Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors.
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.
Choi
M E
ME
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Ballermann
B J
BJ
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001769:Blood; D002196:Capillaries / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D005799:Genes, Dominant; D008969:Molecular Sequence Data; D009024:Morphogenesis; D012333:RNA, Messenger / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000494:pharmacology
D000906:Antibodies; D017931:DNA Primers; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D013936:Thymidine