Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.
TGF-beta1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-beta signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-beta type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-beta type II receptor N-terminal peptide that perturbs TGF-beta ligand-receptor binding increased branching by 70%. Similarly, antisense TGF-beta type II receptor oligodeoxynucleotides (40 microM) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-beta, type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-beta1 did not overcome the stimulatory effects either of TGF-beta type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Using in situ hybridization and immunohistochemistry, both TGF-beta type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-beta type II receptor is an important regulator of epithelial-mesenchymal interaction. Exogenous TGF-beta1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-beta type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-beta1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-beta type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-beta1, whereas TGF-beta1 alone decreased PCNA staining. Thus, abrogation of TGF-beta type II receptor expression prevented TGF-beta1-induced epithelial cell G1 arrest. These results demonstrate, for the first time, that abrogation of the TGF-beta type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-beta signaling upon epithelial cell cycle progression.
Zhao
J
J
Department of Surgery, Pediatrics, and Cardiothoracic Surgery, Childrens Hospital, Los Angeles Research Institute, California, USA.
Bu
D
D
Lee
M
M
Slavkin
H C
HC
Hall
F L
FL
Warburton
D
D
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D019938:Cyclin D1; D016213:Cyclins / Q000096:biosynthesis; D017931:DNA Primers; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000196:embryology*; D051379:Mice; D009024:Morphogenesis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D015513:Oncogene Proteins / Q000096:biosynthesis; D009924:Organ Culture Techniques; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000906:Antibodies; D016213:Cyclins; D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D015513:Oncogene Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II