Mouse Embryonic Palatal Mesenchymal Cells Research Articles

All- trans retinoic acid inhibited chondrogenesis of mouse embryonic palate mesenchymal cells by down-regulation of TGF-β/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-β3 (TGF-β3) 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-β3 signaling pathway. atRA led to an increase in mRNA expression of TGF-β3 and an instantaneous decrease in TGF-β type II receptor (TβRII) 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 Smad7 ΔC mutant or constitutively active TβRII 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-β3 pathway and suggested a role for TβRII /Smad in retinoid-induced cleft palate.

Apoptosis Induced by atRA in MEPM Cells Is Mediated through Activation of Caspase and RAR

We have previously demonstrated that all-trans retinoic (atRA) induced growth inhibition and apoptosis in mouse embryonic palate mesenchymal cells (MEPM). In the present study, we investigated the molecular mechanisms of atRA-induced apoptosis and its putative action pathway. atRA-induced apoptosis is associated with activation of the initiator caspase-9 and the effector caspase-3, but not of the effector caspase-8. A broad caspase inhibitor (z-VAD-fmk), caspase-9 inhibitor z-LEHD-fmk and caspase-3 inhibitor (z-DEVD-fmk) blocked atRA-induced DNA fragmentation and sub-G1 fraction, but not caspase-8 inhibitor z-IETD-fmk. We further showed that atRA dose-dependently promoted mRNA expression of retinoic acid receptor beta (RAR-beta) and gamma. A weaker increase in RAR-alpha mRNA was seen only at the highest concentration of atRA (5 muM). The pan RAR antagonist, BMS493, completely abrogated atRA-induced DNA fragmentation, Sub-G1 fraction, and caspase-3 activation. Taken together, these findings show that caspase-mediated induction of apoptosis by atRA is an RAR-dependent signaling pathway.

Induction of Cell-Cycle Arrest by all-trans Retinoic Acid in Mouse Embryonic Palatal Mesenchymal (MEPM) Cells

all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.

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

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.

Epidermal growth factor and transforming growth factor alpha regulate extracellular matrix production by embryonic mouse palatal mesenchymal cells cultured on a variety of substrata.

Mouse embryonic palatal mesenchymal (MEPM) cells were cultured either on plastic tissue culture dishes or on the surface of three-dimensional collagen gels or within collagen gel matrices in DMEM/F12 medium containing 2.5% donor calf serum. MEPM cells proliferated exponentially when cultured on collagen or on plastic. Cells cultured within collagen gels did not proliferate but remained viable. Addition of 10 ng/ml epidermal growth factor (EGF) or transforming growth factor alpha (TGFα) stimulated the proliferation of those cells cultured on plastic or on collagen but not those cultured within collagen gels. Immunocytochemical analysis revealed that MEPM cells synthesise collagen types I, III, IV, V, VI and IX; fibronectin, heparan sulphate proteoglycan, laminin and tenascin in vitro. These molecules are all present in the developing palate in vivo. EGF and TGFα produced a generalised stimulation of extracellular matrix (ECM) synthesis by MEPM cells in vitro. Biochemical analysis indicated that cells cultured within collagen gels had the highest intrinsic rate of protein synthesis. On all substrata neither EGF nor TGFα markedly altered the types of ECM molecules synthesised but rather caused a general increase in the total amount produced. This stimulation was most marked where the cells were cultured within collagen gels. The lack of stimulation of proliferation of MEPM cells cultured within collagen gels (i.e. in a physiologically-relevant environment) by EGF or TGFα together with the marked stimulation of ECM synthesis suggests that these factors may act as differentiation signals via their effects on ECM production.

The effects of transforming growth factor- β1 on protein production by mouse embryonic palate mesenchymal cells in the presence or absence of serum

Mouse embryonic palatal mesenchyme cells were cultured on a variety of substrata (plastic, on a collagen gel or within a collagen gel). On each substratum TGF- β 1 (1 ng/ml) inhibited cell proliferation. Cells cultured within a collagen gel had the lowest rate of proliferation, but were metabolically the most active in terms of incorporation of [ 3H]-proline into both collagenous and non-collagenous proteins. TGF- β 1, in the presence of 2.5% donor calf serum stimulated the production of fibronectin and the major collagen types I, III and V. However, in serum-free medium, TGF- β 1 induced a large reduction in total collagen production, mainly due to an effect on type I collagen, whilst stimulating production of some non-collagenous proteins. Experiments involving combinations of TGF- β 1 with other growth factors suggested that the different effects of TGF- β 1 on collagen production, in the presence and absence of serum, may be due to an interaction with platelet-derived growth factor.

Immunocytochemical Demonstration of Simultaneous Synthesis of Types I, III and V Collagen and Fibronectin in Mouse Embryonic Palatal Mesenchymal Cells In Vitro

An immunocytochemical study was made on the palatal mesenchymal cells obtained from mouse embryos during palatal development with special reference to synthesis of types of collagen and fibronectin in vitro. Most cells showed positive staining with antibodies against the four proteins examined. The staining for type I collagen was most intense among the four proteins and was distributed perinuclearly. The staining for type III collagen was quite similar as that for type I collagen but less intense, whereas that for type V collagen was weak and its staining pattern was different from those for types I and III collagen in that the surface of the plasma membrane, in addition to the perinuclear cytoplasm, showed weak staining for type V collagen. Antibodies to fibronectin showed perinuclear and extracellular fibrous staining. These data suggest that palatal mesenchymal cells synthesize types I, III, and V collagen and fibronectin simultaneously.