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Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.
Insights
Transforming growth factor-beta 1 (TGF-beta 1) normally induces angiogenesis. However, a dominant-negative mutant of the TGF-beta type II receptor (T beta R-II) blocked TGF-beta 1
Area of Science:
- Cell Biology
- Molecular Biology
- Endocrinology
Background:
- Transforming growth factor-beta 1 (TGF-beta 1) is a key regulator of angiogenesis and capillary morphogenesis.
- Signal transduction of TGF-beta 1 involves heterodimerization of TGF-beta type I receptor (T beta R-I) and TGF-beta type II receptor (T beta R-II).
- Targeting TGF-beta signaling pathways offers potential therapeutic strategies for angiogenesis-related diseases.
Purpose of the Study:
- To investigate the inhibition of TGF-beta 1-mediated angiogenesis in glomerular capillary endothelial cells.
- To explore the use of a transdominant-negative mutant of the TGF-beta type II receptor (T beta R-IIM) for blocking TGF-beta signaling.
- To determine the role of T beta R-II signaling in TGF-beta 1-induced apoptosis and capillary formation.
Main Methods:
- Generation of a transdominant-negative mutant TGF-beta type II receptor (T beta R-IIM) lacking the kinase domain via PCR.
- Stable transfection of glomerular capillary endothelial cells with T beta R-IIM and verification of mRNA and protein expression.
- Assessment of TGF-beta 1's effect on [3H]thymidine incorporation, apoptosis, and capillary formation in wild-type and transfected cells.
Main Results:
- Transfection with T beta R-IIM successfully inhibited TGF-beta 1-induced cell proliferation, as evidenced by altered [3H]thymidine incorporation.
- TGF-beta 1-induced apoptosis and capillary formation in wild-type cells were completely abolished in T beta R-IIM-expressing cells.
- The study demonstrates that T beta R-II signaling is crucial for TGF-beta 1-mediated capillary morphogenesis and associated apoptosis.
Conclusions:
- Interference with TGF-beta type II receptor signaling effectively inhibits TGF-beta 1-induced capillary morphogenesis in glomerular endothelial cells.
- Capillary morphogenesis in vitro is intrinsically linked to apoptosis, a process regulated by T beta R-II signaling.
- This study provides the first evidence that a dominant-negative T beta R-II mutant can block TGF-beta-mediated angiogenesis and apoptosis.