Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

Transforming growth factor-beta (TGF-beta) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF-beta activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF-beta. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up-regulation of TGF-beta superfamily-induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl-2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase-negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP-2/4 type IA receptor (BMPR-IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner.     

XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway.

Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors. TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. However, the events leading from receptor activation to TAK1 activation remain to be identified. A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1. The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein. XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells. Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner. Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor. These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.     

BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway.     

Transforming growth factor-beta1 (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3

The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.     

Antisense targeting of TGF-beta1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-beta1 on bone formation remains elusive. In particular, the role of autocrine TGF-beta1 on human osteoblasts is largely unknown. Here we show the effect of TGF-beta1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-beta1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-beta1. Notably, TGF-beta1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-beta1. Moreover, TGF-beta1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-beta1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.     

BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner

Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast‐like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG‐MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z‐7‐oxozeaenol, dorsomorphin, H‐8). Apoptosis was characterized by caspase‐3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell‐type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG‐MG63 than MG63 cells. 5Z‐7‐oxozeaenol and dorsomorphin eliminated the BMP2‐induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications. J. Cell. Biochem. 113: 3236–3245, 2012. © 2012 Wiley Periodicals, Inc.     

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-beta-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST-YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6-p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1.     

Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.     

A dominant negative TAK1 inhibits cellular fibrotic responses induced by TGF-beta

Transforming growth factor-beta (TGF-beta) is crucially virulent in the progression of fibrotic disorders. TAK1 (TGF-beta activated kinase 1) is one of the mitogen-activated kinase kinase kinase (MAPKKK) that is involved in TGF-beta signal transduction. To elucidate the importance of TAK1 in TGF-beta-induced fibrotic marker expression, we investigated whether dominant negative TAK1 could suppress TGF-beta signaling. Based on the finding that TAB1 (TAK1 binding protein 1) binding to TAK1 is required for TAK1 activation, a minimal portion of TAK1 lacking kinase activity that binds to TAB1 was designed as a TAK1 dominant negative inhibitor (TAK1-DN). The effect of TAK1-DN was assessed in the cells that respond to TGF-beta stimulation and that lead to the increase in production of extracellular matrix (ECM) proteins. TAK1-DN, indeed, decreased the ECM protein production, indicating that TAK1-DN retains the ability to intercept the TGF-beta signaling effectively.     

Structural basis for the interaction of TAK1 kinase with its activating protein TAB1

Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1) is a member of the MAPKKK family of protein kinases, and is involved in intracellular signalling pathways stimulated by transforming growth factor beta, interleukin-1 and tumour necrosis factor-alpha. TAK1 is known to rely upon an additional protein, TAK1-binding protein 1 (TAB1), for complete activation. However, the molecular basis for this activation has yet to be elucidated. We have solved the crystal structure of a novel TAK1 chimeric protein and these data give insight into how TAK1 is activated by TAB1. Our results reveal a novel binding pocket on the TAK1 kinase domain whose shape complements that of a unique alpha-helix in the TAK1 binding domain of TAB1, providing the basis for an intimate hydrophobic association between the protein activator and its target.     

Generation of a conditional mutant allele for Tab1 in mouse

TAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF-beta activating kinase (TAK1). TAK1, a mitogen-activated kinase kinase kinase, is involved in several distinct signaling pathways including non-Smad pathways for TGF-beta superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C-terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre-recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages.