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Bone morphogenic proteins 2 and 4 and their receptors in the adult human cornea
R R Mohan1, W J Kim, R R Mohan
1Eye Institute and the Department of Cell Biology, The Cleveland Clinic Foundation, Ohio, USA.
Purpose:
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.
Insights
Bone morphogenic proteins 2 and 4 (BMP2, BMP4) and their receptors are present in human cornea cells. These proteins influence corneal fibroblast proliferation and apoptosis, suggesting a role in corneal cell regulation.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Transforming growth factor-beta superfamily members, including bone morphogenic proteins (BMPs), play critical roles in tissue development and repair.
- The specific roles of BMP2 and BMP4 in the human cornea, particularly concerning fibroblast behavior, are not fully elucidated.
Purpose of the Study:
- To investigate the expression of BMP2, BMP4, and their receptors in human corneal cells.
- To determine the effects of BMP2 and BMP4 on corneal fibroblast proliferation and apoptosis.
Main Methods:
- Utilized reverse transcription-polymerase chain reaction (RT-PCR), immunoprecipitation, and Western blot for mRNA and protein analysis.
- Employed immunocytochemistry for protein localization and gel shift assay for nuclear factor-kappaB (NF-kappaB) activation.
- Assessed apoptosis via trypan blue exclusion, DNA laddering, CPP32 assay, and electron microscopy; proliferation was quantified by cell counting.
Main Results:
- BMP2 and BMP4, along with their receptor mRNAs (BMPR1A, BMPR1B, BMPR2), were detected in human corneal epithelial, keratocyte, and endothelial cells.
- BMP2 and BMP4 induced NF-kappaB activation, which mediated apoptosis in corneal fibroblasts when inhibited.
- BMP2 and BMP4 promoted corneal fibroblast proliferation independently of NF-kappaB activation.
Conclusions:
- BMP2, BMP4, and their receptors are expressed in the adult human cornea.
- These signaling molecules likely regulate critical corneal cell functions, including keratocyte proliferation and apoptosis.