Transforming Growth Factor-β1 Differentially Regulates Proliferation, Morphology, and Extracellular Matrix Expression by Three Neural Crest-Derived Neuroblastoma Cell Lines

We reported previously (S. L. Rogers, P. J. Gegick, S. M. Alexander, and P. G. McGuire, Dev. Biol. 151, 191-203, 1992) that transforming growth factor-β1 (TGFβ1) inhibited proliferation, up-regulated fibronectin synthesis, and suppressed melanogenesis in a population of quail neural crest cells in vitro. Here, we report that cell lines derived from the parent SK-N-SH neuroblastoma line (R. A. Ross, B. A. Spengler, and J. L. Biedler, J. Natl. Cancer Inst. 71, 741-747, 1983) respond differentially to TGFβ1, and their responses provide further insights into the actions of this growth factor on neural crest subpopulations. The SH-EP cell line exhibits primarily nonneuronal traits and responded to TGFβ1 with increased thymidine uptake after 6 days of culture, increased expression of fibronectin mRNA and protein, and decreased laminin synthesis. Many SH-EP cells also acquired a dramatically elongated morphology, reminiscent of Schwann cells in culture. Thymidine uptake by the neuronal SY5Y cell line was not substantially altered. Neither fibronectin mRNA nor protein was detectable in either TGFβ1-treated or untreated cultures, although laminin synthesis was upregulated by the growth factor. In TGFβ1-treated cultures of the intermediate SH-IN cell line, which has been reported to display both neuronal and nonneuronal characteristics, there was marked flattening of many cells, a steady decrease in thymidine uptake, and increased expression of both fibronectin and laminin. The observed responses of SH-IN cells mimic those observed in primary neural crest cultures and appear to represent similar differentiation toward a mesenchymal phenotype. These results substantiate the idea that closely related but diverging neural crest-derived cell types respond selectively to TGFβ1 and demonstrate that these SK-N-SH-derived cell lines will be useful in experimental approaches that will allow us to infer mechanisms underlying regulation of neural crest differentiation.     

Restriction of neurogenic ability during neural crest cell differentiation

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells. © 1993 John Wiley & Sons, Inc.     

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290–300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281–285 and 320–329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.     

Noncanonical Transforming Growth Factor β (TGFβ) Signaling in Cranial Neural Crest Cells Causes Tongue Muscle Developmental Defects

Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor β (TGFβ) type II receptor in CNC cells in mice (Tgfbr2^fl/fl;Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development. However, it is still unclear how TGFβ signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2^fl/fl;Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFβ-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2^fl/fl;Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFβ-mediated regulation of FGF and BMP signaling during tongue development.     

TGFβ superfamily signaling in the neural crest lineage

The neural crest cell (NCC) lineage is often referred to as the fourth germ layer in embryos, as its wide range of migration and early colonization of multiple tissues and organ systems throughout the developing body is astounding. Many human birth defects are thought to have their origins within the NCC lineage. Exciting recent conditional mouse targeting and transgenic combinatorial suppression approaches have revealed that the TGFβ superfamily is a key signaling pathway within the cardiac and cranial NCC subpopulations. Given the complexity of TGFβ superfamily signaling and that multiple ligand and receptor combinations have already been shown to be expressed within the NCC subpopulations, and the difficulty in transgenically targeting entire signaling cascades, we review several up-to-date transgenic approaches that are revealing unexpected consequences.Key words: TGFβ, neural crest, heart, cranial crest, mouse transgenics     

Tumor Necrosis Factor-Receptor–associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-β Signaling That Affects Neural Crest Formation

The transforming growth factor (TGF)-β superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFβ signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFβ. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor–associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.     

Transforming Growth Factor β Integrates Smad 3 to Mechanistic Target of Rapamycin Complexes to Arrest Deptor Abundance for Glomerular Mesangial Cell Hypertrophy

In many renal diseases, transforming growth factor β (TGFβ)-stimulated canonical Smad 3 and noncanonical mechanistic target of rapamycin (mTOR) promote increased protein synthesis and mesangial cell hypertrophy. The cellular underpinnings involving these signaling molecules to regulate mesangial cell hypertrophy are not fully understood. Deptor has recently been identified as an mTOR interacting protein and functions as an endogenous inhibitor of the kinase activity for both TORC1 and TORC2. Prolonged incubation of mesangial cells with TGFβ reduced the levels of deptor concomitant with an increase in TORC1 and TORC2 activity. Sustained TGFβ activation was required to inhibit association of deptor with mTOR, whereas rapid activation had no effect. Using the mTOR inhibitor PP242, we found that TGFβ-induced both early and sustained activation of TORC1 and TORC2 was necessary for deptor suppression. PP242-induced reversal of deptor suppression by TGFβ was associated with a significant inhibition of TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, expression of siRNA against Smad 3 or Smad 7, which blocks TGFβ receptor-specific Smad 3 signaling, prevented TGFβ-induced suppression of deptor abundance and TORC1/2 activities. Furthermore, overexpression of Smad 3 decreased deptor expression similar to TGFβ stimulation concomitant with increased TORC1 and TORC2 activities. Finally, knockdown of deptor reversed Smad 7-mediated inhibition of protein synthesis and mesangial cell hypertrophy induced by TGFβ. These data reveal the requirement of both early and late activation of mTOR for TGFβ-induced protein synthesis. Our results support that TGFβ-stimulated Smad 3 acts as a key node to instill a feedback loop between deptor down-regulation and TORC1/2 activation in driving mesangial cell hypertrophy.     

Suppression of Transforming Growth Factor β Receptor 2 and Smad5 Is Associated with High Levels of MicroRNA miR-155 in the Oral Mucosa during Chronic Simian Immunodeficiency Virus Infection

Chronic human immunodeficiency virus and simian immunodeficiency virus (HIV and SIV) infections are characterized by mucosal inflammation in the presence of anti-inflammatory cytokines such as transforming growth factor β (TGFβ). The mechanisms for refractiveness to TGFβ are not clear. Here we show that the expression of microRNA miR-155 was significantly upregulated in the oropharyngeal mucosa during chronic SIV infection and was coincident with downregulation of TGFβ receptor 2 (TGFβ-R2) and SMAD5, key TGFβ signaling genes that harbor putative target sites for miR-155. Ectopic expression of miR-155 in vitro was found to significantly downregulate TGFβ-R2 and Smad5 expression, suggesting a role for miR-155 in the suppression of TGFβ-R2 and SMAD5 genes in vivo. The downregulation of TGFβ signaling genes by miR-155 likely contributes to the nonresponsiveness to TGFβ during SIV infection and may inadvertently aid in increased immune activation during HIV and SIV infections.     

Phospho-specific Smad3 signaling: Impact on breast oncogenesis

Members of the TGFβ superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFβ signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFβ signaling regulated through phosphorylation by the TGFβ receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFβ signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.     

Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells

We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P₁ and thereby their signaling on endosomes. TGFβ, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFβ1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFβ-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFβ receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFβ receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFβ receptor internalization and Smad2/3 phosphorylation. TGFβ1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFβ1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFβ receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFβ.     

Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR

The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ₁ as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.