Transforming Growth Factor Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors

Michael E. DeBakey Department of Surgery and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2009; 284(15):9755-63. DOI: 10.1074/jbc.M809223200
Source: PubMed


Transforming growth factor-β (TGFβ) superfamily ligands control a diverse set of cellular processes by activating type I and
type II serine-threonine receptor kinases. Canonical TGFβ signaling is mediated via the TβRI/ALK5 type I receptor that phosphorylates
Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein
(BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGFβ can also lead to the phosphorylation of Smad1, dependent
on ALK1 receptor activity. Here we present data showing that TGFβ can significantly induce Smad1 phosphorylation in several
non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGFβ/activin/nodal (ALK4/5/7) and
BMP (ALK1/2/3/6) type I receptors, we show that in some cell types TGFβ induces Smad1 phosphorylation independently of the
BMP type I receptors. Thus, TGFβ-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell
type-specific manner.

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    • "These pathways are referred to as the canonical " TGF-β–Smad " and " BMP–Smad " pathways, respectively. This simplified view of TGF-β signaling has been complicated by the observation that in vitro TGF-β can sometimes phosphorylate the " BMP Smads " (Smad1/5/9) in a number of cell types, including endothelial cells, fibroblasts and normal and tumor-derived epithelial cells (Bharathy et al. 2008; Goumans et al. 2002; Wrighton et al. 2009). This unexpected phosphorylation pattern occurs when an alternate RI is recruited into the RI-RII receptor complex. "
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    ABSTRACT: Transforming growth factor-β (TGF-β) is an important regulator of cellular homeostasis and disease pathogenesis. Canonical TGF-β signaling occurs through Smad2/3-Smad4 complexes; however, recent in vitro studies suggest that elevated levels of TGF-β may activate a novel mixed Smad complex (Smad2/3-Smad1/5/9), which is required for some of the pro-oncogenic activities of TGF-β. To determine if mixed Smad complexes are evident in vivo, we developed antibodies that can be used with a proximity ligation assay to detect either canonical or mixed Smad complexes in formalin-fixed paraffin-embedded sections. We demonstrate high expression of mixed Smad complexes in the tissues from mice genetically engineered to express high levels of TGF-β1. Mixed Smad complexes were also prominent in 15-16 day gestation mouse embryos and in breast cancer xenografts, suggesting important roles in embryonic development and tumorigenesis. In contrast, mixed Smad complexes were expressed at extremely low levels in normal adult mouse tissue, where canonical complexes were correspondingly higher. We show that this methodology can be used in archival patient samples and tissue microarrays, and we have developed an algorithm to quantitate the brightfield read-out. These methods will allow quantitative analysis of cell type-specific Smad signaling pathways in physiological and pathological processes.
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    • "We found however, that SMAD2/3 phosphorylation increased only after prolonged obstruction (6 weeks). The ability of TGF-β to activate noncanonical SMAD signaling in a Ras/MEK/ERK-dependent manner is well established [34-36], and in keeping with this possibility, we also observed ERK1/2 phosphorylation coinciding with early SMAD1/5/8 phosphorylation. One possibility, therefore, is that increased TGF-β mRNA levels at 10 days reflect production of bioactive TGF-β that signals via the noncanonical (SMAD1/5/8) pathway. "
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    ABSTRACT: Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.
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    • "The EC lines that consistently trigger a TGFβ/pSMAD1/5/8 response are immortalized mouse lines such as MEEC [15] or SVR, bEnd.3 and C166 (Fig. 1). While there is no doubt that TGFβ/pSMAD1 signaling can be detected in many cell types ranging from fibroblasts [35] to cancer cell lines [36], [37], and a variety of immortalized cells of mesenchymal and epithelial origin [38], it does not appear to be a signaling feature of primary human EC. Our findings in this area are in agreement with results from a very recent study [39]. "
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    ABSTRACT: Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.
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