Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion.
BACKGROUND
Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages.


METHODS
To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days.


RESULTS
During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation.


CONCLUSIONS
TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.
Muraoka
Noriko
N
Graduate School of Dental Science, Kyushu University, Fukuoka, Japan.
Shum
Lillian
L
Fukumoto
Satoshi
S
Nomura
Taisei
T
Ohishi
Masamichi
M
Nonaka
Kazuaki
K
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002971:Cleft Lip / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D019938:Cyclin D1 / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008817:Mice, Mutant Strains; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D009924:Organ Culture Techniques; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053782:Transforming Growth Factor beta3; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000378:metabolism
D019408:Platelet Endothelial Cell Adhesion Molecule-1; D012333:RNA, Messenger; C509045:Tgfb3 protein, mouse; D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; D019938:Cyclin D1; D040301:Vascular Endothelial Growth Factor Receptor-2