Paul Nugent

Regulation of TGFβ3 gene expression in embryonic palatal tissue

SummaryThe TGFβ family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGFβ exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGFβs. We therefore examined the expression of mRNAs encoding TGFβ1, TGFβ2, and TGFβ3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGFβ1 (2.5 kb), two transcripts for TGFβ2 (4.4 kb, 6.0 kb), and one transcript for TGFβ3 (3.5 kb). Although steady-state levels of TGFβ1 mRNA showed no changes during development of the palate, TGFβ2 mRNA levels were maximal on both GD13 and GD14 and TGFβ3 mRNA levels transiently increased on GD 13. In addition, levels of TGFβ3 mRNA seemed much higher than either TGFβ1 or TGFβ2. both TGFβ1 and TGFβ2 were able to increase, in a dose-related manner, the expression of TGFβ3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGFβ3 mRNA even in the presence of TGFβ1 or TGFβ2. Elevation of intracellular levels of cAMP in MEPM cells in vitro resulted in a time-dependent transient increase in steady-state levels of TGFβ3 mRNA with maximal expression by 30 min. Furthermore, steady-state levels of TGFβ3 mRNA increased in embryonic palatal tissue in vivo after administration of retinoic acid. These data suggest that TGFβ3 is developmentally regulated in embryonic palatal tissue and that its expression may be modulated via both autoregulatory pathways, other signaling peptides, or the cAMP cascade. Thus, the protein product of one regulatory gene involved in signal transduction during development of the mammalian palate can affect the expression of another regulatory gene.

The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts.

The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.

Effects of dexamethasone on the expression of transforming growth factor‐β in mouse embryonic palatal mesenchymal cells

The central role of TGF‐β in the development of the embryonic palate has been well characterized. TGF‐β inhibits mesenchymal cell proliferation, induces medial edge epithelial cell differentiation, and modulates the expression of extracellular matrix proteins as well as the proteases that act upon them. Mechanisms by which TGF‐β expression itself is regulated are less well understood. Glucocorticoids are recognized in several cellular systems as able to regulate the expression of TGF‐β. This study was therefore designed to examine whether glucocorticoids affect the expression of TGF‐β isoforms in embryonic palatal cells. Based on flow cytometric analysis and viability determination, confluent primary cultures of mouse embryonic palate mesenchymal (MEPM) cells exposed to up to 10−6 M dexamethasone (dex) exhibited no signs of cytotoxicity after 24 hours of exposure. Northern blot analyses revealed that dexamethasone reduced steady‐state mRNA levels of TGF‐β3 in a dose‐dependent manner as early as 4 hours after treatment but had little effect on TGF‐β1 and TGF‐β2 expression up to 24 hours of dex exposure. Dex also reduced the synthesis of both latent and mature forms of TGF‐β protein by approximately four‐fold as determined by the mink lung epithelial cell growth inhibition bioassay. Assessment of the ratio of mature to latent protein found in conditioned medium of control compared to dex‐treated cultures indicated that dexamethasone may reduce the activation of latent TGF‐β to mature biologically active TGF‐β. Dexamethasone inhibited the proliferation of MEPM cells despite the down‐regulation of TGF‐β suggesting that dex‐induced growth inhibition of MEPM cells is not mediated by TGF‐β. These data suggest that dex modulates TGF‐β signaling pathways directly by down‐regulating TGF‐β expression and possibly indirectly by altering the availability of mature TGF‐β necessary to exert its biological effects in the developing palate.

Antisense oligonucleotides to CRABP I and II alter the expression of TGF-beta 3, RAR-beta, and tenascin in primary cultures of embryonic palate cells.

SummaryThe cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor β (TGF-β). These cells also express high levels of TGF-β3, which is also regulated by RA and TGF-β. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 µM or 0.33 µM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-β3, retinoic acid receptor β (RAR-β), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-β3, RAR-β, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-β3, RAR-β, and tenascin gene expression. These results have important implications for our understanding of the role of the CRABPs in retinoid teratology.