Xin-Hua Feng

Molecular Antagonism and Plasticity of Regulatory and Inflammatory T Cell Programs

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-β signaling

Smads are important intracellular signaling effectors for transforming growth factor-β (TGF-β) and related factors. Proper TGF-β signaling requires precise control of Smad functions. In this study, we have identified a novel HECT class ubiquitin E3 ligase, designated Smurf2, that negatively regulates Smad2 signaling. In both yeast two-hybrid and in vitro binding assays, we found that Smurf2 could interact with receptor-activated Smads (R-Smads), including Smad1, Smad2, and Smad3 but not Smad4. Ectopic expression of Smurf2 was sufficient to reduce the steady-state levels of Smad1 and Smad2 but not Smad3 or Smad4. Significantly, Smurf2 displayed preference to Smad2 as its target for degradation. Furthermore, Smurf2 exhibited higher binding affinity to activated Smad2 upon TGF-β stimulation. The ability of Smurf2 to promote Smad2 destruction required the HECT catalytic activity of Smurf2 and depended on the proteasome-dependent pathway. Consistent with these results, Smurf2 potently reduced the transcriptional activity of Smad2. These data suggest that a ubiquitin/proteasome-dependent mechanism is important for proper regulation of TGF-β signaling.

PPM1A Functions as a Smad Phosphatase to Terminate TGFβ Signaling

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.

Microtubule Binding to Smads May Regulate TGFβ Activity

Abstract Smad proteins are intracellular signaling effectors of the TGFβ superfamily. We show that endogenous Smad2, 3, and 4 bind microtubules (MTs) in several cell lines. Binding of Smads to MTs does not require TGFβ stimulation. TGFβ triggers dissociation from MTs, phosphorylation, and nuclear translocation of Smad2 and 3, with consequent activation of transcription in CCL64 cells. Destabilization of the MT network by nocodazole, colchicine, or a tubulin mutant disrupts the complex between Smads and MTs and increases TGFβ-induced Smad2 phosphorylation and transcriptional response in CCL64 cells. These data demonstrate that MTs may serve as a cytoplasmic sequestering network for Smads, controlling Smad2 association with and phosphorylation by activated TGFβ receptor I, and suggest a novel mechanism for the MT network to negatively regulate TGFβ function.

Phospho-control of TGF-β superfamily signaling

Members of the transforming growth factor-β (TGF-β) family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad (de)phosphorylation as a tightly controlled and dynamic event. This article illustrates the essential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensuing physiological responses.

Latent TGFβ1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder

Transforming growth factor β1 (TGFβ1), a potent keratinocyte growth inhibitor, has been shown to be overexpressed in keratinocytes in certain inflammatory skin diseases and has been thought to counteract the effects of other growth factors at the site of inflammation. Surprisingly, our transgenic mice expressing wild‐type TGFβ1 in the epidermis using a keratin 5 promoter (K5.TGFβ1wt) developed inflammatory skin lesions, with gross appearance of psoriasis‐like plaques, generalized scaly erythema, and Koebners phenomenon. These lesions were characterized by epidermal hyperproliferation, massive infiltration of neutrophils, T lymphocytes, and macrophages to the epidermis and superficial dermis, subcorneal microabscesses, basement membrane degradation, and angiogenesis. K5.TGFβ1wt skin exhibited multiple molecular changes that typically occur in human Th1 inflammatory skin disorders, such as psoriasis. Further analyses revealed enhanced Smad signaling in transgenic epidermis and dermis. Our study suggests that certain pathological condition‐induced TGFβ1 overexpression in the skin may synergize with or induce molecules required for the development of Th1 inflammatory skin disorders.

Direct Interaction of c-Myc with Smad2 and Smad3 to Inhibit TGF-β-Mediated Induction of the CDK Inhibitor p15Ink4B

Summary rynck and Feng, 1997; Massague´ et al., 2000; Roberts and Sporn, 1990). Mutations or deregulation of compo- The c-Myc oncogene has been implicated in the gene- nents of the TGF- growth arrest pathway have beensis of diverse human tumors. Ectopic expression of shown to be responsible for the inability of tumor cells the c-Myc gene in cultured epithelial cells causes re- torespondtoTGF- Ink4B.Inactivationofthegeneforp15 ,sistancetotheantiproliferativeeffectsofTGF- .How- Smad4, Smad2, or TGF- receptors can allow cells to ever, little is known about the precise mechanisms of escape from TGF- control and drive progression in ac-Myc-mediated TGF- resistance. In this study, we specific tumor (Barrett et al., 1996; Eppert et al., 1996; reveal that c-Myc physically interacts with Smad2 and Hahn et al., 1996; Riggins et al., 1997; Schutte et al.,Smad3, two specific signal transducers involved in 1996; for review see Massague´ et al., 2000). TGF- signaling. Through its direct interaction with In human cancers, the c-Myc gene is often amplifiedSmads, c-Myc binds to the Sp1-Smad complex on the

Smad7 Antagonizes Transforming Growth Factor β Signaling in the Nucleus by Interfering with Functional Smad-DNA Complex Formation

ABSTRACT Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor β (TGF-β) signaling by inhibiting TGF-β signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-β signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-β target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-β-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-β signaling in the nucleus by a novel mechanism.

Opposed Regulation of Corepressor CtBP by SUMOylation and PDZ Binding

The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. In this study, we demonstrate that CtBP1 was SUMOylated and that its SUMOylation profoundly affected its subcellular localization. SUMOylation occurred at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacked the SUMOylation site and was not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifted CtBP1 from the nucleus to the cytoplasm, while it had little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolished the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 was inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression.

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

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.