A MAP1B-cortactin-Tks5 axis regulates TNBC invasion and tumorigenesis.

The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.

CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-ß (TGF-ß) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-ß signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-ß-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-ß-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-ß-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

Combination therapy with anamorelin and a myostatin inhibitor is advantageous for cancer cachexia in a mouse model.

Cancer cachexia is a multifactorial disease that causes continuous skeletal muscle wasting. Thereby, it seems to be a key determinant of cancer-related death. Although anamorelin, a ghrelin receptor agonist, has been approved in Japan for the treatment of cachexia, few medical treatments for cancer cachexia are currently available. Myostatin (MSTN)/growth differentiation factor 8, which belongs to the transforming growth factor-ß family, is a negative regulator of skeletal muscle mass, and inhibition of MSTN signaling is expected to be a therapeutic target for muscle-wasting diseases. Indeed, we have reported that peptide-2, an MSTN-inhibiting peptide from the MSTN prodomain, alleviates muscle wasting due to cancer cachexia. Herein, we evaluated the therapeutic benefit of myostatin inhibitory D-peptide-35 (MID-35), whose stability and activity were more improved than those of peptide-2 in cancer cachexia model mice. The biologic effects of MID-35 were better than those of peptide-2. Intramuscular administration of MID-35 effectively alleviated skeletal muscle atrophy in cachexia model mice, and the combination therapy of MID-35 with anamorelin increased food intake and maximized grip strength, resulting in longer survival. Our results suggest that this combination might be a novel therapeutic tool to suppress muscle wasting in cancer cachexia.

Transgenically expressed Helicobacter pylori CagA in vascular endothelial cells accelerates arteriosclerosis in mice.

Arteriosclerosis is intimately associated with cardiovascular diseases. Recently, evidence accumulated that infection with Helicobacter pylori cagA-positive strains, which causes gastritis, peptic ulceration, and gastric cancer, is also involved in the development of arteriosclerosis. The cagA-encoded CagA protein is injected into the attached gastric epithelial cells via the type IV secretion system. We previously showed that CagA-containing exosomes are secreted from CagA-injected gastric epithelial cells and enter the systemic blood circulation, delivering CagA into endothelial cells. In the present study, transgenic mice were established in which CagA was selectively expressed in endothelial cells by Cre-loxP system. Treatment of the mice with a high-fat diet revealed that atherogenic lesions were induced in mice expressing CagA in vascular endothelial cells but not in CagA-nonexpressing mice. To investigate the effects of CagA on endothelial cells, we also established conditional CagA-expressing human vascular endothelial cells using the Tet-on system. Upon induction of CagA, a dramatic change in cell morphology was observed that was concomitantly associated with the loss of the endothelial cells to form tube-like structures. Induction of CagA also activated the pro-inflammatory transcription factor STAT3. Thus, exosome-delivered CagA deregulates signals that activates STAT3 in endothelial cells, which accelerates inflammation that promotes arteriosclerosis/atherosclerosis.

TGF-ß signaling in lymphatic vascular vessel formation and maintenance.

Transforming growth factor (TGF)-ß and its family members, including bone morphogenetic proteins (BMPs), nodal proteins, and activins, are implicated in the development and maintenance of various organs. Here, we review its role in the lymphatic vascular system (the secondary vascular system in vertebrates), which plays a crucial role in various physiological and pathological processes, participating in the maintenance of the normal tissue fluid balance, immune cell trafficking, and fatty acid absorption in the gut. The lymphatic system is associated with pathogenesis in multiple diseases, including lymphedema, inflammatory diseases, and tumor metastasis. Lymphatic vessels are composed of lymphatic endothelial cells, which differentiate from blood vascular endothelial cells (BECs). Although TGF-ß family signaling is essential for maintaining blood vessel function, little is known about the role of TGF-ß in lymphatic homeostasis. Recently, we reported that endothelial-specific depletion of TGF-ß signaling affects lymphatic function. These reports suggest that TGF-ß signaling in lymphatic endothelial cells maintains the structure of lymphatic vessels and lymphatic homeostasis, and promotes tumor lymphatic metastasis. Suppression of TGF-ß signaling in lymphatic endothelial cells may therefore be effective in inhibiting cancer metastasis. We highlight recent advances in understanding the roles of TGF-ß signaling in the formation and maintenance of the lymphatic system.

Endothelial-specific depletion of TGF-ß signaling affects lymphatic function.

BACKGROUND: Transforming growth factor (TGF)-ß is a multifunctional cytokine involved in cell differentiation, cell proliferation, and tissue homeostasis. Although TGF-ß signaling is essential for maintaining blood vessel functions, little is known about the role of TGF-ß in lymphatic homeostasis. METHODS: To delineate the role of TGF-ß signaling in lymphatic vessels, TßRIIfl/fl mice were crossed with Prox1-CreERT2 mice to generate TßRIIfl/fl; Prox1-CreERT2 mice. The TßRII gene in the lymphatic endothelial cells (LECs) of the conditional knockout TßRIIiΔLEC mice was selectively deleted using tamoxifen. The effects of TßRII gene deletion on embryonic lymphangiogenesis, postnatal lymphatic structure and drainage function, tumor lymphangiogenesis, and lymphatic tumor metastasis were investigated. RESULTS: Deficiency of LEC-specific TGF-ß signaling in embryos, where lymphangiogenesis is active, caused dorsal edema with dilated lymphatic vessels at E13.5. Postnatal mice in which lymphatic vessels had already been formed displayed dilation and increased bifurcator of lymphatic vessels after tamoxifen administration. Similar dilation was also observed in tumor lymphatic vessels. The drainage of FITC-dextran, which was subcutaneously injected into the soles of the feet of the mice, was reduced in TßRIIiΔLEC mice. Furthermore, Lewis lung carcinoma cells constitutively expressing GFP (LLC-GFP) transplanted into the footpads of the mice showed reduced patellar lymph node metastasis. CONCLUSION: These data suggest that TGF-ß signaling in LECs maintains the structure of lymphatic vessels and lymphatic homeostasis, in addition to promoting tumor lymphatic metastasis. Therefore, suppression of TGF-ß signaling in LECs might be effective in inhibiting cancer metastasis.

Systemic administration of monovalent follistatin-like 3-Fc-fusion protein increases muscle mass in mice.

Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor ß (TGF-ß) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-ß family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.

Blood and lymphatic systems are segregated by the FLCN tumor suppressor.

Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

Peptide-2 from mouse myostatin precursor protein alleviates muscle wasting in cancer-associated cachexia.

Cancer cachexia, characterized by continuous muscle wasting, is a key determinant of cancer-related death; however, there are few medical treatments to combat it. Myostatin (MSTN)/growth differentiation factor 8 (GDF-8), which is a member of the transforming growth factor-ß family, is secreted in an inactivated form noncovalently bound to the prodomain, negatively regulating the skeletal muscle mass. Therefore, inhibition of MSTN signaling is expected to serve as a therapeutic target for intractable muscle wasting diseases. Here, we evaluated the inhibitory effect of peptide-2, an inhibitory core of mouse MSTN prodomain, on MSTN signaling. Peptide-2 selectively suppressed the MSTN signal, although it had no effect on the activin signal. In contrast, peptide-2 slightly inhibited the GDF-11 signaling pathway, which is strongly related to the MSTN signaling pathway. Furthermore, we found that the i.m. injection of peptide-2 to tumor-implanted C57BL/6 mice alleviated muscle wasting in cancer cachexia. Although peptide-2 was unable to improve the loss of heart weight and fat mass when cancer cachexia model mice were injected with it, peptide-2 increased the gastrocnemius muscle weight and muscle cross-sectional area resulted in the enhanced grip strength in cancer cachexia mice. Consequently, the model mice treated with peptide-2 could survive longer than those that did not undergo this treatment. Our results suggest that peptide-2 might be a novel therapeutic candidate to suppress muscle wasting in cancer cachexia.

The evolutionarily conserved deubiquitinase UBH1/UCH-L1 augments DAF7/TGF-ß signaling, inhibits dauer larva formation, and enhances lung tumorigenesis.

Modification of the transforming growth factor ß (TGF-ß) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-ß signaling, suggesting that this mode of regulation of TGF-ß signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-ß signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-ßRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-ß signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-ß signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-ß/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-ß/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-ß/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-ß signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.

PDZK1-interacting protein 1 (PDZK1IP1) traps Smad4 protein and suppresses transforming growth factor-ß (TGF-ß) signaling.

Transforming growth factor (TGF)-ß signaling in humans is stringently regulated to prevent excessive TGF-ß signaling. In tumors, TGF-ß signaling can both negatively and positively regulate tumorigenesis dependent on tumor type, but the reason for these opposite effects is unclear. TGF-ß signaling is mainly mediated via the Smad-dependent pathway, and herein we found that PDZK1-interacting protein 1 (PDZK1IP1) interacts with Smad4. PDZK1IP1 inhibited both the TGF-ß and the bone morphogenetic protein (BMP) pathways without affecting receptor-regulated Smad (R-Smad) phosphorylation. Rather than targeting R-Smad phosphorylation, PDZK1IP1 could interfere with TGF-ß- and BMP-induced R-Smad/Smad4 complex formation. Of note, PDZK1IP1 retained Smad4 in the cytoplasm of TGF-ß-stimulated cells. To pinpoint PDZK1IP1's functional domain, we created several PDZK1IP1 variants and found that its middle region, from Phe40 to Ala49, plays a key role in its Smad4-regulating activity. PDZK1IP1 knockdown enhanced the expression of the TGF-ß target genes Smad7 and prostate transmembrane protein androgen-induced (TMEPAI) upon TGF-ß stimulation. In contrast, PDZK1IP1 overexpression suppressed TGF-ß-induced reporter activities, cell migration, and cell growth inhibition. In a xenograft tumor model in which TGF-ß was previously shown to elicit tumor-promoting effects, PDZK1IP1 gain of function decreased tumor size and increased survival rates. Taken together, these findings indicate that PDZK1IP1 interacts with Smad4 and thereby suppresses the TGF-ß signaling pathway.

TMEPAI family: involvement in regulation of multiple signalling pathways.

The TMEPAI family, composed of TMEPAI and C18ORF1, is known to inhibit transforming growth factor-ß (TGF-ß) signalling via its competition for binding of receptor-regulated Smad with Smad anchor for receptor activation. However, TMEPAI has also been reported to be involved in androgen receptor signalling, phosphatase and tensin homologue deleted on chromosome 10 signalling, and formation of autophagosomes in addition to degradation of TßRI (TGF-ß type I receptor) through lysosomes. Thus, TMEPAI seems to act as a regulator of multiple signalling pathways. A great deal of attention has already been paid to the relationship between the TMEPAI family and tumourigenicity. In this paper, therefore, we describe recent progresses in the understanding of how the TMEPAI family physiologically contributes to cellular functions and diseases.

Two Cases of Thrombocytopenia, Anasarca, Fever, Reticulin Fibrosis/Renal Failure, and Organomegaly (TAFRO) Syndrome with High Serum Procalcitonin Levels, Including the First Case Complicated with Adrenal Hemorrhaging.

Thrombocytopenia, Anasarca, Fever, Reticulin fibrosis/Renal failure, and Organomegaly (TAFRO) syndrome is a recently described systemic inflammatory disorder characterized by thrombocytopenia, anasarca, fever, reticulin fibrosis/renal failure, and organomegaly. It has an acute or subacute onset of unknown etiology, although some pathological features resemble those of multicentric Castleman disease. We here report two cases of TAFRO syndrome. The symptoms and pathological findings in these cases met the 2015 diagnostic criteria. Our cases showed high serum procalcitonin levels, suggesting bacterial infection as an onset trigger. In addition, Case 1 is the first case complicated with adrenal hemorrhaging. Case 2 is the second case of tocilizumab-resistant TAFRO syndrome successfully treated with rituximab.

TMED10 Protein Interferes with Transforming Growth Factor (TGF)-ß Signaling by Disrupting TGF-ß Receptor Complex Formation.

The intensity and duration of TGF-ß signaling determine the cellular biological response. How this is negatively regulated is not well understood. Here, we identified a novel negative regulator of TGF-ß signaling, transmembrane p24-trafficking protein 10 (TMED10). TMED10 disrupts the complex formation between TGF-ß type I (also termed ALK5) and type II receptors (TßRII). Misexpression studies revealed that TMED10 attenuated TGF-ß-mediated signaling. A 20-amino acid-long region from Thr91 to Glu110 within the extracellular region of TMED10 was found to be crucial for TMED10 interaction with both ALK5 and TßRII. Synthetic peptides corresponding to this region inhibit both TGF-ß-induced Smad2 phosphorylation and Smad-dependent transcriptional reporter activity. In a xenograft cancer model, where previously TGF-ß was shown to elicit tumor-promoting effects, gain-of-function and loss-of-function studies for TMED10 revealed a decrease and increase in the tumor size, respectively. Thus, we determined herein that TMED10 expression levels are the key determinant for efficiency of TGF-ß receptor complex formation and signaling.

A case of gradually manifesting McCune-Albright syndrome with a 10-year follow-up.

McCune-Albright syndrome (MAS) is characterized by fibrous dysplasia (FD) of bone, café-au-lait skin pigmentation, and precocious puberty. Here we report a case of a 12-year-old girl with MAS presenting sexual precocity as initial signs, followed by FD of bone with her growth. She was referred to our hospital because of breast budding and abnormal genital bleeding at the age of 2.8 years. On physical examination, her height and weight were greater than two standard deviations of the mean ranges. Hormonal analysis revealed an elevated serum estradiol and suppressed luteinizing hormone and follicle-stimulating hormone production. Her bone age had advanced, and a 16-mm monocystic lesion was observed on her right ovary by pelvic ultrasonography. Considering the clinical and paraclinical findings, precocious pseudopuberty was suspected and periodic observations were started. Her estrogen "flare up" was transient and she had repeated similar episodes three times in the following 7 years. She complained of pain in her right hip at the age of 9.6 years, which was diagnosed as FD of bone by fluorodeoxyglucose-positron emission tomography. Although no café-au-lait skin pigmentation was observed, we made a preliminary diagnosis of MAS. Because clinical evidence for MAS can appear later in the course of recurrent autonomous cysts, careful observation and periodical assessments of patients with suspected MAS is necessary.

Transforming growth factor-ß signaling enhancement by long-term exposure to hypoxia in a tumor microenvironment composed of Lewis lung carcinoma cells.

Transforming growth factor-ß (TGF-ß) is a potent growth inhibitor in normal epithelial cells. However, a number of malignant tumors produce excessive amounts of TGF-ß, which affects the tumor-associated microenvironment by furthering the progression of tumorigenicity. Although it is known that the tumor-associated microenvironment often becomes hypoxic, how hypoxia influences TGF-ß signaling in this microenvironment is unknown. We investigated whether TGF-ß signaling is influenced by long-term exposure to hypoxia in Lewis lung carcinoma (LLC) cells. When the cells were exposed to hypoxia for more than 10 days, their morphology was remarkably changed to a spindle shape, and TGF-ß-induced Smad2 phosphorylation was enhanced. Concomitantly, TGF-ß-induced transcriptional activity was augmented under hypoxia, although TGF-ß did not influence the activity of a hypoxia-responsive reporter. Consistently, hypoxia influenced the expression of several TGF-ß target genes. Interestingly, the expressions of TGF-ß type I receptor (TßRI), also termed activin receptor like kinase-5 (ALK5), and TGF-ß1 were increased under the hypoxic condition. When we monitored the hypoxia-inducible factor-1 (HIF-1) transcriptional activity by use of green fluorescent protein governed by the hypoxia-responsive element in LLC cells transplanted into mice, TGF-ß-induced Smad2 phosphorylation was upregulated in vivo. Our results demonstrate that long-term exposure to hypoxia might alter responsiveness to TGF-ß signaling and affected the malignancy of LLC cells.

The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

Myostatin, a muscle-specific transforming growth factor-ß (TGF-ß), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-ß family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-ß1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

Identification of the minimum peptide from mouse myostatin prodomain for human myostatin inhibition.

Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice.

Roles of TGF-ß family signals in the fate determination of pluripotent stem cells.

Members of the transforming growth factor-ß (TGF-ß) family have been implicated in embryogenesis as well as in the determination of the cell fates of mouse and human embryonic stem (ES) cells, which are characterized by their self-renewal and pluripotency. The cellular responses to TGF-ß family signals are divergent depending on the cellular context and local environment. TGF-ß family signals play critical roles both in the maintenance of the pluripotent state of ES cells by inducing the expression of Nanog, Oct4, and Sox2, and in their differentiation into various cell types by regulating the expression of master regulatory genes. Moreover, multiple lines of evidence have suggested the importance of TGF-ß family signals in establishing induced pluripotent stem (iPS) cells. Since ES and iPS cells have great potential for applications in regenerative medicine, it is critical to figure out the mechanisms underlying their self-renewal, pluripotency, and differentiation. Here, we discuss the roles of TGF-ß family ligands and their downstream signaling molecules, Smad proteins, in the maintenance of the pluripotency and lineage specification of mouse and human ES and iPS cells.

C18 ORF1, a novel negative regulator of transforming growth factor-ß signaling.

Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.