Negative Regulator Of TGF-β Signaling Research Articles

SET8 is a novel negative regulator of TGF-β signaling in a methylation-independent manner

Transforming growth factor β (TGF-β) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-β signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-β in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-β. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-β triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-β signaling.

Putting transcriptional brakes on fibrosis: a negative regulator of TGFβ signaling.

Due to the pleiotropic effect of transforming growth factor β (TGFβ) signaling inhibition, function-specific targeted inhibition of TGFβ signaling is required. In a recent study, Yang et al. found that Krüppel-like factor (KLF)-13 acts as a negative regulator of TGFβ. Thus, activating KLF13 in fibrotic tissues may protect them from fibrosis by decreasing TGFβ signaling.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

Hepatocyte-specific Prominin-1 protects against liver injury-induced fibrosis by stabilizing SMAD7

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFβ signaling during liver fibrosis.

The Transcriptional Cofactor Ski in Thymic Epithelial Cells Regulates Peripheral T Cell Responses

Abstract The thymus is an important site for the establishment and maintenance of an appropriate immune response through positive and negative selection of developing T cells. Cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively, interact with developing thymocytes to mediate this selection process. Developing thymocytes also support the maturation and proliferation of mTECs by secreting transforming growth factor-beta (TGF-β) superfamily cytokines. Our lab has previously shown that deletion of TGF-β signaling in TECs enhanced negative selection and functional maturation of thymocytes, and increased the production of regulatory T cells. To investigate whether the proto-oncogene product Ski (Sloan-Kettering Institute), a negative regulator of TGF-β signaling, is associated with thymocyte development in homeostatic condition and inflammatory conditions, we generated mice that deleted Ski specifically in TECs (Foxn1Cre Ski fl/fl mice). We found that the mTEC population was decreased as predicted, however we did not find any differences in thymocyte development between the Foxn1Cre Skifl/fl mice and control mice. We also found that CD4 T cells purified from Ski deleted mice showed a defect in proliferation, and Th1 and Th17 differentiation in vitro, while regulatory T cell differentiation was normal. Lastly, we found that these mice were significantly protected from developing experimental autoimmune encephalomyelitis (EAE) disease compared to the control mice. Overall, our findings suggest that Ski signaling in TECs regulates peripheral T cell self-reactivity responses and further study may provide evidence for targeting Ski in T cell driven autoimmune diseases such as multiple sclerosis. Supported by grants from NIH grant R01-AI136475 (to SZ) and AAI Intersect fellowship (to HC)

Abstract 1984: Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer

Abstract Background: Cancer stem cells that escape suppression by key pathways such as TGF-β represent one of the mechanisms for poor prognosis in hepatocellular cancer (HCC). Our mouse models with disruption of TGF-β signaling (β2SP+/-/Smad3+/-) develop HCC and phenocopy an epigenetically human cancer stem cell disorder, providing clues towards the role of TGF-β in the switch in stem cell transformation to HCC. An examination of HCC genomics revealed a surprising and strong correlation between TGF-β and Sirtuins (SIRTs) expression. SIRTs are also implicated in cancer stem cell and HCC suppression. Here we hypothesized that the TGF-β pathway harnesses Sirtuin members to maintain stem cell homeostasis, which may mediate the progression of HCC. Methods: Overall Survival (OS) analysis was carried out by using early-stage HCC (TCGA, Firehose Legacy, stage N1, n=186) from cBioportal. ChIP assay was performed in HepG2 cells using anti-Smad3 and normal rabbit IgG. Biotinylated SIRT6 promoter with/without SMAD binding sites was used as probes to pull down the DNA-interacting proteins in HepG2 cells treated with/without TGF-β for 2h. Results: Analysis of TCGA HCC dataset (cBioportal, stage N1, n=186) indicated reduced OS for HCC patients with decreased SIRT6 together with loss of SPTBN1 in the early stage (Median survival months: 40.37 vs 83.18, p=0.0417). • Chip-qPCR and the DNA-protein pulldown assay revealed that Smad3 positively upregulates the SIRT6 mRNA expression (Smad3 vs IgG, SBE1: 3.27±0.53 vs 0.99±0.01, p<0.05; SBE2: 3.32±0.74 vs 1.12±0.3, p<0.05) through direct binding to the SIRT6 promoter in HepG2 cells after TGF-β treatment. • Co-IP analysis revealed directly binding of SIRT6 with β2sp and Smad3. • Overexpression of SIRT6 represses Smad3 and β2sp expression (p<0.01) and results in inhibition of the TGF-β target genes (p<0.05) such as MMP9 (68.2% inhibited), HMGA2 (53.4% inhibited), and PAI-1 (35.4% inhibited); whereas knockdown of SIRT6 increases β2sp expression in HepG2 cells (3 fold, p<0.05). • Similar to mice with disruption of SIRT6 (SIRT6-/-) that develop liver inflammation, steatosis with progression to tumorigenesis, mice with TGF-β signaling defect (Smad3+/-Sptbn1+/-) also develop fatty liver with progression to tumorigenesis under HFD. Interestingly, we found that SIRT6 was significantly downregulated in MEF cells from mice with loss of TGF-β signaling (Smad3+/-Sptbn1+/-). Conclusions: Our findings suggest that the TGF-β signaling participates in positively regulating the expression of SIRT6. In turn, SIRT6 acts as a negative regulator of TGF-β signaling through de-acetylating Smad3 and β2sp. SIRT6 and TGF-β signaling exert integrative yet complex roles in inhibiting hepatic steatosis, inflammation, and HCC that when disrupted lead to poor prognosis in HCC. The data provide new combined driving roles for the two pathways in suppressing NASH and HCC. Citation Format: Xiyan Xiang, Sobia Zaidi, Shuyun Rao, Kazufumi Ohshiro, Zhanhuai Wang, Wilma Jogunoori, Chuxia Deng, Patricia Latham, Lopa Mishra. Crosstalk between sirtuins and TGF-β signaling suppresses liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1984.

Th17 Cells Targeting By MiR-21 Inhibitor Impairs Multiple Myeloma Related Bone Disease

Bone disease (BD) represents a hallmark of multiple myeloma (MM), characterized by classical osteolytic lesions and/or osteoporosis, that can be life threatening and contribute to poor outcomes. The complex interplay among MM conditioned bone effectors such as MM cells, osteoclasts (OCLs) and osteoblasts within BM microenvironment (BMM) supports MMBD. Several studies have shown that the extent of BD is tightly correlated to Th17 cells development and maintenance. Th17 secrete pro OCL cytokines such as RANKL, TNF, and IL-17, contributing to MMBD development. Recent findings showed that microRNAs (miRNAs) are involved in MMBD. MiRNAs are short non-coding RNAs that regulate gene expression at a post transcriptional level. Among others, miR-21 is involved in Th17 differentiation by targeting SMAD-7, a negative regulator of TGF-β signaling, promoting the activation of SMAD-2/3, suppressing IL-2 expression, and ultimately enhancing the activity of the TGF-β signaling pathway. Taken together, these data support a therapeutic strategy of miR-21 targeting to ameliorate MMBD through Th17 inhibition. We have previously demonstrated that miR-21 activity within BMM plays a crucial role in bone resorption/apposition balance through PIAS3 suppression and STAT3 dependent RANKL transcription in BM stromal cells. In the present study, we sought to evaluate the role of miR-21 in Th17 mediated BD. We enriched Th17 from BM CD4+CCR6+ T cells and evaluated miR-21 levels. We found increased miR-21 in MM derived Th17 (MMTh17) as compared to controls, supporting the relevance of miR-21 in MMTh17. To further explore the mechanisms of miR-21 activity in Th17 function, freshly isolated naïve CD4+ T cells were differentiated from healthy donors into Th17 along 18 days of culture in the presence of polarizing cytokines. At the end of culture, T cells expressed the Th17 full maturation markers RORC and IL17. As a model of MMBD in vitro, OCL resorptive activity was evaluated on dentine discs with/without Th17 freshly isolated from MM patients or differentiated in vitro. In both cases, Th17 promoted potent OCL mediated dentin pits generation as compared to controls. To counteract miR-21 activity, we electroporated naïve CD4+ T cells with miR-21 inhibitor (miR21i) or scramble control mirna before starting Th17 differentiation culture. MiR-21 expression levels were assessed by rt pcr before and at the end of differentiation culture to ascertain its effective inhibition. To map signaling pathways differences elicited by miR21i-Th17, we performed a mass spectrometry-based quantitative phosphoproteomics analysis. 1134 phosphorylated proteins were identified, with 386 significant changes as compared to controls. The global-pairwise-normalized data underwent to Ingenuity Pathway Analysis (IPA). Interestingly, analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition included PI3K/AKT and 14-3-3 Signaling. Among Th17 potential regulators, enhanced activity of PDCD4, STAT1, MINK1 and FOXO1 were observed, while ITGAL showed reduced activity. On these bases, we performed WB to validate IPA analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of RORC were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated in the presence of miR-21 antagonism. STAT3 is a master transcription factor for Th17 upon IL-6 stimulation. As PIAS3 and PDCA4 negatively regulate STAT3, we checked phospho-STAT3 expression and found it downregulated as expected. Overall, these data indicated that Th17 differentiation was impaired by miR-21i. In the second set of experiments, we aimed to evaluate Th17 functional impairment by miR-21 antagonism in MMBD. OCLs were plated with dentine discs and exposed to either Th17 alone, miR21i-Th17, or RANKL/MCSF cytokine alone as positive control. Dentine pits and areas were significantly lower in the presence of miR21i-Th17 as compared to Th17 alone and positive control (Fig.1). These data indicated that miR-21 antagonism negatively regulates Th17 mediated OCL activity and support the role of miR-21 inhibition as an innovative treatment tool in MMBD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization.

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

Dysregulated PJA1-TGF-β signaling in cancer stem cell-associated liver cancers.

The transforming growth factor beta (TGF-β) signaling pathway plays important roles in cell differentiation, stem cell modulation, organ lineage, and immune suppression. TGF-β signaling is negatively regulated by the ubiquitin–proteasome pathway. Although mouse models of cancer arising from a defective TGF-β pathway clearly demonstrate the tumor-suppressive role of TGF-β, the underlying mechanism by which a defective TGF-β pathway triggers liver cancer development is poorly understood. This review summarizes key findings from our recent studies connecting TGF-β to hepatic oncogenesis and highlights the vulnerability of TGF-β signaling to PJA1-mediated ubiquitination. TGF-β, together with the chromatin insulator CCCTC-binding factor (CTCF), epigenetically and transcriptionally regulate tumor promoter genes, including IGF2 and TERT, in TGF-β–defective mice and in human liver cancers. Dysfunction of the TGF-β–regulated SPTBN1/SMAD3/CTCF complex increases stem cell–like properties in hepatocellular carcinoma (HCC) cells and enhances tumorigenesis in tumor-initiating cells in a mouse model. PJA1, a novel E3 ubiquitin ligase, is a key negative regulator of TGF-β signaling. PJA1 overexpression is detected in HCCs and is sufficient to suppress SMAD3- and SPTBN1-mediated TGF-β tumor suppressor signaling, promoting HCC proliferation. Dysregulated PJA1-TGF-β signaling activates oncogenic genes and promotes tumorigenesis in human liver cancers. In addition, inhibition of PJA1 by treatment with E3 ligase inhibitors restores TGF-β tumor-suppressor function and suppresses liver cancer progression. These new findings suggest potential therapeutic avenues for targeting dysregulated PJA1-TGF-β signaling via cancer stem cells in liver cancers.

Corrigendum] Ski prevents TGF‑β‑induced EMT and cell invasion by repressing SMAD‑dependent signaling in non‑small cell lung cancer.

Following the publication of the above paper, an interested reader drew to our attention apparent anomalies associated with Figs.2, 3 and 4; essentially, these three figures contained panels exhibiting overlapping data, such that data purportedly relating to different experiments were apparently drawn from the same original sources. [Specifically, the Ski, +TGF‑β1 data panel in Fig.2B, the Mock, +TGF‑β1 data panel in Fig.3A, and the +TGF‑β1, +SIS3 data panel in Fig.4B in the original figures were chosen incorrectly.] Upon investigating this matter with the authors, the authors have realized that they made errors in the compilation of the affected figures. The errors were made inadvertently, and the authors have been able to identify the correct data for each of the figures concerned. The corrected versions of these figures are shown opposite and on the next page. Note that these errors did not affect the overall conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum; furthermore, the authors apologize for any inconvenience caused to the readership of the Journal. [the original article was published in Oncology Reports 34: 87-94, 2015; DOI: 10.3892/or.2015.3961].

The Post-translational Modifications of Smurf2 in TGF-β Signaling.

Smad ubiquitin regulatory factor 2 (Smurf2), an essential negative regulator of TGF-β signaling, ubiquitinates TGF-β receptors (TβRs) and Smad proteins, inducing their proteasomal degradation. Smurf2 plays crucial roles in regulating TGF-β signaling and maintaining normal cellular functions and tissue homeostasis; dysfunction of Smurf2 triggers abnormal TGF-β signaling in pathological states. Smurf2 has been reported as a potentially strong candidate for targeting therapies for related diseases. Recent work has begun to focus on the regulation of Smurf2 itself, and emerging evidence indicates that Smurf2 is regulated by post-translational modifications (PTMs) mechanisms. These mechanisms predominantly regulate the expression level and E3 ligase activity of Smurf2, strongly suggesting that this protein contributes to complicated roles under multiple pathophysiological conditions. In this review, we cover some significant and novel mechanisms of the PTMs that potentially control Smurf2 participation in TGF-β signaling, including ubiquitylation, SUMOylation, neddylation, phosphorylation, and methylation in order to provide a broad view of the depth and sophistication of Smurf2 function in TGF-β regulation, as well as perspectives for future therapeutic directions for its associated diseases.

A schistosome miRNA promotes host hepatic fibrosis by targeting transforming growth factor beta receptor III

MicroRNAs (MiRNAs) derived from parasites, and even from plants, have been detected in body fluids and are known to modulate host genes. In this study, we aimed to investigate if the schistosome miRNAs are involved in the occurrence and progression of hepatic fibrosis during Schistosoma japonicum (S.japonicum) infection. The presence of miRNAs from S.japonicum (sja-miRNAs) in hepatic stellate cells (HSCs) was detected by RNA sequencing. sja-miRNAs were screened by transfecting HSCs with sja-miRNA mimics. The role of sja-miR-2162 in hepatic fibrosis was evaluated by either elevating its expression in naïve mice or by inhibiting its activity in infected mice, through administration of recombinant adeno-associated virus serotype 8 vectors expressing sja-miR-2162 or miRNA sponges, respectively. We identified a miRNA of S.japonicum, sja-miR-2162, that was consistently present in the HSCs of infected mice. Transfection of sja-miR-2162 mimics led to activation of HSC cells invitro, characterized by elevation of collagens and α-SMA. The rAAV8-mediated delivery of sja-miR-2162 to naïve mice induced hepatic fibrosis, while sustained inhibition of sja-miR-2162 in infected mice attenuated hepatic fibrosis. The transforming growth factor beta receptor III (TGFBR3), a negative regulator of TGF-β signaling, was a direct target of sja-miR-2162 in HSCs. This study demonstrated that pathogen-derived miRNAs directly promote hepatic fibrogenesis in a cross-species manner, and their efficient and sustained inhibition might present a promising therapeutic intervention for infectious diseases. A schistosome-specific microRNA, sja-miR-2162, is consistently present in the hepatic stellate cells of mice infected with S.japonicum, where it promotes hepatic fibrosis in the host through cross-species regulation of host fibrosis-related genes. The efficient and sustained inhibition of pathogen-derived micRNAs may represent a novel therapeutic intervention for infectious diseases.

Smad7 Controls Immunoregulatory PDL2/1-PD1 Signaling in Intestinal Inflammation and Autoimmunity

SUMMARYSmad7, a negative regulator of TGF-β signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells. Smad7 deficiency in DCs promotes TGF-β responsiveness and the coinhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs. In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro. The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-β and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention.

Ablation of SUN2-containing LINC complexes drives cardiac hypertrophy without interstitial fibrosis.

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear–­cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but a full understanding of disease etiology in these patients is lacking. Here we show that SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from profibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGFβ signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGFβ signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of profibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.

Abstract 4431: Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy

Abstract Th17 cells play a critical role in Multiple Myeloma (MM) microenvironment by triggering osteoclastogenic activity, that leads to severe skeleton damage. We have recently found that Th17 cells directly promote osteoclast (OCL) differentiation and function via expression of RANKL. It has been demonstrated that miRNAs are involved in the differentiation of Th17 cells. Murugaiyan et al. demonstrated that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Indeed, we have been able to negatively modulate Th17 differentiation by a specific inhibitor of miR-21 (mir21i). The overall effect of mir-21 inhibition is the suppression of OCL resorptive activity, thus attenuating Th17 mediated multiple myeloma bone disease (MMBD). However, the molecular mechanisms underlying these biological effects have not been clarified. On these bases, we aimed to identify proteins involved in the effect of miR21i on Th17 cells. Quantitative phosphoproteomics technologies were applied to study the effects of miR21i in Th17 cells. To unveil the molecular targets responsible for this interaction, Ingenuity's IPA® software was interrogated. This approach allowed to identify several unknown miR-21 target proteins and the related signal transduction pathway.To perfom global phosphoproteome analysis, we performed a mass spectrometry study of phosphopeptides on protein extracts from miR21i- treated Th17 and control Th17 cells, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software. Among analyses of more than 1134 phosphorylation sites, 386 have shown significant changes, as compared to controls. Interestingly, IPA analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition including PI3K/AKT, 14-3-3 and GRANZYME A signaling. Moreover IPA analysis disclosed RANKL among the top upstream proteins. The most up regulated protein was CYTIP and the ones with enhanced activity were PDCD4, STAT1, MINK1, FOXO1, while ITGAL showed reduced activity. To validate these data, we performed WB analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of the master Th17 transcription factor, RORC, were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated by mir21i. As PIAS3 and PDCD4 negatively regulate STAT3, we checked phospho-STAT3 expression and found that it was indeed downregulated, as expected. Overall, this study defines a phosphoproteomic signature of miR-21i treated Th17 cells, providing new insights into molecular impairment of Th17 differentiation by miR21i and suggest innovative molecular targeted approaches to treat MMBD Citation Format: Emanuela Altomare, Marco Rossi, Daniele Caracciolo, Nicola Amodio, Marco Gaspari, Domenico Taverna, Domenica Scumaci, Cirino Botta, Annamaria Gullà, Eugenio Morelli, Paola Critelli, Mariamena Arbitrio, Pierosandro Tagliaferri, Pierfrancesco Tassone. Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4431.

MicroRNA-181c promotes Th17 cell differentiation and mediates experimental autoimmune encephalomyelitis

Among T helper (Th) cell subsets differentiated from naive CD4+ T cells, IL-17-producing Th17 cells are closely associated with the pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and the MS animal model, experimental autoimmune encephalomyelitis (EAE). The modulation of Th17 differentiation offers a potential avenue for treatment. Although a series of microRNAs (miRNAs) that modulate autoimmune disease development have been reported, further studies on miRNA roles in Th17 differentiation and MS pathogenesis are still warranted. Here, we demonstrated that mice with miR-181c knockdown presented with delayed EAE and slowed disease progression, along with a decreased Th17 cell population. We also found that miR-181c was a Th17 cell-associated miRNA and that Smad7, a negative regulator of TGF-β signaling, was a potential target of miR-181c. miR-181c knockdown rendered T cells less sensitive to TGF-β-induced Smad2/3, enhancing the expression of IL-2 which has been reported to inhibit Th17 cell differentiation. Moreover, through the analysis of published miRNA expression profiles from the Gene Expression Omnibus database, increased miR-181c levels were found in peripheral blood from MS patients. Our results identified a novel miRNA that promotes Th17 cell differentiation and autoimmunity, thus miR-181c may serve as a potential treatment target in patients with MS.

Identification and validation a TGF-\u03b2-associated long non-coding RNA of head and neck squamous cell carcinoma by bioinformatics method

BackgroundThe role of transforming growth factorβ (TGF-β)-induced tumor progression in advanced malignancy is well established, but the involvement of long non-coding RNAs (lncRNAs) in TGF-β signaling remains unclear. This study aimed to identify TGF-β-associated lncRNAs in head and neck squamous cell carcinoma (HNSCC).MethodsExpression profiling of lncRNAs was obtained using Gene Expression Omnibus and The Cancer Genome Atlas. Real-time quantitative PCR was used to analyze the expression of EPB41L4A-AS2 in HNSCC cell line. We used bioinformatics resources (DAvID) to conduct Gene Ontology biological processes and KEGG pathways at the significant level. Wound healing assay, cell migration and invasion assays, were used to examine the effects of EPB41L4A-AS2 on tumor cell metastasis in vivo. Protein levels of EPB41L4A-AS2 targets were determined by western blot.ResultsA novel TGF-β-associated lncRNA, EPB41L4A-AS2, was found downregulated by TGF-β and associated with invasion and metastasis. The relationship of EPB41L4A-AS2 with the clinicopathological features and prognosis of HNSCC patients was evaluated. Bioinformatic analyses revealed that EPB41L4A-AS2 may be involved in processes associated with the tumor-associated signaling pathway, especially the TGF-β signaling pathway. Furthermore, a TGF-β-induced epithelial-to-mesenchymal transition (EMT) model was established. Low EPB41L4A-AS2 expression was determined, and overexpression of this gene inhibited cell migration and invasion in the EMT model. Moreover, EPB41L4A-AS2 suppressed TGFBR1 expression.ConclusionsEPB41L4A-AS2 might serve as a negative regulator of TGF-β signaling and as an effective prognostic biomarker and important target in anti-metastasis therapies of HNSCC patients.

Lung epithelial-specific TRIP-1 overexpression maintains epithelial integrity during hyperoxia exposure.

The onset and degree of injury occurring in animals that develop hyperoxic acute lung injury (HALI) is dependent on age at exposure, suggesting that developmentally regulated pathways/factors must underlie initiation of the epithelial injury and subsequent repair. Type II TGFβ receptor interacting protein‐1 (TRIP‐1) is a negative regulator of TGFβ signaling, which we have previously shown is a developmentally regulated protein with modulatory effects on epithelial‐fibroblastic signaling. The aim of this study was to assess if type II alveolar epithelial cells overexpressing TRIP‐1 are protected against hyperoxia‐induced epithelial injury, and in turn HALI. Rat lung epithelial cells (RLE) overexpressing TRIP‐1 or LacZ were exposed to 85% oxygen for 4 days. A surfactant protein C (SPC)‐driven TRIP‐1 overexpression mouse (TRIP‐1AECTg+) was generated and exposed to hyperoxia (>95% for 4 days) at 4 weeks of age to assess the effects TRIP‐1 overexpression has on HALI. RLE overexpressing TRIP‐1 resisted hyperoxia‐induced apoptosis. Mice overexpressing TRIP‐1 in their lung type II alveolar epithelial cells (TRIP‐1AECTg+) showed normal lung development, increased phospho‐AKT level and E‐cadherin, along with resistance to HALI, as evidence by less TGFβ activation, apoptosis, alveolar macrophage influx, KC expression. Taken together, these findings point to existence of a TRIP‐1 mediated molecular pathway affording protection against epithelial/acute lung injury.

Vasorin: a newly identified regulator of ovarian folliculogenesis.

Members of the TGF-β superfamily take part in the control of folliculogenesis. Vasorin (Vasn) is a newly identified negative regulator of TGF-β signaling whose possible involvement in ovarian physiology has never been studied. Here, we demonstrate that Vasn is expressed in the ovary by somatic cells of follicles, and that its expression is up-regulated by LH. We established a conditional knockout (cKO) mouse model in which Vasn is deleted specifically in granulosa cells of growing follicles from the secondary stage onwards. Using this model, we show that, upon hormonal stimulation, follicle ovulation size is almost 2-fold higher. This enhanced ovulatory response is associated with overactivation of the TGF-β signaling pathway and a lower number of atretic antral follicles. Of importance, we demonstrate that the number of primordial follicles is reduced in prepubertal cKO mouse ovaries, which suggests that the production of VASN by growing follicles protects the ovarian reserve. Finally, analysis of systemic KO mice revealed that the ovarian reserve is almost 2.5-fold higher, which implies that Vasn may also play a role in primordial follicle formation. Overall, our findings reveal that Vasn is a new regulator that exerts an effect on several key ovarian functions, including folliculogenesis, maintenance of the ovarian reserve, and ovulation.-Rimon-Dahari, N., Heinemann-Yerushalmi, L., Hadas, R., Kalich-Philosoph, L., Ketter, D., Nevo, N., Galiani, D., Dekel, N. Vasorin: a newly identified regulator of ovarian folliculogenesis.

MiR-146a-5p acts as a negative regulator of TGF-β signaling in skeletal muscle after acute contusion.

Growing evidence suggests the importance of microRNAs (miRNAs) in stress signaling pathways. Transforming growth factor-β (TGF-β) is a potent cytokine that promotes the development of skeletal muscle fibrosis after acute contusion. However, how miRNAs are involved in TGF-β signaling and confer the robustness of TGF-β-induced fibrotic response remains to be fully elucidated. Here, we demonstrated that miR-146a-5p (miR-146) levels were reduced in a fibrotic mouse model after acute muscle contusion. It was also found that TGF-β treatment decreased the expression of miR-146 in vitro in a dose- and time-dependent manner. In addition, overexpression of Smad3 and Samd4, two key players in TGF-β signaling, suppressed the expression of miR-146 in muscle cells. Overexpression of miR-146 inhibited the expressions of fibrosis markers both in vitro and in vivo. Moreover, increase in the expression of miR-146 in muscle cells was able to attenuate the effect of TGF-β on the expressions of fibrosis markers. Mechanistic analysis revealed that Smad4 is a direct target of miR-146 in muscle cells. Furthermore, the anti-fibrotic effect of miR-146 could be blocked by overexpression of Smad4 in vivo. These results suggest that Smad4 is down-regulated by miR-146 in skeletal muscle. Taken together, our results indicate that the anti-fibrotic miR-146 is a component of TGF-β signaling. It is down-regulated by Smad protein, and can inhibit the expression of Smad4. Our study suggests that miR-146 might have a therapeutic potential to reduce skeletal muscle fibrosis after injury.