Transforming Growth Factor- 2 Induces Expression of Biologically Active Bone Morphogenetic Protein-1 in Human Trabecular Meshwork Cells
ABSTRACT There are limited studies on the factors that regulate the processing of transforming growth beta-2 (TGF-β2) and extracellular matrix (ECM) proteins into their mature form. BMP1 is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of this study was to determine whether: (a) cultured human TM cells express BMP1, (b) BMP1 expression is regulated by TGF-β2, (c) BMP1 is biologically active, and (d) BMP1 regulates LOX activity.
Primary human TM cells were isolated and subjected to qPCR and Western immunoblotting (WB) for BMP1. BMP1 immunolocalization was performed in TM tissues. qPCR was used to determine BMP1 mRNA expression and WB results was used to determine BMP1 protein expression. BMP1 activity was measured in TM cells treated with TGF-β2 or with a combination of TGF-β2 /UK383367. Lysyl oxidase (LOX) enzyme activity was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/β-aminoprorionitrile (BAPN).
Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-β2 increased mRNA expression compared with their controls (p<0.05). An ELISA showed TGF-β2 induced BMP1 secretion compared to their controls in all cell strains (p<0.05). Secreted BMP1 stimulated LOX enzymatic activity in TM cells.
BMP1 is expressed in the human TM. TGF-β2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms resulting in TM stiffness and resistance to ECM degradation.
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ABSTRACT: Abstract Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary risk factor associated with POAG. Increased aqueous humor (AH) outflow resistance through the trabecular meshwork (TM) results in elevated IOP in POAG patients. Resistance to AH outflow is associated with increased accumulation of extracellular matrix (ECM) proteins in the TM. In addition, levels of transforming growth factor-beta2 (TGF-β2) are elevated in the AH and TM tissue of POAG patients. Elevated levels of TGF-β2 in other tissues have been associated with fibrosis and increased tissue stiffness. However, locally produced effectors that maintain homeostatic relationships must also be present. Bone morphogenetic proteins (BMPs) serve this purpose in the TM as they inhibit TGF-β2-induced ECM changes in TM cells. This review article first describes the TGF-β superfamily of growth factors including BMPs and their canonical and noncanonical signaling pathways. The article then addresses the role of TGF-β2 in the pathophysiology of POAG as related to the ECM and ECM crosslinking enzymes. This is followed by a discussion of potential homeostatic control mechanisms of TGF-β2 signaling in the TM including the inhibitory role of BMP-4 and BMP-7. We then describe the relationship of TGF-β2 and BMPs in TM fibrosis including the role of antagonists. Lastly, in future directions, we identify potential future studies that explore new and unique cellular interactions within the TM for potential therapeutic interventions.Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics 02/2014; 30. DOI:10.1089/jop.2013.0220 · 1.42 Impact Factor
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ABSTRACT: Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian Tolloid (mTLD), mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days, by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1) and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). BMP-1/mTLD and PCPE-1 were also immunolocalized in two cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: BMP-1/mTLD and PCPE-1 are up-regulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.Investigative Ophthalmology & Visual Science 09/2014; 55(10). DOI:10.1167/iovs.13-13800 · 3.66 Impact Factor
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ABSTRACT: The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-β superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-β co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-β was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-β co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .Cellular and Molecular Life Sciences CMLS 09/2014; 72(5). DOI:10.1007/s00018-014-1733-x · 5.86 Impact Factor