Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear

Background: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. Methods: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. Results: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). Conclusions: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.     

Pro-metastasis function of TGFbeta mediated by the Smad pathway

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.     

Sonic hedgehog regulates otic capsule chondrogenesis and inner ear development in the mouse embryo

Development of the cartilaginous capsule of the inner ear is dependent on interactions between otic epithelium and its surrounding periotic mesenchyme. During these tissue interactions, factors endogenous to the otic epithelium influence the differentiation of the underlying periotic mesenchyme to form a chondrified otic capsule. We report the localization of Sonic hedgehog (Shh) protein and expression of the Shh gene in the tissues of the developing mouse inner ear. We demonstrate in cultures of periotic mesenchyme that Shh alone cannot initiate otic capsule chondrogenesis. However, when Shh is added to cultured periotic mesenchyme either in combination with otic epithelium or otic epithelial-derived fibroblast growth factor (FGF2), a significant enhancement of chondrogenesis occurs. Addition of Shh antisense oligonucleotide (AS) to cultured periotic mesenchyme with added otic epithelium decreases levels of endogenous Shh and suppresses the chondrogenic response of the mesenchyme cells, while supplementation of Shh AS-treated cultures with Shh rescues cultures from chondrogenic inhibition. We demonstrate that inactivation of Shh by targeted mutation produces anomalies in the developing inner ear and its surrounding capsule. Our results support a role for Shh as a regulator of otic capsule formation and inner ear development during mammalian embryogenesis.     

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.     

Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling

Background: Retinoic acid (RA) is essential for inner ear development. However, exposure to excess RA at a critical period leads to inner ear defects. These defects are associated with disruption in epithelial–mesenchymal interactions. METHODS: This study investigates the role of Dlx5 in the epithelial–mesenchymal interactions that guide otic capsule chondrogenesis, as well as the effect of excess in utero RA exposure on Dlx5 expression in the developing mouse inner ear. Control of Dlx5 by Fgf3 and Fgf10 under excess RA conditions is investigated by examining the developmental window during which Fgf3 and Fgf10 are altered by in utero RA exposure and by testing the ability of Fgf3 and Fgf10 to mitigate the reduction in chondrogenesis and Dlx5 expression mediated by RA in high‐density cultures of periotic mesenchyme containing otic epithelium, a model of epithelial–mesenchymal interactions in which chondrogenic differentiation of periotic mesenchyme ensues in response to induction by otic epithelium. RESULTS: Dlx5 deletion alters expression of TGFβ₁, important for otic capsule chondrogenesis, in the developing inner ear and compromises the ability of cultured periotic mesenchyme containing otic epithelium, harvested from Dlx5 null embryos, to differentiate into cartilage when compared with control cultures. Downregulation in Dlx5 ensues as a consequence of in utero RA exposure in association with inner ear dysmorphogenesis. This change in Dlx5 is noted at embryonic day 10.5 (E10.5), but not at E9.5, suggesting that Dlx5 is not a direct RA target. Before Dlx5 downregulation, Fgf3 and Fgf10 expression is modified in the inner ear by excess RA, with the ability of exogenous Fgf3 and Fgf10 to rescue chondrogenesis and Dlx5 expression in RA‐treated cultures of periotic mesenchyme containing otic epithelium supporting these fibroblast growth factors (FGFs) as intermediary genes by which RA mediates its effects. CONCLUSIONS : Disruption in an Fgf3, ‐10/Dlx5 signaling cascade is operant in molecular mechanisms of inner ear teratogenesis by excess RA. Birth Defects Res (Part B) 2008. ©2008 Wiley‐Liss, Inc.     

GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor

The cascade of phosphorylation is a pivotal event in transforming growth factor beta (TGFbeta) signaling. Reversible phosphorylation regulates fundamental aspects of cell activity. TGFbeta-induced Smad7 binds to type I receptor (TGFbeta type I receptor; TbetaRI) functioning as a receptor kinase antagonist. We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TbetaRI. Blocking Smad7 expression by RNA interference inhibits association of GADD34-PP1c complex with TbetaRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TbetaRI for dephosphorylation. SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7-GADD34 complex by controlling the specific subcellular localization of PP1c. Importantly, GADD34-PP1c recruited by Smad7 inhibits TGFbeta-induced cell cycle arrest and mediates TGFbeta resistance in responding to UV light irradiation. The dephosphorylation of TbetaRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFbeta signaling.     

Inhibition of TGFbeta1-mediated PAI-1 induction by oltipraz through selective interruption of Smad 3 activation

Transforming growth factor-beta1 (TGFbeta1) induces plasminogen activator inhibitor-1 (PAI-1) as a major target protein. PAI-1 is associated with fibrosis, thrombosis, and metabolic disorders. TGFbeta1 induces PAI-1 via phosphorylation and nuclear translocation of Smads. Oltipraz inhibits TGFbeta1 expression and also regenerates cirrhotic liver. Nevertheless, whether oltipraz modulates TGFbeta1-mediated cell signaling is unclear. First, this study examined the effect of oltipraz on PAI-1 expression in cirrhotic rat liver. The cells immunochemically stained with anti-PAI-1 antibody accumulated around and within fibrous nodules in cirrhotic liver, which was notably decreased by oltipraz treatment. Next, whether oltipraz inhibits TGFbeta1-mediated Smads activation or Smad-mediated PAI-1 induction was determined in L929 fibroblasts. Oltipraz inhibited the ability of TGFbeta1 to induce PAI-1, as indicated by repression of TGFbeta1-mediated luciferase induction from the plasmid comprising the human PAI-1 promoter and of TGFbeta1-induced Smad-DNA-binding activity. TGFbeta1 induced nuclear transport of receptor-regulated Smad 2 and Smad 3, of which oltipraz selectively inhibited the transport and phosphorylation of Smad 3, thereby reducing formation of Smad 3/4 complex in the nucleus. In summary, oltipraz inhibits PAI-1 induction via a decrease in the formation of Smad 3/4 complex due to selective interruption of Smad 3 activation, indicating that oltipraz regulates the cellular responses downstream of ligand-activated TGFbeta1 receptor.     

TGFbeta protein processing and activity through TCR triggering of primary CD8+ T regulatory cells

In general, TGFbeta is synthesized as a procytokine that requires proteolytic activation, release of the mature cytokine from its noncovalently associated latent-associated peptide, and binding to TGFbetaRII to mediate suppressive activity. We tracked this process in mice containing primed CD8 regulatory T cells (Tregs) by immunoblotting in primary whole cell lysates for pro-TGFbeta, latent-associated peptide and mature TGFbeta. Generation of CD8 Tregs promoted processing of the 50 kDa pro-TGFbeta protein into a 12.5 kDa mature TGFbeta species in vivo. Despite the inability to detect mature TGFbeta in the sera of mice with primed CD8 Tregs and in the synthetic culture medium of stimulated CD8 Tregs, we demonstrated engagement of TGFbetaRII through immunoblotting for Smad2 phosphorylation. This process relied on continual TCR triggering, which also induced Smad3 phosphorylation. To understand the movement of mature TGFbeta, we showed that in contrast to IFN-gamma, mature TGFbeta does not remain a soluble cytokine but is likely to be rapidly adsorbed by neighboring cells. These data show the exquisite local control directed toward TGFbeta by the immune system and underscore the fine specificity involved in its detection.     

Effects of TGFbeta and bFGF on the differentiation of human bone marrow stromal fibroblasts

Adipocytes and osteoblasts have common origins from fibroblastic stem cells. Consequently, modulation of the processes of adipogenesis and osteogenesis has implications for the possible treatment of metabolic bone diseases, such as osteoporosis, in which medullary fat accumulates and trabecular bone volume decreases. It is likely that the balance between these two systems is affected by particular endogenous growth factors which are known to affect bone metabolism. We have therefore investigated the effects of transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on cultured human bone marrow (HBM) fibroblastic cells to observe the effects on adipogenesis and osteogenesis. In the absence of fetal calf serum (FCS), TGFbeta caused a dose-dependent increase in cell growth and alkaline phosphatase activity (AP); however, in the presence of FCS growth was inhibited at high concentrations and AP unaffected. TGFbeta increased matrix proteoglycan and collagen synthesis. bFGF inhibited AP and increased colony number and size, while Dex treatment increased AP activity and colony number, and both factors in combination resulted in an additive increase in growth. Dex-induced adipocyte formation was accelerated but not increased by bFGF. A significant inhibition of adipogenesis by TGFbeta was observed within 7 days. These results demonstrate the importance of biological factors known to be involved in bone remodelling in the regulation of osteogenesis and adipogenesis.     

Transforming growth factor beta-1 up-regulates clusterin synthesis in thyroid epithelial cells

Porcine epithelial cells in primary culture seeded on plastic substratum form a monolayer containing pseudo-vesicles. When cultured in the presence of thyreotropin (TSH) thyrocytes adopted a follicular-like structure and formed clusters. Transforming growth factor beta-1 (TGFbeta1) induced a rapid spreading of the TSH-treated cells only. At the same time, TGFbeta1 enhanced clusterin protein and mRNA expression. The increase of clusterin synthesis was proportional to the TGFbeta1 concentration in the culture medium. Tunicamycin abolished the up-regulation of whole clusterin synthesis and morphological changes. The activator protein-1 binding site partly directed the TGFbeta1-stimulated clusterin expression. Phorbol ester caused rapid spreading of the cells with disappearance of vesicular and follicular structures. It decreased clusterin mRNA and protein expression, but increased Mr 45, 000 protein secretion in both TSH-treated and nontreated cells. Up-regulation of clusterin expression may be a marker of TGFbeta-mediated thyrocyte dedifferentiation.     

Integrin signaling and cell spreading mediated by phorbol 12-myristate 13-acetate treatment

Spreading of SNU16mAd gastric carcinoma cells was previously shown to be regulated via a signaling network from transforming growth factor beta1 (TGFbeta1) to integrins signaling, through a mediation of protein kinase C delta (PKCdelta). However, in the previous study, the roles of PKCdelta appeared complicated. In this study to clarify the roles of PKCdelta in the spreading of the gastric carcinoma cells, we questioned if PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment could mimic the TGFbeta1 effects. An acute PMA treatment increased phosphorylations of focal adhesion (FA) kinase, paxillin, c-Src, and cofilin, just as TGFbeta1 did. Furthermore, cell spreading mediated by TGFbeta1- or acute PMA treatment correlated with activation of RhoA, which regulates actin reorganization and FA formation. However, stress fiber formation was prominent in TGFbeta1-treated cells, compared to cortical actin organization in PMA-treated cells. Altogether, these observations indicate that acute PMA treatment could mimic the TGFbeta1 mechanisms for cell spreading through subtly different effects on actin reorganization.