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Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype*

Open AccessPublished:July 30, 2010DOI:https://doi.org/10.1074/jbc.M110.151696
      The transforming growth factor-beta (TGF-β) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-β1 (LAP-TGF-β1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-β stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-β1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.

      Introduction

      NRP-1 was initially discovered as a semaphorin co-receptor and vascular permeability factor/vascular endothelial growth factor (VPF
      The abbreviations used are: VPF
      vascular permeability factor
      BMP
      bone morphogenetic proteins
      NRP
      neuropilin
      PSC
      pancreatic tumor stromal cells
      MEF
      mouse embryonic fibroblasts.
      /VEGF) co-receptor (
      • He Z.
      • Tessier-Lavigne M.
      ,
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      ). Recently, NRP-1 was shown to bind and activate latency-associated protein (LAP)-TGF-β1 and enhance regulatory T cell (Treg) activity (
      • Glinka Y.
      • Prud'homme G.J.
      ). The extracellular domain of NRP-1 contains three structural motifs: two cubilin (CUB) homology domains (a1, a2), two coagulation factor V/VIII homology domains (b1, b2), and a meprin/A5-protein/PTPmu (MAM) domain (c) (
      • Bagri A.
      • Tessier-Lavigne M.
      • Watts R.J.
      ). The relatively short (about 40 amino acids) cytoplasmic domain lacks kinase motifs. Interestingly, NRP-1 has a similar intracellular domain as TGF-β receptor III (TGF-βRIII/β-glycan), and its homolog endoglin, with the PSD-95/Disc-large/ZO-1 (PDZ) binding motif (supplemental Fig. S1). Therefore, we hypothesize that NRP-1 may also serve as a TGF-β co-receptor that regulates TGF-β signaling.
      TGF-β is one member of a superfamily of secreted proteins, which also includes activins and bone morphogenetic proteins (BMPs). TGF-β signaling is one of the most diversified signaling cascades, controlling many aspects of cell behavior, including cell division, differentiation, motility, and death. TGF-β receptors include type I (TβRI), type II (TβRII), and type III (TβRIII). TβRI and TβRII, which are serine/threonine kinase receptors, constitute a hetertetrameric core receptor complex, and TβRIII modulates signaling by regulating ligand binding to the core receptor complex. There are at least seven TβRIs (activin receptor-like kinase 1–7, ALK1–7), five TβRIIs (TGF-βRII, ActRIIA, ActRIIB, AMHRII, and BMPRII), and two TβRIIIs (β-glycan and endoglin). ALK-2, ALK3, ALK4, ALK5, and ALK6 are also known as ActR-I, BMPR-IA, ActR-IB, TGF-βRI, and BMPR-IB, respectively (
      • Persson U.
      • Izumi H.
      • Souchelnytskyi S.
      • Itoh S.
      • Grimsby S.
      • Engström U.
      • Heldin C.H.
      • Funa K.
      • ten Dijke P.
      ).
      Upon TGF-β binding, TβRII activates and phosphorylates the TβRI, which then phosphorylates the receptor-regulated Smad (R-Smad) proteins (including Smad1, 2, 3, 5, and 8). Subsequently, the phosphorylated R-Smad protein forms a heteromeric complex with the co-Smad (Smad4) and translocates to the nucleus to regulate the target gene transcription (
      • Itoh S.
      • ten Dijke P.
      ,
      • Kang J.S.
      • Liu C.
      • Derynck R.
      ,
      • Massagué J.
      • Gomis R.R.
      ,
      • Massagué J.
      • Seoane J.
      • Wotton D.
      ). In addition to this canonical Smad pathway, TGF-β also activates Smad-independent signaling transduction pathways in a cell-type-specific manner, including Rho-ROCK1, Cdc42/Rac1- p21-activated kinase-2 (PAK2), c-Abl, and the mammalian targets of rapamycin (mTOR), JNK, and p38 MAPK (). There are also R-Smad-dependent, but Smad4-independent pathways that mediate TGF-β signaling (
      • Davis B.N.
      • Hilyard A.C.
      • Lagna G.
      • Hata A.
      ,
      • Descargues P.
      • Sil A.K.
      • Sano Y.
      • Korchynskyi O.
      • Han G.
      • Owens P.
      • Wang X.J.
      • Karin M.
      ,
      • He W.
      • Dorn D.C.
      • Erdjument-Bromage H.
      • Tempst P.
      • Moore M.A.
      • Massagué J.
      ). The effects of TGF-β are highly dependent on cell type. For example, TGF-β inhibits epithelial cell proliferation but increases endothelial cell proliferation. As TGF-β signaling is transduced from the cell membrane, receptor expression levels and combinations on each cell type contribute to the outcome.
      Initially, TGF-β was believed to activate only Smad2/3 through ALK5. Some cells, like endothelial cells, sequentially express ALK1 and ALK5, active Smad1/5/8, and Smad2/3, upon TGF-β binding. Recently, TGF-β was shown can active Smad1/5/8 and Smad2/3 in several types of cell including non-cancerous epithelial cells, fibroblasts and cancer cells (
      • Daly A.C.
      • Randall R.A.
      • Hill C.S.
      ,
      • Liu I.M.
      • Schilling S.H.
      • Knouse K.A.
      • Choy L.
      • Derynck R.
      • Wang X.F.
      ,
      • Wrighton K.H.
      • Lin X.
      • Yu P.B.
      • Feng X.H.
      ). Activated Smad1/5/8 and Smad2/3 have different, and occasionally opposite, functions. The mechanisms that regulate the diversity of responses downstream from TGF-β are unknown. Here, we report that NRP-1 can promote divergent signaling that leads to differential Smad1/5 and Smad2/3 activation and downstream myofibroblast phenotypes. Hence, NRP-1 can control Smad1/5 and Smad2/3 signaling counterbalances and regulates diversified TGF-β signaling.

      DISCUSSION

      We demonstrate that NRP-1, like β-glycan and endoglin, can regulate TGF-β signaling by interacting with TGF-βRII. These proteins have similar structures: a short cytoplasmic C-terminal tail containing a PDZ binding domain that binds to GAIP-interacting protein, C terminus (GIPC). NRP-1 and TβRIII are expressed in almost all kinds of cells, but levels vary. In addition to its role in neural and vascular development, NRP-1 is overexpressed in cancers and correlates with cancer progression and poor prognosis (
      • Bagri A.
      • Tessier-Lavigne M.
      • Watts R.J.
      ,
      • Ellis L.M.
      ,
      • Klagsbrun M.
      • Takashima S.
      • Mamluk R.
      ). Correspondingly, TβRIII expression was down-regulated in most cancer types (
      • Gatza C.E.
      • Blobe G.C.
      ). Although NRP-1 and TβRIII are dysregulated in malignancies, mutated forms of the proteins have not been found in tumors.
      TGF-β was thought to act exclusively through TβRII and TβRI (ALK5) receptor complexes, as well as intracellular Smad2/Smad3, to mediate the signaling. In endothelial cells, which specifically express ALK-1, it was shown that TGF-β, through ALK1 active Smad1/5/8, regulates cell proliferation and migration (
      • Goumans M.J.
      • Valdimarsdottir G.
      • Itoh S.
      • Rosendahl A.
      • Sideras P.
      • ten Dijke P.
      ). Recently, TGF-β was found to activate Smad1/5 in normal and cancerous epithelial cells and fibroblasts (
      • Daly A.C.
      • Randall R.A.
      • Hill C.S.
      ,
      • Liu I.M.
      • Schilling S.H.
      • Knouse K.A.
      • Choy L.
      • Derynck R.
      • Wang X.F.
      ,
      • Wrighton K.H.
      • Lin X.
      • Yu P.B.
      • Feng X.H.
      ), and all of the results indicated that TGF-β-induced Smad1/5 phosphorylation depends on ALK5. Moreover, BMP type I receptors (ALK1/2/3/6) also participated in TGF-β-induced phosphorylation of Smad1/5 (
      • Daly A.C.
      • Randall R.A.
      • Hill C.S.
      ,
      • Wrighton K.H.
      • Lin X.
      • Yu P.B.
      • Feng X.H.
      ). These results indicated that TGF-β, like other TGF-β family proteins, acts through multiple possible receptor combinations and regulates the complicated TGF-β signaling. While most cells express several TβRIs, it is possible that phosphorylation of Smad proteins is activated by different homodimeric TβRIs or a heterodimeric TβRI on TGF-β binding.
      Knockdown TβRII blocks Smad1/5 and Smad2/3 signaling, and the regulatory role of NRP-1 also was inhibited. This, together with the results that NRP-1 and TβRII co-immunoprecipitation, indicates that NRP-1 regulates TGF-β signaling through TβRII. The impairment of BMPS signaling after knockdown NRP-1 also suggests that NRP-1, like β-glycan and endoglin, plays a role in BMP signaling, likely through the regulation of BMPRII. These findings also point out the complexity of TGF-β signaling initiation on the cell membrane. Given the cell-type specific expression patterns of TGF-β superfamily receptors, our observations regarding the novel role of NRP-1 in fibroblasts reveals an intriguing area of new investigation.
      Physiologically, TGF-β signaling maintains tissue homeostasis; in pathogenesis, the deregulation of TGF-β signaling causes fibrosis, tumorigenesis, and metastasis. TGF-β plays an important role in fibrotic diseases in most organs (
      • Branton M.H.
      • Kopp J.B.
      ), as well as stromal cell activation in tumor tissue. TGF-β has a dual role in the control of fibroblast activation. On one hand, it activates Smad1/5, which controls Id-1, a functional protein that can maintain a quiescent state and be a marker for quiescent stromal cells; on the other hand, it activates Smad2/3, which induces α-SMA expression, an active stromal cell/myofibroblast marker. Id-1 is abundantly exressed in quiescent stellate cells and diminished during activation (
      • Mann D.A.
      • Smart D.E.
      ,
      • Vincent K.J.
      • Jones E.
      • Arthur M.J.
      • Smart D.E.
      • Trim J.
      • Wright M.C.
      • Mann D.A.
      ). Id-1−/− mice were susceptible to bleomycin-induced lung injury and fibrosis, and fibroblasts from Id-1−/− mice showed enhanced responses to TGF-β stimulation (
      • Lin L.
      • Zhou Z.
      • Zheng L.
      • Alber S.
      • Watkins S.
      • Ray P.
      • Kaminski N.
      • Zhang Y.
      • Morse D.
      ). Also, Id-1 up-regulation was an early event in the fibroblast after TGF-β stimulation. Id-1, a known Smad1 response gene, was up-regulated by phospho-Smad1 and can be suppressed by the Smad3/4 response gene ATF3 (
      • Kang Y.
      • Chen C.R.
      • Massagué J.
      ). Hence, Id-1 induced by TGF-β may act as a negative regulator to inhibit fibrosis progression. While Smad2/3 is known for inducing α-SMA expression, Smad3 also induces ATF-3 expression. ATF-3, together with Smad3/4, inhibits Id-1 expression and overcomes the quiescent effect of Id-1. Furthermore, Smad3−/− mice were resistant to TGF-β-mediated pulmonary fibrosis (
      • Bonniaud P.
      • Kolb M.
      • Galt T.
      • Robertson J.
      • Robbins C.
      • Stampfli M.
      • Lavery C.
      • Margetts P.J.
      • Roberts A.B.
      • Gauldie J.
      ). These findings suggest that the functions of Smad1/5/8 and Smad2/3 may counteract each other, and the fibrotic process is involved in gaining of Smad2/3 signaling whereas diminishing of Smad1/5/8 signaling. It has been shown that during fibroblast activation, the expression of TβRI, TβRII, and TβRIII were dysregulated (
      • Roulot D.
      • Sevcsik A.M.
      • Coste T.
      • Strosberg A.D.
      • Marullo S.
      ,
      • Friedman S.L.
      • Yamasaki G.
      • Wong L.
      ). It is possible that up-regulation of NRP-1, as well as the TGF-β receptors, induces activation of the stellate cell by modulating TGF-β signaling.
      Here, we demonstrated that NRP-1 is a co-receptor of TGF-β and that it regulates the TGF-β canonical signaling in the Smad proteins phosphorylation. Interestingly, in the stromal/fibroblast cell, knocking down NRP-1 up-regulates TGF-β-induced Smad1/5 phosphorylation and down-regulates Smad/2/3 phosphorylation. The Id-1 protein, which is transcriptionally controlled by phospho-Smad1/5, is a major protein in maintaining the quiescent state of fibroblasts. Phospho-Smad2/3 controls α-SMA expression, a marker of fibroblasts activation. Thus, NRP-1 controls two aspects of TGF-β signaling: down-regulating of the Smad1/5 signaling which inhibits fibrosis progression and up-regulating of the Smad2/3 signaling, which promotes fibrosis; both reinforce fibrosis. During the activation, the up-regulated NRP-1 shifts the TGF-β signaling from Smad1/5/8 to Smad2/3, from maintenance of the quiescent state to activation of the cells (Fig. 8). NRP-1 might also regulate TGF-β non-Smad signaling, such as collagen production (
      • Cao S.
      • Yaqoob U.
      • Das A.
      • Shergill U.
      • Jagavelu K.
      • Huebert R.C.
      • Routray C.
      • Abdelmoneim S.
      • Vasdev M.
      • Leof E.
      • Charlton M.
      • Watts R.J.
      • Mukhopadhyay D.
      • Shah V.H.
      ). Like endoglin, NRP-1 expression is also increased during fibroblast activation. According to our results, NRP-1 up-regulation worsens the fibrosis, but endoglin up-regulation seems to attenuate fibrosis (
      • Rodriguez-Barbero A.
      • Obreo J.
      • Alvarez-Munoz P.
      • Pandiella A.
      • Bernabéu C.
      • López-Novoa J.M.
      ,
      • Guo B.
      • Slevin M.
      • Li C.
      • Parameshwar S.
      • Liu D.
      • Kumar P.
      • Bernabeu C.
      • Kumar S.
      ,
      • Obreo J.
      • Díez-Marques L.
      • Lamas S.
      • Düwell A.
      • Eleno N.
      • Bernabéu C.
      • Pandiella A.
      • López-Novoa J.M.
      • Rodríguez-Barbero A.
      ). The mechanism of NRP-1 up-regulation during fibroblasts activation is unclear. NRP-1 also functions as a cell adhesion molecular (
      • Shimizu M.
      • Murakami Y.
      • Suto F.
      • Fujisawa H.
      ) and it is possible that NRP-1 is up-regulated by the similar mechanisms as the integrins during the cell activation. TGF-β also controls NRP-1 and TβRIII expression. Endoglin is up-regulated by TGF-β (
      • Obreo J.
      • Díez-Marques L.
      • Lamas S.
      • Düwell A.
      • Eleno N.
      • Bernabéu C.
      • Pandiella A.
      • López-Novoa J.M.
      • Rodríguez-Barbero A.
      ), while β-glycan and NRP-1 is down-regulated by TGF-β (
      • Hempel N.
      • How T.
      • Cooper S.J.
      • Green T.R.
      • Dong M.
      • Copland J.A.
      • Wood C.G.
      • Blobe G.C.
      ,
      • Schramek H.
      • Sarközi R.
      • Lauterberg C.
      • Kronbichler A.
      • Pirklbauer M.
      • Albrecht R.
      • Noppert S.J.
      • Perco P.
      • Rudnicki M.
      • Strutz F.M.
      • Mayer G.
      ) (supplemental Fig. S3).
      Figure thumbnail gr8
      FIGURE 8The schematic illustration of NRP-1 function in regulating TGF-β signaling. TGF-β induced both Smad1/5/8 and Smad2/3 phosphorylation in the fibroblast cells. Without NRP-1 (left panel), Smad1/5/8 is more phosphorylated and Smad2/3 less phosphorylated, and the corresponding gene expression controlled by the Smad proteins (e.g. Smad1/Id-1, Smad3/a-SMA, PAI-1) caused the cell to enter a less activated state (more quiescent). With NRP-1 (right panel), Smad1/5/8 is less phosphorylated, and Smad2/3 is more phosphorylated, and the corresponding gene expression controlled by the Smad proteins caused the cell to enter a more activated state (less quiescent).
      The mechanism through which NRP-1 controls the Smad1/5 and Smad2/3 phosphorylation counterbalance is still largely unknown. It is possible that NRP-1 binds TβRII, presents TGF-β more favorably to ALK4/5/7 than ALK1/2/3/6, or the existence of NRP-1 in the TGF-β receptor complex changes the TβRI's conformation, mediating phosphorylation of Smad2/3 rather than Smad1/5/8. Furthermore, NRP-1 may recruit other proteins to the complex.
      The cell growth arrest observed upon knockdown of NRP-1 is likely not TGF-β-dependent. In the present of SB431542 (which inhibited the TGF-β-Smad signaling), knocking down NRP-1 still induces cell growth arrest (supplemental Fig. S4). It has been previously shown that NRP-1 has TGF-β-independent functions such acting as a semaphorin 3 (SEMA3) and VEGF co-receptor (
      • He Z.
      • Tessier-Lavigne M.
      ,
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      ,
      • Gatza C.E.
      • Blobe G.C.
      ), mediating cell adhesion (
      • Shimizu M.
      • Murakami Y.
      • Suto F.
      • Fujisawa H.
      ) and binding galectin-1 (
      • Hsieh S.H.
      • Ying N.W.
      • Wu M.H.
      • Chiang W.F.
      • Hsu C.L.
      • Wong T.Y.
      • Jin Y.T.
      • Hong T.M.
      • Chen Y.L.
      ), forming receptor complexes with platelet-derived growth factor receptors (PDGFRs) and modulating PDGF signaling (
      • Ball S.G.
      • Bayley C.
      • Shuttleworth C.A.
      • Kielty C.M.
      ). It also can regulate endothelial cell survival independent of VEGF receptors (
      • Wang L.
      • Dutta S.K.
      • Kojima T.
      • Xu X.
      • Khosravi-Far R.
      • Ekker S.C.
      • Mukhopadhyay D.
      ). Finally, NRP-1 is known to promote vascular and neural development, immune responses (
      • Romeo P.H.
      • Lemarchandel V.
      • Tordjman R.
      ), and cancer progression. TGF-β is also well known for participating in these processes. Consequently, the role of NRP-1 in the regulation of TGF-β signaling in these processes needs to be defined in the future.

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