Inhibition of Smad signaling is implicated in cleft palate induced by all-trans retinoic acid

The effect of all-trans retinoic acid (atRA) on palatal fusion and the underlying mechanisms were investigated using organ culture. Compared with control group, the atRA-treated group (1 μM and 5 μM) had more medial edge epithelium (ME) remaining within the midline epithelial seam (MES). At 10 μM atRA, the opposing shelves were not in contact at the culture end (72 h). Cell death detection by TUNEL and laminin immunohistochemistry demonstrated that atRA (5 μM) induced apoptosis in mesenchyme and inhibited degradation of basal lamina within MES. Notably, migration and apoptosis of ME cells and degradation of basal lamina within MES markedly represented vehicle control palatal shelves in culture. Additionally, apoptosis was not detected in mesenchyme of control palatal shelves. Immunoblotting analysis revealed that Smad2 and Smad3 were endogenously activated and expression of Smad7 was inhibited during the fusion process. In contrast, atRA treatment abrogated phosphorylation of Smad2 and Smad3 and inducible expression of Smad7 in ME. From these data, it is assumed that inhibition of Smad pathway by atRA in ME may play a critical role in abrogation of the ME cell apoptosis and degradation of the basal laminin, which might contribute to failure of palatal fusion.     

Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.     

All-trans retinoic acid inhibited chondrogenesis of mouse embryonic palate mesenchymal cells by down-regulation of TGF-beta/Smad signaling

Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-beta3 (TGF-beta3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-beta3 signaling pathway. atRA led to an increase in mRNA expression of TGF-beta3 and an instantaneous decrease in TGF-beta type II receptor (TbetaRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7deltaC mutant or constitutively active TbetaRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-beta3 pathway and suggested a role for TbetaRII /Smad in retinoid-induced cleft palate.     

Cleft lip with or without cleft palate: associations with transforming growth factor alpha and retinoic acid receptor loci.

The first association study of cleft lip with or without cleft palate (CL/P), with candidate genes, found an association with the transforming growth-factor alpha (TGFA) locus. This finding has since been replicated, in whole or in part, in three independent studies. Here we extend our original analysis of the TGFA TaqI RFLP to two other TGFA RFLPs and seven other RFLPs at five candidate genes in 117 nonsyndromic cases of CL/P and 113 controls. The other candidate genes were the retinoic acid receptor (RARA), the bcl-2 oncogene, and the homeobox genes 2F, 2G, and EN2. Significant associations with the TGFA TaqI and BamHI RFLPs were confirmed, although associations of clefting with previously reported haplotypes did not reach significance. Of particular interest, in view of the known teratogenic role of retinoic acid, was a significant association with the RARA PstI RFLP (P = .016; not corrected for multiple testing). The effect on risk of the A2 allele appears to be additive, and although the A2A2 homozygote only has an odds ratio of about 2 and recurrence risk to first-degree relatives (lambda 1) of 1.06, because it is so common it may account for as much as a third of the attributable risk of clefting. There is no evidence of interaction between the TGFA and RARA polymorphisms on risk, and jointly they appear to account for almost half the attributable risk of clefting.     

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.     

Effect of tamoxifen and retinoic acid on bradykinin induced proliferation in MCF‐7 cells

Chemopreventive approaches for the treatment of breast cancer have been validated clinically and with in vitro studies. The combined action of tamoxifen/all‐trans retinoic acid was advantageous in MCF‐7 cells, reducing cell proliferation, Bcl‐2 and c‐Myc protein levels and increasing E‐Cadherin protein levels and Gap junctional Intercellular Communication. We further investigated their combined effect in the presence of bradykinin, a pro‐inflammatory agent, previously reported to contribute to the proliferation of breast cancer cells. Bradykinin increased MCF‐7 cell proliferation, c‐Myc levels and ERK1/2 activity. The co‐incubation of bradykinin‐MCF‐7 cells with tamoxifen/all‐trans retinoic acid reduced cell proliferation, ERK1/2 activity, as well as Bcl‐2, c‐Myc, and bradykinin receptor‐2 levels, without altering the enhanced E‐cadherin levels induced by tamoxifen/all‐trans retinoic acid. We showed that the anti‐tumoral effect of tamoxifen/all‐trans retinoic acid is beneficial in MCF‐7 breast cancer cells grown in a bradykinin‐pro‐mitogenic environment, an effect that might be, at least in part, through the MAPK pathway and B2‐bradykinin receptor inhibition. J. Cell. Biochem. 106: 473–481, 2009. © 2008 Wiley‐Liss, Inc.     

Genetics of cleft lip and cleft palate in China.

During the past 10 years, 60 cases of cleft lip with or without cleft palate [CL(P)] were recorded among 45,072 newborns at Shanghai International Peace Maternity and Infant Hospital, China. The incidence was 1.33 per 1,000 births. The family histories of 163 CL(P) patients were analyzed. The incidences of CL(P) in the first-, second-, and third-degree relatives of CL(P) patients were 11/246 (4.47%), 10/1,032 (0.97%), and 6/1,727 (0.35%), respectively. Of the 163 probands, three had a history of consanguinity of the parents (1.8%), in contrast to 0.77% in the general population. These data are suggestive of multifactorial inheritance. The heritability of CL(P) in our study calculated by Falconer's formula was 77.6%.     

Inhibition of neuronal cell death after retinoic acid-induced down-regulation of P2X7 nucleotide receptor expression

Apoptosis is a major mechanism for cell death in the nervous system during development. P2X₇ nucleotide receptors are ionotropic ATP receptors that mediate cell death under pathological conditions. We developed an in vitro protocol to investigate the expression and functional responses of P2X₇ nucleotide receptors during retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y neuroblastoma cells. Neuronal differentiation was examined measuring cellular growth arrest and neuritic processes elongation. We found that SH-SY5Y cells treated for 5 days with RA under low serum content exhibited a neuron-like phenotype with neurites extending more than twice the length of the cell body and cell growth arrest. Concurrently, we detected the abolishment of intracellular-free calcium mobilization and the down-regulation of P2X₇ nucleotide receptor protein expression that protected differentiated cells from neuronal cell death and reduced caspase-3 cleavage-induced by P2X₇ nucleotide receptor agonist. The role of P2X₇ nucleotide receptors in neuronal death was established by selectively antagonizing the receptor with KN-62 prior to its activation. We assessed the involvement of protein kinases and found that p38 signaling was activated in undifferentiated after nucleotide stimulation, but abolished by the differentiating RA pretreatment. Importantly, P2X₇ receptor-induced caspase-3 cleavage was blocked by the p38 protein kinase specific inhibitor PD169316. Taken together, our results suggest that RA treatment of human SH-SY5Y cells leads to decreased P2X₇ nucleotide receptor protein expression thus protecting differentiated cells from extracellular nucleotide-induced neuronal death, and p38 signaling pathway is critically involved in this protection of RA-differentiated cells.     

Retinoic acid, GABA-ergic, and TGF-beta signaling systems are involved in human cleft palate fibroblast phenotype

During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.