:Drug Synergism; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007124:Immunoenzyme Techniques; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta
D000894:Anti-Inflammatory Agents, Non-Steroidal; C525718:BMP2 protein, human; C525809:BMP4 protein, human; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017931:DNA Primers; D016328:NF-kappa B; D010455:Peptides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D017978:Receptors, Growth Factor; C118258:SN50 peptide; D016212:Transforming Growth Factor beta; D052004:Bone Morphogenetic Protein Receptors