Smad Transcription Factors Research Articles

Transcriptional regulators ensuring specific gene expression and decision-making at high TGFβ doses.

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.

TMIC-43. BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF NOVEL GLIOBLASTOMA TME SUBTYPES USING TRANSCRIPTOMIC ANALYSIS AND NETWORK MODELLING TO IDENTIFY NOVEL TARGETS OF VULNERABILITY

Abstract New precision medicine therapies are urgently required for glioblastoma (GBM). Recently, we developed a novel GBM classification system, identifying three patient clusters uniquely characterized by tumor microenvironment (TME) composition: TMELow, TMEMedium, and TMEHigh1. Our objective now is to further investigate these subtypes employing transcriptomic analysis and network modelling approaches to identify novel subtype-specific targets of vulnerability. We analyzed transcriptomic data from >600 GBM samples from publicly available and in-house datasets. All samples underwent TME subtyping. Next, we performed differential gene expression (using DESeq2, edgeR) and pathway analysis (using PROGENy). The ‘TRANSFAC’ tool 2 was used to identify potential transcription factors enriched in each TME subtype. TMELow tumours manifested highly upregulated NLGN3 (P=5.777e-5) and HES5 (P=2.233e-3) expression compared to TMEHigh. Moreover, TMEHigh compared to TMELow tumours were enriched for genes associated with tissue remodelling, and showed elevated MMP7 (P=3.051e-6), CLCL13 (P= 3.843e-4), and CXCL5 expression (P= 1.592e-6). PROGENy analysis suggested that the WNT, TRAIL, and JAK/STAT pathways were significantly enriched in TMEHigh, while the PI3K pathway was significantly upregulated in TMEMedium. Additionally, VEGF pathway activity was significantly upregulated in TMELow. TRANSFAC analysis measured by regulatory score (a measure of a transcription factor effect on gene expression) revealed that in TMELow, key transcription factors includes ETS2, NANOG, and MECP2 (regulatory score: 1.97, 1.45, and 1.4, respectively). In TMEMedium, transcription factors HIF1A, PPARA, and CDX2 were identified (regulatory score: 2.52, 2.08 and 1.96, respectively). In TMEHigh, TRANSFAC indicated activiation of transcription factors SMAD3, ETS2, and NFKB1 regulatory score: 2.56, 2.41, 2.24, respectively). Overall these findings highlight distinct molecular signatures across TME subtypes, and provide a deeper understanding of associated molecular mechanisms. Ongoing work is focused on validating key subtype specific genes and associated pathways using spatial proteomics. Prioritised targets will ultimately be interrogated in vivo, as novel subtype-specific treatments. 1. White et al. Ann Oncol. 2023;34(3):300–314. 2. Matys et al. Nucleic Acids Res. 2003;31.

Smad transcription factors as mediators of 7 transmembrane G protein-coupled receptor signalling.

The Smad transcription factors are well known for their role at the core of transforming growth factor-β (TGF-β) signalling. However, recent evidence shows that the Smad transcription factors play a vital role downstream of other classes of receptors including G protein-coupled receptors (GPCR). The versatility of Smad transcription factors originated from the two regions that can be differently activated by the TGF-β receptor superfamily or through the recruitment of intracellular kinases stimulated by other receptors classes such as GPCRs. The classic GPCR signalling cascade is further expanded to conditional adoption of the Smad transcription factor under the stimulation of Akt, demonstrating the unique involvement of the Smad transcription factor in GPCR signalling pathways in disease environments. In this review, we provide a summary of the signalling pathways of the Smad transcription factors as an important downstream mediator of GPCRs, presenting exciting opportunities for discovering new therapeutic targets for diseases.

MIKC*-type MADS transcription factors control JINGUBANG expression and the degree of pollen dormancy in Arabidopsis.

While pollen dormancy has been proposed to play a necessary role in sexual reproduction, it remains poorly understood. Here, we used traditional pollen germination assays to characterize dormancy. Our results underscore variation in the degree of dormancy between individual pollen grains. In addition, we provide evidence that JINGUBANG (JGB), previously defined as a negative regulator of pollen germination in Arabidopsis (Arabidopsis thaliana), is responsible for the uneven degrees of pollen dormancy, as asynchronous pollen germination in vitro reflected varied expression levels of JGB. We identified five cis-acting elements, including four CArG-boxes and the previously uncharacterized element ERE7, as essential for the initiation and enhancement of JGB expression. A 10-bp sequence between CArG-box 3 and ERE7, likely the result of an inverse DNA loop formed between CArG-box 3 and CArG-box 4, was required for robust gene expression. In addition, the pollen-specific AtMIKC*-type MADS transcription factors AGAMOUS-LIKE 30 (AGL30), AGL65, AGL66, AGL94, and AGL104 activated JGB transcription. Notably, the transactivation levels differed among the obligate AtMIKC* heterodimers tested. Our results indicate that distinct AtMIKC* complexes formed in individual pollen grains direct pollen dormancy to uneven degrees, which is likely an adaptive trait that ensures broader pollen dispersal under adverse environmental conditions.

VCAM-1 targeted nanocarriers of shRNA-Smad3 mitigate endothelial-to-mesenchymal transition triggered by high glucose concentrations and osteogenic factors in valvular endothelial cells

Endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VEC) is a key process in the development and progression of calcific aortic valve disease (CAVD). High expression of the Smad3 transcription factor is crucial in the transition process. We hypothesize that silencing Smad3 could hinder EndMT and provide a novel treatment for CAVD. We aimed at developing nanoparticles encapsulating short-hairpin (sh)RNA sequences specific for Smad3 targeted to the aortic valve. We synthesized VCAM-1-targeted lipopolyplexes encapsulating shRNA-Smad3 plasmid (V-LPP/shSmad3) and investigated their potential to reduce the EndMT of human VEC. VEC incubation in a medium containing high glucose concentrations and osteogenic factors (HGOM) triggers EndMT and increased expression of Smad3. Exposed to lipopolyplexes, VEC took up efficiently the V-LPP/shSmad3. The latter reduced the EndMT process in VEC exposed to HGOM by downregulating the expression of αSMA and S100A4 mesenchymal markers and increasing the expression of the CD31 endothelial marker. In vivo, V-LPP/shSmad3 accumulated in the aortic root and aorta of a murine model of atherosclerosis complicated with diabetes, without affecting the liver and kidney function. The results suggest that targeting activated VEC with lipopolyplexes to silence Smad3 could be an effective, novel treatment for CAVD mediated by the EndMT process.

Cyclophilin D, regulator of the mitochondrial permeability transition, impacts bone development and fracture repair

Mitochondrial Permeability Transition Pore (MPTP) and its key positive regulator, Cyclophilin D (CypD), control activity of cell oxidative metabolism important for differentiation of stem cells of various lineages including osteogenic lineage. Our previous work (Sautchuk et al., 2022) showed that CypD gene, Ppif, is transcriptionally repressed during osteogenic differentiation by regulatory Smad transcription factors in BMP canonical pathway, a major driver of osteoblast (OB) differentiation. Such a repression favors closure of the MPTP, priming OBs to higher usage of mitochondrial oxidative metabolism. The physiological role of CypD/MPTP regulation was demonstrated by its inverse correlation with BMP signaling in aging and bone fracture healing in addition to the negative effect of CypD gain-of-function (GOF) on bone maintenance. Here we show evidence that CypD GOF also negatively affects bone development and growth as well as fracture healing in adult mice. Developing craniofacial and long bones presented with delayed ossification and decreased growth rate, respectively, whereas in fracture, bony callus volume was diminished. Given that Genome Wide Association Studies showed that PPIF locus is associated with both body height and bone mineral density, our new data provide functional evidence for the role of PPIF gene product, CypD, and thus MPTP in bone growth and repair.

Trait association and prediction through integrative k-meranalysis.

Genome-wide association study (GWAS) with single nucleotide polymorphisms (SNPs) has been widely used to explore genetic controls of phenotypic traits. Alternatively, GWAS can use counts of substrings of length k from longer sequencing reads, k-mers, as genotyping data. Using maize cob and kernel color traits, we demonstrated that k-mer GWAS can effectively identify associated k-mers. Co-expression analysis of kernel color k-mers and genes directly found k-mers from known causal genes. Analyzing complex traits of kernel oil and leaf angle resulted in k-mers from both known and candidate genes. A gene encoding a MADS transcription factor was functionally validated by showing that ectopic expression of the gene led to less upright leaves. Evolution analysis revealed most k-mers positively correlated with kernel oil were strongly selected against in maize populations, while most k-mers for upright leaf angle were positively selected. In addition, genomic prediction of kernel oil, leaf angle, and flowering time using k-mer data resulted in a similarly high prediction accuracy to the standard SNP-based method. Collectively, we showed k-mer GWAS is a powerful approach for identifying trait-associated genetic elements. Further, our results demonstrated the bridging role of k-mers for data integration and functional gene discovery.

Development of an automated high-content immunofluorescence assay of pSmads quantification: Proof-of-concept with drugs inhibiting the BMP/TGF-β pathways.

Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-β) are members of the TGF-β superfamily, known for their roles in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-β receptors, respectively, which induces the phosphorylation of Smad (pSmad) transcription factors. This pathway is generally studied with Western blot and luciferase bioluminescence assay, which presents some limitations. In this work, we developed and optimized a high-throughput assay to study pSmad pathways using immunofluorescence (IF) as an alternative to Western blot. We aimed to overcome the technical challenges usually faced in the classical IF assay in image acquisition, analysis, and quantification. We used C2C12 cells as a cellular model. The cells were stimulated with BMP-2 and TGF-β1 that were delivered either in solution (soluble) or via a biomaterial presenting the growth factor (GF), that is in a "matrix-bound" manner. Image acquisition parameters, analysis methods, and quantification of pSmads using IF were optimized for cells cultured on two types of supports: on bare glass and on a biomimetic coating made by self-assembly of the biopolymers hyaluronic acid and poly(l-lysine), which was crosslinked and then loaded with the GFs. We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-β1. The detection limit of the IF-based assay was found to be similar to Western blot. Additionally, we provide a proof-of-concept for drug testing using inhibitors of BMP and TGF-β receptors, under conditions where specific signaling pathways are engaged via the ligand/receptor interactions. Altogether, our findings offer perspectives for future mechanistic studies on cell signaling and for studies at the single cell level using imaging methods.

Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- β.

The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-β was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl2 and TGF-β was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (-447/ + 657), P2 (-103/ + 657), and P3 (-267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-β is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-β, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (-80/-52) and SMAD (+163/+194) regions with EMSA.

Analysis of the DNA-binding properties of TGF-β-activated Smad complexes unveils a possible molecular basis for cellular context-dependent signaling.

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that modulates a wide variety of cellular responses by regulating target gene expression. It principally transmits signals via receptor-activated transcription factors Smad2 and Smad3, which form trimeric complexes with Smad4 upon activation and regulate gene expression by binding to genomic DNA. Here, we examined the mechanisms by which TGF-β regulates the transcription of target genes in a cell context-dependent manner by screening a double-stranded DNA oligonucleotide library for DNA sequences bound to endogenous activated Smad complexes. Screening was performed by cyclic amplification of selected targets (CASTing) using an anti-Smad2/3 antibody and nuclear extracts isolated from three cell lines (A549, HepG2, and HaCaT) stimulated with TGF-β. The preference of the activated Smad complexes for conventional Smad-binding motifs such as Smad-binding element (SBE) and CAGA motifs was different in HepG2 than in the other two cell lines, which may indicate the distinct composition of the activated Smad complexes. Several transcription factor-binding motifs other than SBE or CAGA, including the Fos/Jun-binding motifs, were detected in the enriched sequences. Reporter assays using sequences containing these transcription factor-binding motifs together with Smad-binding motifs indicated that some of the motifs may be involved in cell type-dependent transcriptional activation by TGF-β. The results suggest that the CASTing method is useful for elucidating the molecular basis of context-dependent Smad signaling.

Transsynaptic BMP Signaling Regulates Fine-Scale Topography between Adjacent Sensory Neurons.

Sensory axons projecting to the central nervous system are organized into topographic maps that represent the locations of sensory stimuli. In some sensory systems, even adjacent sensory axons are arranged topographically, forming "fine-scale" topographic maps. Although several broad molecular gradients are known to instruct coarse topography, we know little about the molecular signaling that regulates fine-scale topography at the level of two adjacent axons. Here, we provide evidence that transsynaptic bone morphogenetic protein (BMP) signaling mediates local interneuronal communication to regulate fine-scale topography in the nociceptive system of Drosophila larvae. We first show that the topographic separation of the axon terminals of adjacent nociceptors requires their common postsynaptic target, the A08n neurons. This phenotype is recapitulated by knockdown of the BMP ligand, Decapentaplegic (Dpp), in these neurons. In addition, removing the Type 2 BMP receptors or their effector (Mad transcription factor) in single nociceptors impairs the fine-scale topography, suggesting the contribution of BMP signaling originated from A08n. This signaling is likely mediated by phospho-Mad in the presynaptic terminals of nociceptors to ensure local interneuronal communication. Finally, reducing Dpp levels in A08n reduces the nociceptor-A08n synaptic contacts. Our data support that transsynaptic BMP signaling establishes the fine-scale topography by facilitating the formation of topographically correct synapses. Local BMP signaling for synapse formation may be a developmental strategy that independently regulates neighboring axon terminals for fine-scale topography.

Smad4 and FoxH1 potentially interact to regulate cyp19a1a promoter in the ovary of ricefield eel (Monopterus albus)

BackgroundCyp19a1a is a key enzyme in the pathway that converts androgens into estrogen and is regulated by TGF-β signaling. Smad4 and FoxH1 are downstream effectors of TGF-β signaling and may play important roles in ovarian development in M. albus.MethodsWe investigated the expression pattern of the Smad4 and FoxH1 using qRT‒PCR and immunofluorescence, then tested the changes of smad4 and foxh1 by qRT‒PCR after ovary incubation with FSH in vitro, and analysed the regulation of cyp19a1a transcription by Smad4 and FoxH1 by dual-luciferase reporter assays.ResultsWe found that Smad4 encoded a putative protein of 449 amino acids and harbored the three conserved domains typical of this protein family. Smad4 and foxh1 exhibited similar expression patterns during ovarian development and after FSH incubation, with Pearson’s coefficients of 0.873 and 0.63–0.81, respectively. Furthermore, Smad4, FoxH1 and Cyp19a1a colocalized in the granulosa cells and theca cells of ovaries during the mid-to-late vitellogenic stage. Smad4 repressed cyp19a1a activity via SBE1 (− 1372/−1364) and SBE2 (− 415/−407) in the cyp19a1a promoter, whereas mutating SBE1 or SBE2 restored cyp19a1a promoter activity. Co-overexpression of Smad4 and FoxH1 significantly reduced cyp19a1a promoter activity.ConclusionsThis study provides new insights into the potential functions of transcription factors Smad4 and FoxH1 in ovarian development and the transcriptional regulation mechanism of cyp19a1a in M. albus, which will reveal Smad4/FoxH1-mediated TGF-β signaling in reproduction and the regulation of the cyp19a1a.Plain English summaryAromatase, encoded by cyp19a1a, is involved in ovarian development and plays an important role in the quality of eggs, as well the sex ratio, of the teleost fish, M. albus. The research on the transcriptional regulation of cyp19a1a has contributed to the understanding of its role in ovarian development. In previous study, it was shown that FoxH1 inhibits cyp19a1a transcription. In the present study, Smad4 was confirmed as a cyp19a1a transcriptional repressor and Smad4 may also coordinate with FoxH1 to repress cyp19a1a transcription. At present, we provide a new perspective for the transcriptional regulation of cyp19a1a by transcription factors Smad4 and FoxH1 in teleost fish ovary. In the future, the regulatory networks of Smad4 and FoxH1 will be further studied and the gene editing technology will be applied to screen specific regulatory factors of cyp191a1a gene, so as to alter the female cycle and modulate the sex ratio of the eggs production.

C-phycocyanin reinforces autophagy to block pulmonary fibrogenesis by inhibiting lncIAPF biogenesis

Pulmonary fibrosis is a chronic and irreversible progressive lung disease caused by various factors, such as age and environmental pollution. With countries stepping into an aging society and the seriousness of environmental pollution caused by global industrialization, the incidence of pulmonary fibrosis is annually increasing. However, no effective drug is available for pulmonary fibrosis treatment. C-phycocyanin (C-PC), extracted from blue-green algae, has good water solubility and antioxidation. This study elucidated that C-PC reinforces autophagy to block pulmonary fibrogenesis by inhibiting long noncoding RNA (lncRNA) biogenesis in vivo and in vitro. Cleavage under targets and release using nuclease (CUT & RUN)-PCR, co-immunoprecipitation (Co-IP), and nuclear–cytoplasmic separation experiments clarified that C-PC blocked the nuclear translocation of activating transcription factor 3 (ATF3) to prevent the binding between ATF3 and transcription factor Smad3, thereby hindering lncIAPF transcription. Human antigen R (HuR) truncation experiment and RNA binding protein immunoprecipitation (RIP) were then performed to identify the binding domain with lncIAPF in the 244–322 aa of HuR. lncIAPF exerted its profibrogenic function through the binding protein HuR, a negative regulator of autophagy. In summary, C-PC promoted autophagy via down-regulating the lncIAPF–HuR-mediated signal pathway to alleviate pulmonary fibrosis, showing its potential as a drug for treating pulmonary fibrosis. Exploring how C-PC interacts with biological molecules will help us understand the mechanism of this drug and provide valuable target genes to design new drugs.

FRMD6 determines the cell fate towards senescence: involvement of the Hippo-YAP-CCN3 axis

Cellular senescence, a hallmark of aging, is pathogenically linked to the development of aging-related diseases. This study demonstrates that FRMD6, an upstream component of the Hippo/YAP signaling cascade, is a key regulator of senescence. Proteomic analysis revealed that FRMD6 is upregulated in senescent IMR90 fibroblasts under various senescence-inducing conditions. Silencing FRMD6 mitigated the senescence of IMR90 cells, suggesting its requirement in senescence. Conversely, the overexpression of FRMD6 alone induced senescence in cells and in lung tissue, establishing a causal link. The elevated FRMD6 levels correlated well with increased levels of the inhibitory phosphorylated YAP/TAZ. We identified cellular communication network factor 3 (CCN3), a key component of the senescence-associated secretory phenotype regulated by YAP, whose administration attenuated FRMD6-induced senescence in a dose-dependent manner. Mechanistically, FRMD6 interacted with and activated MST kinase, which led to YAP/TAZ inactivation. The expression of FRMD6 was regulated by the p53 and SMAD transcription factors in senescent cells. Accordingly, the expression of FRMD6 was upregulated by TGF-β treatment that activates those transcription factors. In TGF-β-treated IMR90 cells, FRMD6 mainly segregated with p21, a senescence marker, but rarely segregated with α-SMA, a myofibroblast marker, which suggests that FRMD6 has a role in directing cells towards senescence. Similarly, in TGF-β-enriched environments, such as fibroblastic foci (FF) from patients with idiopathic pulmonary fibrosis, FRMD6 co-localized with p16 in FF lining cells, while it was rarely detected in α-SMA-positive myofibroblasts that are abundant in FF. In sum, this study identifies FRMD6 as a novel regulator of senescence and elucidates the contribution of the FRMD6-Hippo/YAP-CCN3 axis to senescence.

Genome-wide analysis of Smad and Schnurri transcription factors in C. elegans demonstrates widespread interaction and a function in collagen secretion.

Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the Transforming Growth Factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. A functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.

Critical role of Gα12 and Gα13 proteins in TGF-β-induced myofibroblast differentiation.

Myofibroblast differentiation, characterized by accumulation of cytoskeletal and extracellular matrix proteins by fibroblasts, is a key process in wound healing and pathogenesis of tissue fibrosis. Transforming growth factor-β (TGF-β) is the most powerful known driver of myofibroblast differentiation. TGF-β signals through transmembrane receptor serine/threonine kinases that phosphorylate Smad transcription factors (Smad2/3) leading to activation of transcription of target genes. Heterotrimeric G proteins mediate a distinct signaling from seven-transmembrane G protein coupled receptors, not commonly linked to Smad activation. We asked if G protein signaling plays any role in TGF-β-induced myofibroblast differentiation, using primary cultured human lung fibroblasts. Activation of Gαs by cholera toxin blocked TGF-β-induced myofibroblast differentiation without affecting Smad2/3 phosphorylation. Inhibition of Gαi by pertussis toxin, or siRNA-mediated combined knockdown of Gαq and Gα11 had no significant effect on TGF-β-induced myofibroblast differentiation. A combined knockdown of Gα12 and Gα13 resulted in a drastic inhibition of TGF-β-stimulated expression of myofibroblast marker proteins (collagen-1, fibronectin, smooth-muscle α-actin), with siGα12 being significantly more potent than siGα13. Mechanistically, a combined knockdown of Gα12 and Gα13 resulted in a substantially reduced phosphorylation of Smad2 and Smad3 in response to TGF-β, which was accompanied by a significant decrease in the expression of TGFβ receptors (TGFBR1, TGFBR2) and of Smad3 under siGα12/13 conditions. In conclusion, our study uncovers a novel role of Gα12/13 proteins in the control of TGF-β signaling and myofibroblast differentiation.

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development.

MADS transcription factors are master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily of MADS transcription factors is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. SEPALLATAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro. To date, the MADS complexes characterized in angiosperm floral organ development contain at least 1 SEPALLATA protein. Whether DNA binding by SEPALLATA-containing dimeric MADS complexes is sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization-impaired SEPALLATA mutant proteins. Here, we used a combination of genome-wide-binding studies, high-resolution structural studies of the SEP3/AGAMOUS (AG) tetramerization domain, structure-based mutagenesis and complementation experiments in Arabidopsis (Arabidopsis thaliana) sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers can bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild-type-binding sites genome-wide, tetramerization is required not only for floral meristem determinacy but also for floral organ identity in the second, third, and fourth whorls.

Abstract 1147: Crosstalk Between Alk5 And Mtorc1 Signaling Promotes VSMC Differentiation And The Therapeutic Effect Of Rapamycin

Background: Vascular smooth muscle cells (VSMC) phenotypic switching contributes to vascular repair and remodeling but also to pathologies including intimal hyperplasia. The mTORC1 inhibitor rapamycin is an effective drug-eluting stent agent that promotes VSMC differentiation. TGFβ also promotes VSMC differentiation through SMAD transcription factors. We investigated unexpected convergence between these pathways in VSMC plasticity. Methods: We assessed the interactions between rapamycin and the TGFβ1 (ALK5) signaling in human coronary artery SMCs and in vascular remodeling using inducible SMC-specific knockout mice (ALK5iKO). Results: Genome-wide histone H3K27 acetylation analysis unexpectedly identified SMAD binding elements as the motif most enriched after rapamycin treatment of hCASMCs. Treatment with rapamycin promoted rapid phosphorylation of SMAD2/3. Extensive signaling studies revealed that this phosphorylation required ALK5 activity but not TGF-β ligand. Surprisingly, ALK5 and SMAD2/3 were required for rapamycin-induced differentiation. We determined that rapamycin relieves FKBP12 inhibition of ALK5 to promote ligand independent Smad signaling, and FKBP12 knockdown was sufficient to induce contractile genes. Notably, rapamycin treatment induced an interaction between TET2 and SMAD2/3, and these SMADs were required for differentiation-associated chromatin remodeling, including DNA hydroxymethylation and H3K27 acetylation at contractile genes, suggesting that SMADs and TET2 function in concert at SBE- and CArG-containing promoter regions. Furthermore, ALK5iKO mice exhibited severe intimal hyperplasia after carotid artery injury compared to controls and were entirely resistant to the therapeutic effect of rapamycin, despite persistent inhibition of mTORC1. Consistent with in vitro findings, treatment with rapamycin elevated pSmad3 staining post-injury in the medial layer of control but not ALK5iKO mice. Conclusions: We report the surprising observation that rapamycin requires FKBP12/ALK5/SMAD2/3 signaling in order to promote TET2-dependent chromatin remodeling and SMC differentiation. Understanding these mechanisms may reveal new therapeutic strategies for treating vascular diseases.

Gadolinium retention effect on macrophages - a potential cause of MRI contrast agent Dotarem toxicity.

Gadolinium is a component of the MRI contrast agent Dotarem. Although Dotarem is the least toxic among MRI contrasts used, gadolinium present in Dotarem accumulates for many years in various organs and tissues exerting toxic effects. We showed previously that gadolinium remains in macrophages for at least 7days after exposure to Dotarem. However, very little is known about the effect of gadolinium retention on the immune cells such as macrophages. We studied the effect of 1-day and 7-day retention of gadolinium on various functions and molecular pathways of macrophages. Gadolinium retention for 7days decreased macrophage adhesion and motility and dysregulated the expression of adhesion and fibrotic pathway-related proteins such as Notch1 and its ligand Jagged1, adhesion/migration-related proteins PAK1 and Shp1, immune response-related transcription factors Smad3 and TCF19, and chemokines CXCL10 and CXCL13, and dysregulated the mRNA expression of fibrosis-related genes involved in extracellular matrix (ECM) synthesis, such as Col6a1, Fibronectin, MMP9, and MMP12. It also completely (below a level of detection) shut down the transcription of anti-inflammatory M2 macrophage polarization marker theArg-1.Such changes, if they occur in MRI patients, can be potentially detrimental to the patient's immune system and immune response-related processes.

EgMADS3 directly regulates EgLPAAT to mediate medium-chain fatty acids (MCFA) anabolism in the mesocarp of oil palm.

EgMADS3, a pivotal transcription factor, positively regulates MCFA accumulation via binding to the EgLPAAT promoter, advancing lipid content in mesocarp of oil palm. Lipids function as the structural components of cell membranes, which serve as permeable barriers to the external environment of cells. The medium-chain fatty acid in the stored lipids of plants is an important renewable energy. Most research on MCFA production in plant lipid synthesis is based on biochemical methods, and the importance of transcriptional regulation in MCFA synthesis and its incorporation into TAGs needs further research. Oil palm is the most productive oil crop in the world and has the highest productivity among the main oil crops. In this study, the MADS transcription factor (EgMADS3) in the mesocarp of oil palm was characterized. Through the VIGS-virus induced gene silencing, it was determined that the potential target gene of EgMADS3 was related to the biosynthesis of medium-chain fatty acid (MCFA). Transient transformation in protoplasts and qRT-PCR analysis showed that EgMADS3 positively regulated the expression of EgLPAAT. The results of the yeast one-hybrid assays and EMSA indicated the interaction between EgMADS3 and EgLPAAT promoter. Through genetic transformation and fatty acid analysis, it is concluded that EgMADS3 directly regulates the mid-chain fatty acid synthesis pathway of the potential target gene EgLPAAT, thus promotes the accumulation of MCFA and improves the total lipid content. This study is innovative in the functional analysis of the MADS family transcription factor in the metabolism of medium-chain fatty acids (MCFA) of oil palm, provides a certain research basis for improving the metabolic pathway of chain fatty acids in oil palm, and improves the synthesis of MCFA in plants. Our results will provide a reference direction for further research on improving the oil quality through biotechnology of oil palm.