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The central role of TGF-beta in the development of the embryonic palate has been well characterized. TGF-beta 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-beta expression itself is regulated are less well understood. Glucocorticoids are recognized in several cellular systems as able to regulate the expression of TGF-beta. This study was therefore designed to examine whether glucocorticoids affect the expression of TGF-beta 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-beta 3 in a dose-dependent manner as early as 4 hours after treatment but had little effect on TGF-beta 1 and TGF-beta 2 expression up to 24 hours of dex exposure. Dex also reduced the synthesis of both latent and mature forms of TGF-beta 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-beta to mature biologically active TGF-beta. Dexamethasone inhibited the proliferation of MEPM cells despite the down-regulation of TGF-beta suggesting that dex-induced growth inhibition of MEPM cells is not mediated by TGF-beta. These data suggest that dex modulates TGF-beta signaling pathways directly by down-regulating TGF-beta expression and possibly indirectly by altering the availability of mature TGF-beta necessary to exert its biological effects in the developing palate.

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Signaling pathways utilized by EGF, cAMP, and TGF beta have been demonstrated to play critical roles in normal palate development. Stimulation of these pathways has been shown in palate cells and numerous other systems to affect cell growth. Because proper regulation of cell growth is critical to palate development, we speculate that fine regulation of palatal cell growth may be accomplished through crosstalk between these signaling pathways. We therefore set out to determine the effects of cAMP and TGF beta on EGF-induced cell proliferation in murine embryonic palate cells. We found that both TGF beta and cAMP inhibited the proliferative response of cells to treatment with EGF, whereas H89, a serine/ threonine protein kinase inhibitor with selectivity towards cAMP-dependent protein kinase, increased the cells' proliferative response to EGF. Genestein, a selective inhibitor of tyrosine kinases, at high doses abrogated the cells' proliferative response to EGF, confirming that EGF's ability to induce cell proliferation is critically dependent upon tyrosine kinase activity. Lower doses of genestein, however, actually enhanced cellular response to EGF. The data suggest that both the TGF beta- and cAMP-mediated signaling pathways may be involved in modulation of the effects of EGF on palate cell growth in vivo.

The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1-mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09-17.4 micrograms/g embryo) were applied to chick embryos at 18 days of incubation (DI). Embryos also received two doses of BrdU at 3 mg/200 microliters (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested. AfB1-mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T cells through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo.

Murine embryonic palate mesenchyme (MEPM) cells are responsive to a number of endogenous factors found in the local embryonic tissue environment. Recently, it was shown that activation of the cyclic AMP (cAMP) or the transforming growth factor beta (TGFbeta) signal transduction pathways modulates the proliferative response of MEPM cells to epidermal growth factor (EGF). Since the mitogen-activated protein kinase (MAPK) cascade is a signal transduction pathway that mediates cellular responsiveness to EGF, we examined the possibility that several signaling pathways which abrogate EGF-stimulated proliferation do so via the p42/p44 MAPK signaling pathway. We demonstrate that EGF stimulates MAPK phosphorylation and activity in MEPM cells maximally at 5 minutes. Tyrosine phosphorylation and activation of MAPK was unaffected by treatment of MEPM cells with TGFbeta or cholera toxin. Similarly, TGFbeta altered neither EGF-induced MAPK tyrosine phosphorylation nor activity. However, the calcium ionophore, A23187, significantly increased MAPK phosphorylation which was further increased in the presence of EGF, although calcium mobilization reduced EGF-induced proliferation. Despite the increase in phosphorylation, we could not demonstrate induction of MAPK activity by A23187. Like EGF, phorbol ester, under conditions which activate PKC isozymes in MEPM cells, increased MAPK phosphorylation and activity but was also growth inhibitory to MEPM cells. The MEK inhibitor, PD098059, only partially abrogated EGF-induced phosphorylation. Likewise, depletion of PKC isozymes partially abrogated EGF-induced MAPK phosphorylation. Inhibition of both MEK and PKC isozymes resulted in a marked decrease in MAPK activity, confirming that EGF uses multiple pathways to stimulate MAPK activity. These data indicate that the MAPK cascade does not mediate signal transduction of several agents that inhibit growth in MEPM cells, and that there is a dissociation of the proliferative response and MAP kinase activation. Furthermore, other signaling pathways known to play significant roles in differentiation of palatal tissue converge with the MAPK cascade and may use this pathway in the regulation of alternative cellular processes.

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