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.     

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.     

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ-like cell differentiation

Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic-like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4-GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4-GFP ES cell line in culture (MPC 85.5 ± 5.1% vs MEF 84.1 ± 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA-1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4-GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem-like cells, expressing Mvh, Stra-8, Hsp90-α, integrinβ1 and α6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare.     

Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway

Transforming growth factor-beta (TGF-beta) and its family are potent and multi-functional cytokines that affect various fundamental biological events. TGF-beta has a unique signaling pathway that is carried by Smad family, and many recent studies showed the extensive crosstalk between Smad pathway and other signaling pathway. There were also clear evidences for the involvement of oxidative events in TGF-beta signaling pathway. To elucidate the role of oxidative events in carrying TGF-beta signals, we examined the effect of various antioxidants on TGF-beta activities in osteoblastic cell line. Among the examined compounds, we found nordihydroguaiaretic acid (NDGA) has a unique and strong inhibitory effect on various TGF-beta activities. Since the majority of TGF-beta activities are mediated by Smad, we questioned whether NDGA blocks the Smad signaling pathway. The result showed that NDGA inhibits the translocation of Smad2 to the nucleus. Further study revealed the strong inhibitory effect of NDGA on the phosphorylation of Smad2. This result may be important for designing chemical modulators of TGF-beta and its family related events and may provide new insights into the action mechanism of antioxidant.     

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

INTRODUCTIONAsaderivativeofvitaminA,RAcaninhibittheproliferati0nofmanymalignantcel1sallde1icitdifferentiationinsometumorce1lsI1-5].RecentstudieshaveshownthatRAmodu1atessynthesisofover4Oproteinsthroughitsnucleicrecept0r[6].Forinstance,RAinducesthesynthesis0ffibronectin(FN)incertaintumorcellsandactivatesthegenecodingfOrB1sllbunitoflaminin(LN)causingitsexpressi0n[7,8].FNandLN'areknownt0bethemostimportantcomponentsofnon-c0llageng1ycoproteinsintheextracellu1armatrixandareclose1yrelatedt…     

Differential responses to retinoic acid and endocrine disruptor compounds of subpopulations within human embryonic stem cell lines

The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1–60, Tra-1–80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy.     

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.     

Requirement of Smad3 for mast cell growth

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.     

Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling

Vertebrate muscle morphogenesis is a complex developmental process, which remains quite yet unexplored at cellular and molecular level. In this work, we have found that sculpturing programmed cell death is a key morphogenetic process responsible for the formation of individual foot muscles in the developing avian limb. Muscle fibers are produced in excess in the precursor dorsal and ventral muscle masses of the limb bud and myofibers lacking junctions with digital tendons are eliminated via apoptosis. Microsurgical experiments to isolate the developing muscles from their specific tendons are consistent with a role for tendons in regulating survival of myogenic cells. Analysis of the expression of Raldh2 and local treatments with retinoic acid indicate that this signaling pathway mediates apoptosis in myogenic cells, appearing also involved in tendon maturation. Retinoic acid inhibition experiments led to defects in muscle belly segmentation and myotendinous junction formation. It is proposed that heterogeneous local distribution of retinoids controlled through Raldh2 and Cyp26A1 is responsible for matching the fleshy and the tendinous components of each muscle belly.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

Restraint of Fgf8 signaling by retinoic acid signaling is required for proper heart and forelimb formation

Cardiomelic or heart–hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb.