Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-β (TGF-β) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and β-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Prostate is a male sex-accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of phosphorylated Smad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1.

Smad1, Smad5 and Smad9 (also known as Smad8) are activated by phosphorylation by bone morphogenetic protein (BMP)-bound type I receptor kinases. We examined the role of Smad1, Smad5, and Smad9 by creating constitutively active forms (Smad(DVD)). Transcriptional activity of Smad9(DVD) was lower than that of Smad1(DVD) or Smad5(DVD), even though all three Smad(DVD)s associated with Smad4 and bound to the target DNA. The linker region of Smad9 was sufficient to reduce transcriptional activity. Smad9 expression was increased by the activation of BMP signaling, similar to that of inhibitory Smads (I-Smads), and Smad9 reduced BMP activity. In contrast to I-Smads, however, Smad9 did not inhibit the type I receptor kinase and suppressed the constitutively active Smad1(DVD). Smad9 formed complexes with Smad1 and bound to DNA but suppressed the transcription of the target gene. Taken together, our findings suggest that Smad9 is a new type of transcriptional regulator in BMP signaling.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown.

To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells.

BMP2 expression is spatiotemporally correlated with embryo implantation and is crucial for endometrial decidualization and fertility in mice. BMP2 has been reported to increase the mesenchymal adhesion molecule N-cadherin and enhance cell invasion in cancer cells; moreover, studies suggest that N-cadherin promotes placental trophoblast invasion. However, whether BMP2 can promote trophoblast cell invasion during placentation remains unknown. The objective of our study was to investigate the effects of BMP2 on human trophoblast cell invasion and the involvement of N-cadherin and SMAD signaling. Primary and immortalized (HTR8/SVneo) cultures of human extravillous trophoblast (EVT) cells were used as study models. Treatment with recombinant human BMP2 increased HTR8/SVneo cell transwell Matrigel invasion as well as N-cadherin mRNA and protein levels, but had no significant effect on cell proliferation. Likewise, BMP2 treatment enhanced primary human EVT cell invasion and N-cadherin production. Basal and BMP2-induced invasion were attenuated by small interfering RNA-mediated downregulation of N-cadherin in both HTR8/SVneo and primary EVT cells. Intriguingly, BMP2 induced the phosphorylation/activation of both canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in HTR8/SVneo and primary EVT cells. Knockdown of SMAD2/3 or common SMAD4 totally abolished the effects of BMP2 on N-cadherin upregulation in HTR8/SVneo cells. Upregulation of SMAD2/3 phosphorylation and N-cadherin were totally abolished by type I receptor activin receptor-like kinases 2/3 (ALK2/3) inhibitor DMH1; moreover, knockdown of ALK2 or ALK3 inhibited N-cadherin upregulation. Interestingly, activation of SMAD2/3 and upregulation of N-cadherin were partially attenuated by ALK4/5/7 inhibitor SB431542 or knockdown of ALK4, but not ALK5. Our results show that BMP2 promotes trophoblast cell invasion by upregulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling.

Members of the transforming growth factor-beta (TGF-beta) family transmit signals from membrane to nucleus via intracellular proteins known as Smads. A subclass of Smad proteins has recently been identified that antagonize, rather than transduce, TGF-beta family signals. Smad7, for example, binds to and inhibits signaling downstream of TGF-beta receptors. Here we report that the C-terminal MAD homology domain of murine Smad7 (mSmad7) is sufficient for both of these activities. In addition, we show that mSmad7 interacts with activated bone morphogenetic protein (BMP) type I receptors (BMPR-Is), inhibits BMPR-I-mediated Smad phosphorylation, and phenocopies the effect of known BMP antagonists when overexpressed in ventral cells of Xenopus embryos. Xenopus Smad7 (XSmad7, previously termed Smad8) and mSmad7 are nearly identical within their bioactive C-domain, but have quite distinct N-domains. We found that XSmad7, similar to mSmad7, interacted with BMP and TGF-beta type I receptors and inhibited receptor-mediated phosphorylation of downstream signal-transducing Smads. However, XSmad7 is a less efficient inhibitor of TbetaR-I-mediated responses in mammalian cells than is mSmad7. Furthermore, overexpression of XSmad7 in Xenopus embryos produces patterning defects that are not observed following overexpression of mSmad7, suggesting that mSmad7 and XSmad7 may preferentially target distinct signaling pathways. Our results are consistent with the possibility that the C-domain of antagonistic Smads is an effector domain whereas the N-domain may confer specificity for distinct signaling pathways.

FSH is an essential regulator of mammalian reproduction. Its synthesis by pituitary gonadotrope cells is regulated by multiple endocrine and paracrine factors, including TGFβ superfamily ligands, such as the activins and inhibins. Activins stimulate FSH synthesis via transcriptional regulation of its β-subunit gene (Fshb). More recently, bone morphogenetic proteins (BMPs) were shown to stimulate murine Fshb transcription alone and in synergy with activins. BMP2 signals via its canonical type I receptor, BMPR1A (or activin receptor-like kinase 3 [ALK3]), and SMAD1 and SMAD5 to stimulate transcription of inhibitor of DNA binding proteins. Inhibitor of DNA binding proteins then potentiate the actions of activin-stimulated SMAD3 to regulate the Fshb gene in the gonadotrope-like LβT2 cell line. Here, we report the unexpected observation that BMP2 also stimulates the SMAD2/3 pathway in these cells and that it does so directly via ALK3. Indeed, this novel, noncanonical ALK3 activity is completely independent of ALK4, ALK5, and ALK7, the type I receptors most often associated with SMAD2/3 pathway activation. Induction of the SMAD2/3 pathway by ALK3 is dependent upon its own previous activation by associated type II receptors, which phosphorylate conserved serine and threonine residues in the ALK3 juxtamembrane glycine-serine-rich domain. ALK3 signaling via SMAD3 is necessary for the receptor to stimulate Fshb transcription, whereas its activation of the SMAD1/5/8 pathway alone is insufficient. These data challenge current dogma that ALK3 and other BMP type I receptors signal via SMAD1, SMAD5, and SMAD8 and not SMAD2 or SMAD3. Moreover, they suggest that BMPs and activins may use similar intracellular signaling mechanisms to activate the murine Fshb promoter in immortalized gonadotrope-like cells.

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte marker genes and participate in skeletal muscle regeneration. Molecular circuits that regulate the myogenic commitment of MABs are still poorly characterized. The critical role of bone morphogenetic protein (BMP) signalling during proliferation and differentiation of adult myogenic precursors, such as satellite cells, has recently been established. We evaluated whether BMP signalling impacts on the myogenic potential of embryonic and adult MABs both in vitro and in vivo. Addition of BMP inhibited MAB myogenic differentiation, whereas interference with the interactions between BMPs and receptor complexes induced differentiation. Similarly, siRNA-mediated knockdown of Smad8 in Smad1/5-null MABs or inhibition of SMAD1/5/8 phosphorylation with Dorsomorphin (DM) also improved myogenic differentiation, demonstrating a novel role of SMAD8. Moreover, using a transgenic mouse model of Smad8 deletion, we demonstrated that the absence of SMAD8 protein improved MAB myogenic differentiation. Furthermore, once injected into α-Sarcoglycan (Sgca)-null muscles, DM-treated MABs were more efficacious to restore α-sarcoglycan (αSG) protein levels and re-establish functional muscle properties. Similarly, in acute muscle damage, DM-treated MABs displayed a better myogenic potential compared with BMP-treated and untreated cells. Finally, SMADs also control the myogenic commitment of human MABs (hMABs). BMP signalling antagonists are therefore novel candidates to improve the therapeutic effects of hMABs.

NOTCH and bone morphogenetic protein (BMP)/SMAD signaling play key regulatory roles in mammalian ovarian development. The study aimed to investigate interregulatory mechanisms between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells (GCs). The results showed that NOTCH2 silence reduced the mRNA expression of SMAD1, SMAD5, SMAD8 (also known as SMAD9) and Mg2+/Mn2+- dependent Protein Phosphatase 1A (PPM1A), which are effectors of BMP/SMAD signaling pathway (P < 0.01). Overexpressing NOTCH2 intracellular sequence increased the mRNA expression of BMPR1A, SMAD1, SMAD4, SMAD5, SMAD8 and PPM1A (P < 0.01). Meanwhile, treating GCs with BMP4 inhibited the mRNA expression of its downstream gene SMAD1 and steroidogenesis genes STAR and CYP11A1 in the presence of follicular stimulating hormone (FSH) (P < 0.01). Moreover, BMP4 inhibited the mRNA expression of NOTCH signaling pathway target gene HES1 (P < 0.05), while the increase in NOTCH2 may be due to negative feedback of HES1. By and large, these results indicated that NOTCH2 up-regulated key genes of BMP/SMAD signaling in bovine follicle GCs, while BMP4 inhibited its downstream signaling factors and NOTCH signaling pathway target gene HES1. This study suggests there are complex synergistic and antagonistic effects between the two signaling pathways, which jointly participate in regulating bovine follicular development through regulating follicular GCs.

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

The spatiotemporal coordination of multiple morphogens is essential for embryonic patterning yet poorly understood. During neural crest (NC) formation, dynamic bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and WNT signals cooperate by acting on mesoderm and ectoderm. Here, we show that Fhl3, a scaffold LIM domain protein, modulates BMP gradient interpretation during NC induction. During gastrulation, low BMP signaling neuralizes the neural border (NB) ectoderm, while Fhl3 enhances Smad1 intracellular response in underlying paraxial mesoderm, triggering the high WNT8 signals needed to pattern the NB. During neurulation, fhl3 activation in NC ectoderm promotes simultaneous high BMP and BMP-dependent WNT activity required for specification. Mechanistically, Fhl3 interacts with Smad1 and promotes Smad1 binding to wnt8 promoter in a BMP-dependent manner. Consequently, differential Fhl3 expression in adjacent cells ensures a finely tuned coordination of BMP and WNT signaling at several stages of NC development, starting by positioning the NC-inducing mesoderm center under competent NB ectoderm.

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

Renal tubular epithelial-mesenchymal transition (EMT) is the main pathological change in diabetic renal tubulointerstitial fibrosis. Mounting evidence indicates that the inhibitor of differentiation 2 (Id2) protein acts as a negative regulatory factor in organ fibrosis and can inhibit or reverse the process of fibrosis. However, its specific regulatory mechanism is not clear. Diabetes mellitus (DM) rat models were established by injecting rats with streptozotocin and sacrificing them after 16 weeks. Rat renal tubular epithelial cells (NRK-52E) were cultured with normal and high glucose. Immunohistochemical analysis, double immunofluorescence staining, co-immunoprecipitation, Western blot analysis, and real-time polymerase chain reaction were used to determine the expression of Id2, Twist, Smad1/5/8, E-cadherin, α-smooth muscle actin (α-SMA), and collagen Ⅳ. The results showed that bone morphogenetic protein-7 (BMP-7) upregulated the expression of Id2 against high-glucose-induced EMT and extracellular matrix secretion. Moreover, only the simultaneous knockdown of Smad1, Smad5, and Smad8 downregulated the expression of Id2, which was not altered by the individual knockdown of Smad1, Smad5, and Smad8. Basic helix-loop-helix (bHLH) transcription factors were essential for Id2 to regulate the role of downstream target genes, and Twist was a bHLH transcription factor. Therefore, the expression of Twist was examined in this study. Twist was found to be highly expressed in the kidney of DM rats and renal tubular epithelial cells cultured with high glucose. The overexpression of Id2 did not alter the expression of Twist, but the interaction between Id2 and Twist was enhanced. In conclusion, the data showed the specific mechanism underlying Id2 negative regulation in diabetic renal tubulointerstitial fibrosis.

Hematopoietic stem cells (HSCs) that sustain lifelong blood production are created during embryogenesis. They emerge from a specialized endothelial population, termed hemogenic endothelium (HE), located in the ventral wall of the dorsal aorta (DA). In Xenopus, we have been studying the gene regulatory networks (GRNs) required for the formation of HSCs, and critically found that the hemogenic potential is defined at an earlier time point when precursors to the DA express hematopoietic as well as endothelial genes, in the definitive hemangioblasts (DHs). The GRN for DH programming has been constructed and, here, we show that bone morphogenetic protein (BMP) signaling is essential for the initiation of this GRN. BMP2, -4, and -7 are the principal ligands expressed in the lineage forming the HE. To investigate the requirement and timing of all BMP signaling in HSC ontogeny, we have used a transgenic line, which inducibly expresses an inhibitor of BMP signaling, Noggin, as well as a chemical inhibitor of BMP receptors, DMH1, and described the inputs from BMP signaling into the DH GRN and the HE, as well as into primitive hematopoiesis. BMP signaling is required in at least three points in DH programming: first to initiate the DH GRN through gata2 expression, then for kdr expression to enable the DH to respond to vascular endothelial growth factor A (VEGFA) ligand from the somites, and finally for gata2 expression in the DA, but is dispensable for HE specification after hemangioblasts have been formed.

Receptor-regulated SMAD (R-SMAD: SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8) proteins are key transcription factors of the transforming growth factor-β (TGF-β) superfamily of cytokines. MAN1, an integral protein of the inner nuclear membrane, is a SMAD cofactor that terminates TGF-β superfamily signals. Heterozygous loss-of-function mutations in MAN1 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. MAN1 interacts with MAD homology 2 (MH2) domains of R-SMAD proteins using its C-terminal U2AF homology motif (UHM) domain and UHM ligand motif (ULM) and facilitates R-SMAD dephosphorylation. Here, we report the structural basis for R-SMAD recognition by MAN1. The SMAD2-MAN1 and SMAD1-MAN1 complex structures show that an intramolecular UHM-ULM interaction of MAN1 forms a hydrophobic surface that interacts with a hydrophobic surface among the H2 helix, the strands β8 and β9, and the L3 loop of the MH2 domains of R-SMAD proteins. The complex structures also show the mechanism by which SMAD cofactors distinguish R-SMAD proteins that possess a highly conserved molecular surface.