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* This work was supported, in whole or in part, by National Institutes of Health Grants CA78383, HL072178, and HL70567. This work was also supported by a Mayo Clinic Pancreatic Cancer SPORE Pilot grant, a grant from the American Cancer Society as well as the Bruce and Martha Atwater Foundation. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Methods.
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.
). 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) (
). 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 (
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 (
). 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 (
). 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 (
). 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.
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 (
). 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 (
), 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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
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 (
), 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.