Transforming Growth Factor-β Signaling Research Articles

Molecular Mechanisms of Dermal Aging and Antiaging Approaches.

The dermis is primarily composed of the extracellular matrix (ECM) and fibroblasts. During the aging process, the dermis undergoes significant changes. Collagen, which is a major component of ECM, becomes fragmented and coarsely distributed, and its total amount decreases. This is mainly due to increased activity of matrix metalloproteinases, and impaired transforming growth factor-β signaling induced by reactive oxygen species generated during aging. The reduction in the amount of collagen hinders the mechanical interaction between fibroblasts and the ECM, and consequently leads to the deterioration of fibroblast function and further decrease in the amount of dermal collagen. Other ECM components, including elastic fibers, glycosaminglycans (GAGs), and proteoglycans (PGs), also change during aging, ultimately leading to a reduction in the amount of functional components. Elastic fibers decrease in intrinsically aged skin, but accumulate abnormally in photoaged skin. The changes in the levels of GAGs and PGs are highly diverse, and previous studies have reported conflicting results. A reduction in the levels of functional dermal components results in the emergence of clinical aging features, such as wrinkles and reduced elasticity. Various antiaging approaches, including topicals, energy-based procedures, and dermal fillers, can restore the molecular features of dermal aging with clinical efficacy. This review summarizes the current understanding of skin aging at the molecular level, and associated treatments, to put some of the new antiaging technology that has emerged in this rapidly expanding field into molecular context.

Molecular mechanisms of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy is a genetic disorder characterized by the risk of life-threatening arrhythmias, myocardial dysfunction and fibrofatty replacement of myocardial tissue. Mutations in genes that encode components of desmosomes, the adhesive junctions that connect cardiomyocytes, are the predominant cause of arrhythmogenic cardiomyopathy and can be identified in about half of patients with the condition. However, the molecular mechanisms leading to myocardial destruction, remodelling and arrhythmic predisposition remain poorly understood. Through the development of animal, induced pluripotent stem cell and other models of disease, advances in our understanding of the pathogenic mechanisms of arrhythmogenic cardiomyopathy over the past decade have brought several signalling pathways into focus. These pathways include canonical and non-canonical WNT signalling, the Hippo-Yes-associated protein (YAP) pathway and transforming growth factor-β signalling. These studies have begun to identify potential therapeutic targets whose modulation has shown promise in preclinical models. In this Review, we summarize and discuss the reported molecular mechanisms underlying the pathogenesis of arrhythmogenic cardiomyopathy.

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation.

Fibrosis is a hallmark in the pathogenesis of various diseases, with very limited therapeutic solutions. A key event in the fibrotic process is the expression of contractile proteins, including α-smooth muscle actin (αSMA) by fibroblasts, which become myofibroblasts. Here, we report the results of a high-throughput screening of a library of approved drugs that led to the discovery of haloperidol, a common antipsychotic drug, as a potent inhibitor of myofibroblast activation. We show that haloperidol exerts its antifibrotic effect on primary murine and human fibroblasts by binding to sigma receptor 1, independent from the canonical transforming growth factor-β signaling pathway. Its mechanism of action involves the modulation of intracellular calcium, with moderate induction of endoplasmic reticulum stress response, which in turn abrogates Notch1 signaling and the consequent expression of its targets, including αSMA. Importantly, haloperidol also reduced the fibrotic burden in 3 different animal models of lung, cardiac, and tumor-associated fibrosis, thus supporting the repurposing of this drug for the treatment of fibrotic conditions.

Identification of blood microRNA alterations in patients with severe active alopecia areata.

Alopecia areata (AA) is a common inflammatory disease characterized by cellular infiltration of T cells targeting the anagen-stage hair follicle. Lack of efficacious treatment for AA may be due to little knowledge about its exact cellular mechanism. Studies have demonstrated that microRNAs (miRNAs) play an important role in the regulation of inflammatory skin diseases such as atopic dermatitis and psoriasis. However, little is known about the role of miRNAs in AA. The present study aimed to explore the blood miRNAs alterations in patients with severe active AA. We constructed a bipartite miRNA-messenger RNA (mRNA) regulatory network by the validatedmiRNA-mRNA relationships. Subsequently, the miRNA-miRNA synergistic network was formed in consideration of the Gene Ontology function enrichment of coregulated target genes. Lastly, the functional network was identified by the ingenuity pathway analysis. By using an Agilent microarray that covers 2549 human miRNAs, we identified 36 significantly differentially expressed miRNAs in severe active AA patients. miRNA target gene prediction and functional annotation analysis showed significant enrichment in several pathways including the ribosome, cancer, cell cycle, insulin signaling, transforming growth factor-βsignaling, and p53 signaling pathways. Analysis of the three kinds of network showed that miR-185-5p, miR-125b-5p, and miR-186-5p might play important and synergistic roles in the active phase of AA. According to the receiver operating characteristic curve analysis, several miRNAs were selected for the quantitative real-time polymerase chain reaction validation. Among the miRNAs, miR-210 and miR-1246 had high prediction with high accuracy. Blood dysregulated miRNAs are potentially associated with the severe active AA. These miRNAs could function synergistically and might be promising targets for the development of novel treatments for AA in the future.

Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses.

SummaryBackgroundRecessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer‐prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders.ObjectivesTo investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC.MethodsWe conducted RNA‐Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies.ResultsInterdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA‐Seq data. In addition, enzyme‐linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB.ConclusionsOur results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury‐sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development.Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix‐synthetic capacity.This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor‐β signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention.It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3'UTRs, including those involved in the transforming growth factor-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.

Integrative Genomics Analysis Identifies ACVR1B as a Candidate Causal Gene of Emphysema Distribution.

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10-5 in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P < 0.05), with the strongest enrichment observed in CD4+, CD8+, and regulatory T cells. We selected a putative causal variant, rs7962469, associated with ACVR1B expression in lung tissue for additional functional investigation, and reporter assays confirmed allele-specific regulatory activity for this variant in human bronchial epithelial and Jurkat immune cell lines. ACVR1B expression levels exhibit a nominally significant association with emphysema distribution. EABD-associated loci are preferentially enriched in regulatory elements of multiple cell types, most notably T-cell subsets. Multiple EABD loci colocalize to regulatory elements that are active across multiple tissues and cell types, and functional analyses confirm the presence of an EABD-associated functional variant that regulates ACVR1B expression, indicating that transforming growth factor-β signaling plays a role in the EABD phenotype. Clinical trial registered with www.clinicaltrials.gov (NCT00608764).

Expression analysis of selected miR-206 targets from the transforming growth factor-β signaling pathway in breast cancer.

Breast cancer as a molecularly heterogeneous malignancy is associated with dysregulation of several signaling pathways, including transforming growth factor-β (TGF-β) signaling. On the other hand, several recent studies have demonstrated the role of microRNAs (miRNAs) in breast cancer pathogenesis. In the current study, we performed a computerized search to find miR-206 target genes that are functionally linked to the TGF-β signaling pathway. We selected LEF1, Smad2, and Snail2 genes to assess their expression in 65 breast cancer samples and their adjacent noncancerous tissues (ANCTs) in correlation with expression levels of miR-206 as well as clinicopathological characteristics of patients. miR-206 was significantly downregulated in (Estrogen receptor) ER-positive breast cancer samples compared with their corresponding ANCTs. Association analysis between expression levels of genes and demographic features of patients showed significant association between expressions of SMAD2 and LEF1 genes and body mass index ( P values of 0.03 and 0.02, respectively). miR-206 low-expression levels were associated with TNM stage, mitotic rate, and lymph node involvement ( P values of 0.02, 0.01, and 0.01 respectively). In addition, SMAD2 high-expression levels were associated with HER2 status ( P = 0.02). Consequently, our data highlight the role of TGF-β signaling dysregulation in the pathogenesis of breast cancer and warrant further evaluation of miRNAs and messenger RNA coding genes in this pathway to facilitate detection of cancer biomarkers and therapeutic targets.

The motor protein Myo1c regulates transforming growth factor-β–signaling and fibrosis in podocytes

Transforming growth factor-β (TGF-β) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-β signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-β-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-β-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-β signaling through downregulation of canonical and non-canonical TGF-β pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-β signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-β responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-β-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.

Expression profiling of long noncoding RNAs associated with vasculogenic mimicry in osteosarcoma.

Osteosarcoma (OS) is the most common highly malignant bone tumor in teens. Vasculogenic mimicry (VM) is defined as de novo extracellular matrix-rich vascular-like networks formed by highly aggressive tumor cells. We previously reported the presence of VM and it is an unfavorable prognostic factor in OS patients. Long noncoding RNAs (lncRNAs) are aberrantly expressed in OS and involved in cancer cell VM. However, lncRNAs in VM formation of OS have not been investigated. We, therefore, profiled the expression of lncRNAs in highly aggressive OS cell line 143B compared with its parental poorly aggressive cell line HOS. The differentially expressed (DE) lncRNAs and messenger RNA (mRNAs) were subjected to constructed lncRNA-mRNA coexpressed network. The top-ranked hub gene lncRNA n340532 knockdown 143B cells were used for in vitro and in vivo VM assays. The annotation of DE lncRNAs was performed according to the coexpressed mRNAs by Gene Ontology and pathway analysis. A total of 1360 DE lncRNAs and 1353 DE mRNAs were screened out. lncRNA MALAT1 and FTX, which have known functions related to VM formation and tumorigenesis were identified in our data. The coexpression network composed of 226 lncRNAs and 118 mRNAs in which lncRNA n340532 had the highest degree number. lncRNA n340532 knockdown reduced VM formation in vitro. The suppression of n340532 also exhibited potent anti-VM and antimetastasis effect in vivo, suggesting its potential role in OS VM and metastasis. Furthermore, n340532 coexpressed with 10 upregulation mRNAs and 3 downregulation mRNAs. The enriched transforming growth factor-β signaling pathway, angiogenesis and so forth were targeted by those coexpressed mRNAs, implying n340532 may facilitate VM formation in OS through these pathways and gene functions. Our findings provide evidence for the potential role of lncRNAs in VM formation of OS that could be used in the clinic for anti-VM therapy in OS.

Berberine attenuates hypoxia-induced pulmonary arterial hypertension via bone morphogenetic protein and transforming growth factor-β signaling.

Hypoxia-induced excessive pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in the pathology of pulmonary arterial hypertension (PAH). Berberine (BBR) is reported as an effective antiproliferative properties applied in clinical. However, the effect of BBR on PAH remains unclear. In the present study, we elucidated the protective effects of BBR against abnormal PASMC proliferation and vascular remodeling in chronic hypoxia-induced hearts. Furthermore, the potential mechanisms of BBR were investigated. For this purpose, C57/BL6 mice were exposed to chronic hypoxia for 4 weeks to mimic severe PAH. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased the right ventricular systolic pressure (RVSP), the right ventricle/left ventricle plus septum RV/(LV + S) weight ratio, and the median width of pulmonary arterioles. BBR attenuated the elevations in RVSP and RV/(LV + S) and mitigated pulmonary vascular structure remodeling. BBR also suppressed the hypoxia-induced increases in the expression of proliferating cell nuclear antigen (PCNA) and of α-smooth muscle actin. Furthermore, administration of BBR significantly increased the expression of bone morphogenetic protein type II receptor (BMPR-II) and its downstream molecules P-smad1/5 and decreased the expression of transforming growth factor-β (TGF-β) and its downstream molecules P-smad2/3. Moreover, peroxisome proliferator-activated receptor γ expression was significantly decreased in the hypoxia group, and this decrease was reversed by BBR treatment. Our study demonstrated that the protective effect of BBR against hypoxia-induced PAH in a mouse model may be achieved through altered BMPR-II and TGF-β signaling.

Metabolic Syndrome Interferes with Packaging of Proteins within Porcine Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC‐derived EVs. To test this, we compared the protein cargo of EVs to their parent MSCs in pigs with metabolic syndrome (MetS) and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean‐ or MetS‐diet (n = 5 each), and their EVs isolated. Following liquid chromatography mass spectrometry proteomic analysis, proteins were classified based on cellular component, molecular function, and protein class. Five candidate proteins were validated by Western blot. Clustering analysis was performed to identify primary functional categories of proteins enriched in or excluded from EVs. Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean‐ and MetS‐EVs, respectively. Differential expression analysis revealed that 146 proteins were upregulated and 273 downregulated in Lean‐EVs versus Lean‐MSCs, whereas 787 proteins were upregulated and 185 downregulated in MetS‐EVs versus MetS‐MSCs. Proteins enriched in both Lean‐ and MetS‐EVs participate in vesicle‐mediated transport and cell‐to‐cell communication. Proteins enriched exclusively in Lean‐EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor‐β signaling. Contrarily, proteins enriched only in MetS‐EVs are linked to proinflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production. Coculture with MetS‐EVs increased renal tubular cell inflammation. MetS alters the protein cargo of porcine MSC‐derived EVs, selectively packaging specific proinflammatory signatures that may impair their ability to repair damaged tissues. stem cells translational medicine 2019;8:430–440

Expression Characteristics of Genes Hypermethylated and Downregulated in Rat Liver Specific to Nongenotoxic Hepatocarcinogens.

This study examined hypermethylated and downregulated genes specific to carbon tetrachloride (CCl4) by Methyl-Seq analysis combined with expression microarray analysis in the liver of rats treated with CCl4 or N-nitrosodiethylamine (DEN) for 28 days, by excluding those with DEN. Among 52 genes, Ldlrad4, Proc, Cdh17, and Nfia were confirmed to show promoter-region hypermethylation by methylation-specific quantitative PCR analysis on day 28. The transcript levels of these 4 genes decreased by real-time reverse transcription-PCR analysis in the livers of rats treated with nongenotoxic hepatocarcinogens for up to 90 days compared with untreated controls and genotoxic hepatocarcinogens. Immunohistochemically, LDLRAD4 and PROC showed decreased immunoreactivity, forming negative foci, in glutathione S-transferase placental form (GST-P)+ foci, and incidences of LDLRAD4− and PROC− foci in GST-P+ foci induced by treatment with nongenotoxic hepatocarcinogens for 84 or 90 days were increased compared with those with genotoxic hepatocarcinogens. In contrast, CDH17 and NFIA responded to hepatocarcinogens without any relation to the genotoxic potential of carcinogens. All 4 genes did not respond to renal carcinogens after treatment for 28 days. Considering that Ldlrad4 is a negative regulator of transforming growth factor-β signaling, Proc participating in p21WAF1/CIP1 upregulation by activation, Cdh17 inducing cell cycle arrest by gene knockdown, and Nfia playing a role in a tumor-suppressor, all these genes may be potential in vivo epigenetic markers of nongenotoxic hepatocarcinogens from the early stages of treatment in terms of gene expression changes. LDLRAD4 and PROC may have a role in the development of preneoplastic lesions produced by nongenotoxic hepatocarcinogens.

Local administration of halofuginone via osmotic infusion pumps inhibits transforming growth fac-tor-β signaling in subchondral bone

Objective To explore the impact of inhibition of transforming growth factor-β(TGF-β) signaling by local administration of halofuginone (HF) via osmotic infusion pumps on osteoarthritis (OA) pathogenesis and its underlying mechanism. Methods Knee OA models were induced by the anterior cruciate ligament transection (ACLT) in 30 3-month-old male SD rats. They were randomized by random number table into 3 equal groups (n=10): Sham, Vehicle+ACLT and HF+ACLT ones. Specific admin-istration of drugs was achieved via osmotic infusion pumps directly implanted in subchondral bone. Safranin O and fast green, H&E, immunofluorescence staining, CT-based microangiography and bone micro-CT (μCT) were used to measure alterations in articular cartilage and subchondral bone [BV(bone volume)/TV (tissue volume), Tb.Pf (trabecular pattern factor), Tb.N (trabecular number), SBP.Th(subchondral bone plate.Th), pSmad2/3, Nestin, and OARSI (Osteoarthritis Research Society International) scoring]. Results Knee OA models and drug administration devices in subchondral bone were successfully established in rats. Sham and HF+ACLT groups had greater subchondral BV/TV(0.381±0.060 mm3 and 0.322±0.060 mm3), SBP.Th (0.570±0.042 mm and 0.521±0.122 mm) and Tb.N (4.871±0.214 mm-1 and 4.364±0.466 mm-1) than Vehicle+ACLT group did (0.229±0.063) mm3, 0.324±0.165 mm and 2.978±0.804 mm-1, respectively); Sham and HF+ACLT groups had less subchondral Tb.Pf (-0.880±0.210 mm-1 and -0.377±0.259 mm-1), lower expression of pSmad2/3 (90.2±40.0 and 90.8±34.5) and Nestin (16.9±5.8 and 18.5±4.7) and OARSI scores (1.2±0.7 and 2.5±1.9) than Vehicle+ACLT group did (0.057±0.535 mm-1, 142.7±37.0, 25.9±7.4 and 5.4±2.8, respectively). All the above differences were statistically significant (P 0.05). Conclusion Subchondral administration of HF can inhibit TGF-β induced erroneous recruitment of mesenchymal stem cells in subchondral bone, thus attenuating OA progression. Key words: Osteoarthritis, knee; Transforming growth factor-β; Mesenchymal stem cells; Subchondral bone

TCEA3 promotes differentiation of C2C12 cells via an Annexin A1-mediated transforming growth factor-β signaling pathway.

TCEA3 is a member of the transcription elongation factor family that not only promotes transcription but may also participate in other cytoplasmic processes. However, its mechanisms of action remain unclear. Our previous study indicated that TCEA3 may affect muscle differentiation. In this study, we investigated the expression and localization of TCEA3 in C2C12 cells and examined the role of TCEA3 in differentiation, its interaction with other cell proteins, and mechanisms of action. We found that the expression of TCEA3 increased gradually with an increase in the number of differentiation days and that it is mainly expressed in the cytoplasm of C2C12 cells, of which it promotes differentiation. Coimmunoprecipitation experiments and western blot analysis revealed that TCEA3 interacts with Annexin A1 (ANXA1), which is located in the cytoplasm and also promotes cell differentiation. Collectively, our results indicate that TCEA3 promotes cell differentiation by interacting with ANXA1 and affecting transforming growth factor-β signaling pathways.

Uncovering the mechanism whereby dietary nicotinic acid increases the intramuscular fat content in finishing steers by RNA sequencing analysis

In our previous study, we found that a higher dosage of nicotinic acid (NA) in the diet dramatically increases intramuscular fat (IMF) content and improves meat quality in finishing steers. We hypothesised that increased IMF results from the regulation of genes associated with adipogenesis. To address this hypothesis, RNA-seq was used to investigate gene-expression profiles of longissimus muscles from the same 16 cattle that were also used in our previous study and treated with or without dietary NA. Four cDNA libraries were constructed and sequenced. The repeatability and reproducibility of RNA-seq data were confirmed by quantitative reverse-transcription polymerase-chain reaction. In total, 123 differentially expressed genes (DEGs) were identified between longissimus muscles treated and those not treated with dietary NA. Of the 123 DEGs, 117 genes were upregulated by the NA treatment. These DEGs were enriched in 21 pathways, including the extracellular matrix (ECM) –receptor interaction, PPAR signalling pathway, adipocytokine signalling pathway and transforming growth factor-β signalling pathway, all of which are associated with lipid metabolism. Furthermore, candidate genes related to adipocyte differentiation and adipogenesis (PLIN1, PLIN2, ADPN, LEP, LCN2 and SOCS3), lipid metabolism (FABP4, RBP4, GAL, ANXA1, ANXA2 and PTX3) and fatty acid synthesis and esterification (ELOVL6, ACSM1, SOT1 and PTGIS) were upregulated in the NA group. Three genes involved in glucose metabolism (PGAM1, UGDH and GLUT3) were also transcriptionally upregulated. However, MYH4 that encodes glycolytic Type IIb muscle fibres was downregulated by dietary NA. These gene expression results indicated a confirmation of our hypothesis that dietary NA increases the IMF content of longissimus muscle through upregulating the expression of the genes related to adipocyte differentiation, adipogenesis and lipid and glucose metabolism.

Upregulation of bone morphogenetic protein 2 ( Bmp2) in dorsal root ganglion in a rat model of bone cancer pain.

Bone cancer pain is one of the most severe and intractable complications in patients suffering from primary or metastatic bone cancer and profoundly compromises the quality of life. Emerging evidence indicates that the dorsal root ganglion play an integral role in the modulation of pain hypersensitivity. However, the underlying molecular mechanisms during dorsal root ganglion-mediated bone cancer pain remain elusive. In this study, RNA-sequencing was used to detect the differentially expressed genes in dorsal root ganglion neurons of a rat bone cancer pain model established by intratibial inoculation of Walker 256 breast cancer cells. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the differentially expressed genes (fold change > 1.5; false discovery rate < 0.05) were enriched in the bone morphogenetic protein (BMP) signaling pathway, transforming growth factor-β signaling pathway, and positive regulation of cartilage development. Importantly, serum deprivation-response protein (Sdpr), hephaestin (Heph), transthyretin (Ttr), insulin receptor substrate 1 (Irs1), connective tissue growth factor (Ctgf ), and Bmp2 genes were associated with bone pain and degeneration. Of note, Bmp2, a pleiotropic and secreted molecule mediating pain and inflammation, was one of the most significantly upregulated genes in dorsal root ganglion neurons in this bone cancer pain model. Consistent with these data, upregulation of Bmp2 in the bone cancer pain model was validated by immunohistochemistry, real-time quantitative polymerase chain reaction, and western blotting. Importantly, intrathecal administration of siRNA significantly reduced Bmp2 transcription and ameliorated bone cancer pain in rat as shown by paw withdrawal mechanical threshold and spontaneous and movement-evoked pain-like behaviors. In conclusion, we have characterized the comprehensive gene expression profile of dorsal root ganglion from a bone cancer pain rat model by RNA-sequencing and identified Bmp2 as a potential therapeutic target for bone cancer pain treatment.

Fanconi anemia

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure, increased susceptibility to leukemia and cancer, and genomic instabilities. Protein products encoded by 22 FA genes, identified till date, cooperate in a molecular pathway called the FA pathway to repair DNA interstrand cross-links induced by chemotherapeutic agents, such as mitomycin C and cisplatin. An accumulating number of studies have shown several new functional aspects of the FA pathway, particularly in the context of the pathogenesis of bone marrow failure. This review focuses on the following topics: (1) aldehydes as intrinsic interstrand cross-linkers; (2) cytokine-induced hematopoietic stress; (3) increased transforming growth factor-β signaling; (4) mitochondrial functions of FA proteins. These findings are expected to offer new therapeutic opportunities for bone marrow failure in FA.

Circulating extra-cellular RNAs, myocardial remodeling, and heart failure in patients with acute coronary syndrome

Given high on-treatment mortality in heart failure (HF), identifying molecular pathways that underlie adverse cardiac remodeling may offer novel biomarkers and therapeutic avenues. Circulating extracellular RNAs (ex-RNAs) regulate important biological processes and are emerging as biomarkers of disease, but less is known about their role in the acute setting, particularly in the setting of HF. We examined the ex-RNA profiles of 296 acute coronary syndrome (ACS) survivors enrolled in the Transitions, Risks, and Actions in Coronary Events Center for Outcomes Research and Education Cohort. We measured 374 ex-RNAs selected a priori, based on previous findings from a large population study. We employed a two-step, mechanism-driven approach to identify ex-RNAs associated with echocardiographic phenotypes (left ventricular [LV] ejection fraction, LV mass, LV end-diastolic volume, left atrial [LA] dimension, and LA volume index) then tested relations of these ex-RNAs with prevalent HF (N=31, 10.5%). We performed further bioinformatics analysis of microRNA (miRNAs) predicted targets' genes ontology categories and molecular pathways. We identified 44 ex-RNAs associated with at least one echocardiographic phenotype associated with HF. Of these 44 exRNAs, miR-29-3p, miR-584-5p, and miR-1247-5p were also associated with prevalent HF. The three microRNAs were implicated in the regulation p53 and transforming growth factor-β signaling pathways and predicted to be involved in cardiac fibrosis and cell death; miRNA predicted targets were enriched in gene ontology categories including several involving the extracellular matrix and cellular differentiation. Among ACS survivors, we observed that miR-29-3p, miR-584-5p, and miR-1247-5p were associated with both echocardiographic markers of cardiac remodeling and prevalent HF. miR-29c-3p, miR-584-5p, and miR-1247-5p were associated with echocardiographic phenotypes and prevalent HF and are potential biomarkers for adverse cardiac remodeling in HF.

Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure.

Hemodynamic load regulates cardiac remodeling. In contrast to pressure overload (increased afterload), hearts subjected to volume overload (VO; preload) undergo a distinct pattern of eccentric remodeling, chamber dilation, and decreased extracellular matrix content. Critical profibrotic roles of cardiac fibroblasts (CFs) in postinfarct remodeling and in response to pressure overload have been well established. Little is known about the CF phenotype in response to VO. The present study characterized the phenotype of primary cultures of CFs isolated from hearts subjected to 4 wk of VO induced by an aortocaval fistula. Compared with CFs isolated from sham hearts, VO CFs displayed a "hypofibrotic" phenotype, characterized by a ~50% decrease in the profibrotic phenotypic markers α-smooth muscle actin, connective tissue growth factor, and collagen type I, despite increased levels of profibrotic transforming growth factor-β1 and an intact canonical transforming growth factor-β signaling pathway. Actin filament dynamics were characterized, which regulate the CF phenotype in response to biomechanical signals. Actin polymerization was determined by the relative amounts of G-actin monomers versus F-actin. Compared with sham CFs, VO CFs displayed ~78% less F-actin and an increased G-actin-to-F-actin ratio (G/F ratio). In sham CFs, treatment with the Rho kinase inhibitor Y-27632 to increase the G/F ratio resulted in recapitulation of the hypofibrotic CF phenotype observed in VO CFs. Conversely, treatment of VO CFs with jasplakinolide to decrease the G/F ratio restored a more profibrotic response (>2.5-fold increase in α-smooth muscle actin, connective tissue growth factor, and collagen type I). NEW & NOTEWORTHY The present study is the first to describe a "hypofibrotic" phenotype of cardiac fibroblasts isolated from a volume overload model. Our results suggest that biomechanical regulation of actin microfilament stability and assembly is a critical mediator of cardiac fibroblast phenotypic modulation.