Connective Tissue Growth Factor Gene Research Articles

Effects of connective tissue growth factor on prostate cancer bone metastasis and osteoblast differentiation.

Previous studies have demonstrated that connective tissue growth factor (CTGF) is expressed at increased levels in prostate cancer bone metastasis mouse models and patients with prostate cancer which metastasizes to the bone; however, the underlying molecular mechanism(s) remain unknown. The present study investigated the function of CTGF in osteoblast differentiation and its effect on prostate cancer bone metastasis by analyzing CTGF gene expression and transcription at different levels of invasion, metastasis of prostate cancer cells, and the influence of CTGF on proliferation and xenotransplantation. A mouse model demonstrating bone metastasis was used to investigate the function(s) of CTGF in bone metastasis and osteoblast differentiation. Results demonstrated that CTGF expression was increased in association with high bone metastasis in prostate cancer cells, and its expression was significantly decreased in whole cell lysates. CTGF expression in prostate cancer cells with high levels of bone metastasis was increased 1.9-fold compared with prostate cancer cells with low levels of bone metastasis. The expression of CTGF in mesenchymal cells was markedly increased compared with epithelial cells. Results indicated that the increased expression of CTGF does not affect the proliferation of tumor cells and possesses no influence on tumor volume. Control and CTGF plasmids were transfected into RM1 cells and led to 4 and 17% bone lesions, respectively. Increased expression of CTGF significantly enlarged the tumor area in the bone metastatic position compared with the control. Positive areas of alkaline phosphatase were significantly decreased as the concentration of CTGF increased. The results of the present study demonstrated that CTGF promotes prostate carcinoma to metastasize in the bone by dysregulating osteoblast differentiation.

NNMT activation can contribute to the development of fatty liver disease by modulating the NAD+ metabolism

Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD)+, these NNMT-overexpressing mice exhibit fatty liver deterioration following increased expression of the genes mediating fatty acid uptake and decreased very low-density lipoprotein secretion. NNMT overactivation decreased the NAD+ content in the liver and also decreased gene activity related to fatty acid oxidation by inhibiting NAD+–dependent deacetylase Sirt3 function. Moreover, the transgenic mice showed liver fibrosis, with the induction of inflammatory and fibrosis genes. Induced NNMT expression decreased the tissue methylation capacity, thereby reducing methylation of the connective tissue growth factor (CTGF) gene promoter, resulting in increased CTGF expression. These data indicate that NNMT links the NAD+ and methionine metabolic pathways and promotes liver steatosis and fibrosis. Therefore, targeting NNMT may serve as a therapeutic strategy for treating fatty liver and fibrosis.

The effects of platelet-rich plasma on hypertrophic scars fibroblasts.

We hypothesised that a feedback mechanism of the transforming growth factor (TGF)-β1 signalling pathway, triggered by high-level TGF-β1, activates platelet-rich plasma (PRP) release to reduce connective tissue growth factor (CTGF) production and expression of CTGF mRNA in hypertrophic scar dermal fibroblasts. Primary dermal fibroblasts were isolated from cultures of hypertrophic scars. Cells were cultured after addition of serum-free Dulbecco's modified Eagle's medium supplemented with 5% (wt/vol) PRP or platelet-poor plasma (PPP). At 1, 4, 6, 8, 11, and 13 days after addition of PRP or PPP, the TGF-β1 and CTGF levels in supernatants were determined using solid-phase enzyme-linked immunosorbent assays. Quantitative reverse transcription polymerase chain reactions were performed to quantify TGF-β1 and CTGF mRNA expression levels. TGF-β1 mRNA expression in the PRP groups was lower than in the PPP groups from 4 to 13 days of culture, and there was statistically significant difference (P < .01). CTGF level and mRNA expression in the PRP groups was lower than in the PPP groups, and there were statistically significant differences (P < .01). Although further experiments will focus on clarifying the second messenger of the TGF-β1 negative feedback mechanism, the in vitro data presented show that PRP can potentially reduce CTGF and CTGF gene transcription by triggering the TGF-β1 signalling negative feedback mechanism.

Essential role of connective tissue growth factor (CTGF) in transforming growth factor-\u03b21 (TGF-\u03b21)-induced myofibroblast transdifferentiation from Graves\u2019 orbital fibroblasts

Connective tissue growth factor (CTGF) associated with transforming growth factor-β (TGF-β) play a pivotal role in the pathophysiology of many fibrotic disorders. However, it is not clear whether this interaction also takes place in GO. In this study, we investigated the role of CTGF in TGF-β-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts. By Western blot analysis, we demonstrated that TGF-β1 induced the expression of CTGF, fibronectin, and alpha-smooth muscle actin (α-SMA) in Graves’ orbital fibroblasts. In addition, the protein levels of fibronectin and α-SMA in Graves’ orbital fibroblasts were also increased after treatment with a recombinant human protein CTGF (rhCTGF). Moreover, we transfected the orbital fibroblasts with a small hairpin RNA of CTGF gene (shCTGF) to knockdown the expression levels of CTGF, which showed that knockdown of CTGF significantly diminished TGF-β1-induced expression of CTGF, fibronectin and α-SMA proteins in Graves’ orbital fibroblasts. Furthermore, the addition of rhCTGF to the shCTGF-transfected orbital fibroblasts could restore TGF-β1-induced expression of fibronectin and α-SMA proteins. Our findings demonstrate that CTGF is an essential downstream mediator for TGF-β1-induced extracellular matrix production and myofibroblast transdifferentiation in Graves’ orbital fibroblasts and thus may provide with a potential therapeutic target for treatment of GO.

CELL‐MATRIX INTERACTIONS AND MECHANOSENSITIVE PATHWAYS IN UTERINE LEIOMYOMA (FIBROID) PROLIFERATION

BackgroundUterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in &gt;25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth.MethodsCells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively.ResultsSignificant increases in transcripts for ECM gene Collagen 1 (COL1A1) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor (CTGF), alpha smooth muscle actin (ACTA2), Endothelin 1 (EDN1), and plasminogen activator inhibitor 1 (SERPINE1) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 (PTGS2) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium.ConclusionsBased on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unique mechanisms of connective tissue growth factor regulation in airway smooth muscle in asthma: Relationship with airway remodelling.

Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non‐asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)‐β increased CTGF expression both in NA‐ and A‐ASM cells but the expression was higher in A‐ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter‐luciferase reporter constructs into NA‐ and A‐ASM cells indicated that no region of the CTGF promoter (−1500 to +200 bp) displayed enhanced activity in the presence of TGF‐β. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF‐β in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post‐transcriptional regulation in A‐ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A‐ASM may contribute to airway remodelling in asthma.

Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression

AimsLysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Main methodsHepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson’s trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Key findingsSilymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. SignificanceSilymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF.

Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts.

Perspectives on whether the functions of MAS, a G protein-coupled receptor, are beneficial or deleterious in the heart remain controversial. MAS gene knockout reduces coronary vasodilatation leading to ischemic injury. G protein signaling activated by MAS has been implicated in progression of adaptive cardiac hypertrophy to heart failure and fibrosis. In the present study, we observed increased expression of MAS, connective tissue growth factor (CTGF) and collagen genes in failing (HF) human heart samples when compared to non-failing (NF). Expression levels of MAS are correlated with CTGF in HF and NF leading to our hypothesis that MAS controls CTGF production and the ensuing expression of collagen genes. In support of this hypothesis we show that the non-peptide MAS agonist AR234960 increases both mRNA and protein levels of CTGF via ERK1/2 signaling in HEK293-MAS cells and adult human cardiac fibroblasts. MAS-mediated CTGF expression can be specifically blocked by MAS inverse agonist AR244555 and also by MEK1 inhibition. Expression of CTGF gene was essential for MAS-mediated up-regulation of different collagen subtype genes in HEK293-MAS cells and human cardiac fibroblasts. Knockdown of CTGF by RNAi disrupted collagen gene regulation by the MAS-agonist. Our data indicate that CTGF mediates the profibrotic effects of MAS in cardiac fibroblasts. Blocking MAS-CTGF-collagen pathway should be considered for pharmacological intervention for HF.

Regulatory role of miR-18a to CCN2 by TGF-β1 signaling pathway in pulmonary injury induced by nano-SiO2.

This research is designed to investigate the regulatory effect of miR-18a to the target gene connective tissue growth factor (CTGF, or CCN2), by participating in TGF-β1 signaling pathway and explore the pathogenic mechanism of miR-18a in pulmonary injury induced by nano-SiO2 based on our early study. miR-18a and expression of TGF-β1 in Chinese hamster lung (CHL) fibroblasts cells stimulated by supernatants of NR8383 cells exposed to 40μg/ml nano-SiO2 for 24h demonstrated 1.58±0.22-fold and 1096.00±2.60pg/ml increase compared with blank control group analyzed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression increase of miR-18a and reduction of CCN2 mRNA expression levels and protein gray value ratio detected by Western blotting in CHL cells transfect miR-18a mimics for 48h. The reverse of CHL cell transfection miR-18a inhibit is also true. The result of miR-18a and CCN2 binding sites tested by luciferase reporter gene assay shows that the report relative fluorescence value of miR-18a mimics wild type on CCN2 is 0.50±0.02 with the control of mimics NC and mutant relative fluorescence report value 0.86±0.04 (P<0.05). Expression levels of miR-18a, CCN2 mRNA, and protein gray value ratio decreased in CHL cells treated by TGF-β1, respectively, and vice versa treated by TGF-β1corepressor. The results suggest that CCN2 is the target gene regulated by miR-18a and miR-18a participates in TGF-β1 signaling pathway by regulating the expression of CCN2 negatively through CCN2 3'UTR site, and thus may be involved in the development process of pulmonary injury.

Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Identification of target gene of venous thromboembolism in patients with lymphoma via microarray analysis.

Patients with lymphoma are at high risk of developing venous thromboembolism (VTE). The purpose of the present study was to identify the target gene associated with VTE for patients with lymphoma. Microarray data was downloaded from the gene expression omnibus database (GSE17078), which comprised the control group, 27 normal blood outgrowth endothelial cell (BOEC) samples, and the case group, 3 BOEC samples of venous thrombosis with protein C deficiency. Differentially expressed genes (DEGs) were identified by the Limma package of R. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed via the database for annotation, visualization and integrated discovery. Differentially coexpressed pairs were identified by the DCGL package of R. The subsequent protein-protein interaction (PPI) networks and gene coexpression networks were constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins database, and were visualized by Cytoscape software. A total of 110 DEGs were obtained, including 73 upregulated and 37 downregulated genes. GO and KEGG pathway enrichment analyses identified 132 significant GO terms and 9 significant KEGG pathways. In total, 97 PPI pairs for PPI network and 309 differential coexpression pairs for the gene coexpression network were obtained. Additionally, the connective tissue growth factor (CTGF) gene was closely connected with other genes in the two networks. A total of 2 KEGG pathways were associated with VTE and CTGF may be the target gene of VTE in patients with lymphoma. The present study may identify the molecular mechanism of VTE, but additional clinical study is required to validate the results.

Ocular surface cytokine profile in chronic Stevens-Johnson syndrome and its response to mucous membrane grafting for lid margin keratinisation

BackgroundTo study the tear cytokine and the conjunctival and oral mucosal marker profile in chronic ocular Stevens-Johnson syndrome (SJS) and their alteration following mucous membrane grafting (MMG) for lid margin...

Genome-wide DNA methylation analysis of articular chondrocytes identifies TRAF1, CTGF, and CX3CL1 genes as hypomethylated in osteoarthritis.

The aim of this study is to identify osteoarthritis (OA)-associated differentially methylated genes in human articular chondrocytes from patients with OA. DNA methylation profiling of articular chondrocytes from OA patients, rheumatoid arthritis (RA) patients, and controls was performed, and candidate genes were chosen for validation of gene demethylation status. The mRNA expression levels of candidate genes in chondrocytes were detected by real-time quantitative PCR. Chondrocytes from OA and RA group were treated with 5-Aza-2-deoxycytidine (5-Aza), and then the mRNA expression levels were detected. Forty-five genes with significant methylation differences between OA and control group were identified. Tumor necrosis factor receptor-associated factor 1 (TRAF1), connective tissue growth factor (CTGF), and chemokine (C-X3-C motif) ligand 1(CX3CL1) genes were hypomethylated in chondrocytes of OA and RA patients, which verified by bisulfite sequencing analysis. The mRNA expression level of TRAF1 and CTGF was significantly increased in OA and RA group (p<0.05), while the expression level of CX3CL1 was only increased in OA group (p<0.05). For the chondrocytes from OA and RA treated with 5-Aza, the mRNA expression level of TRAF1 and CTGF was highly increased (p<0.05). It is the first time to show that TRAF1, CTGF, and CX3CL1 genes were hypomethylated in OA chondrocytes and have a consistent correlation with mRNA expression, which suggests that epigenetic changes in the methylation status of TRAF1, CTGF, and CX3CL1 contribute to the pathology of OA.

HIF-1α mediates visfatin-induced CTGF expression in vascular endothelial cells.

Visfatin is an adipokine that functions as a mediator of endothelial dysfunction and cardiovascular diseases. Connective tissue growth factor (CTGF) is a key factor in vascular remodeling and atherosclerosis. However, the association between visfatin and CTGF is unclear. Therefore the study was to test the hypothesis that visfatin could modulate the expression of CTGF in vascular endothelial cells. In our study, cultured endothelial cell line EA.Hy926 cells were treated with different concentrations of visfatin for different times. The CTGF gene expression was analyzed by real-time PCR, and the protein expression of CTGF was assessed by Western Blot. The results showed that 100ng/mL concentration of visfatin could induce CTGF mRNA expression after 6 hours treatment, which peaked at 24 hours. And 100ng/mL concentration of visfatin also increased CTGF protein production after 12 hours treatment in EA.Hy926 cells. The expression of transforming growth factor-β1 (TGF-β1) mRNA was almost unaffected in cells treated with visfatin, whereas the expression of hypoxia inducible factor-1α (HIF-1α) was increased significantly. Moreover, knockdown of HIF-1α by its specific shRNA inhibits the effect of visfatin on CTGF expression. In conclusion, the up-regulation of CTGF expression by visfatin might be mediated via HIF-1α -dependent pathway, but not the TGF-β1 pathway in EA.Hy926 cells.

Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis

Cardiac hypertrophy is highly prevalent in patients with type 2 diabetes mellitus. Experimental evidence has implied that pregnant women with type 2 diabetes mellitus and their children are at an increased risk of cardiovascular diseases. Our previous mouse model study revealed that maternal type 2 diabetes mellitus induces structural heart defects in their offspring. This study aims to determine whether maternal type 2 diabetes mellitus induces embryonic heart hypertrophy in a murine model of diabetic embryopathy. The type 2 diabetes mellitus embryopathy model was established by feeding 4-week-old female C57BL/6J mice with a high-fat diet for 15 weeks. Cardiac hypertrophy in embryos at embryonic day 17.5 was characterized by measuring heart size and thickness of the right and left ventricle walls and the interventricular septum, as well as the expression of β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, desmin, and adrenomedullin. Cardiac remodeling was determined by collagen synthesis and fibronectin synthesis. Fibrosis was evaluated by Masson staining and determining the expression of connective tissue growth factor, osteopontin, and galectin-3 genes. Cell apoptosis also was measured in the developing heart. The thicknesses of the left ventricle walls and the interventricular septum of embryonic hearts exposed to maternal diabetes were significantly thicker than those in the nondiabetic group. Maternal diabetes significantly increased β-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, and desmin expression, but decreased expression of adrenomedullin. Moreover, collagen synthesis was significantly elevated, whereas fibronectin synthesis was suppressed, in embryonic hearts from diabetic dams, suggesting that cardiac remodeling is a contributing factor to cardiac hypertrophy. The cardiac fibrosis marker, galectin-3, was induced by maternal diabetes. Furthermore, maternal type 2 diabetes mellitus activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P < .05). Maternal type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy.

Cyclic tensile strain combined with icariin promotes the osteogenic differentiation of adipose-derived stem cells

Objective To study the roles of icariin and cyclic tensile strain (CTS) in promoting the osteogenic differentiation of adipose-derived stem cells (ASCs) and the molecular mechanisms involved. Methods ASCs were isolated from Sprague-Dawley rats and treated either with icariin (10-7 mol/L) or with 1000 μ, 2000 μ or 3000 μ of CTS for 7 days, or with icariin plus CTS at 2000 μ for three days. Alkaline phosphatase (ALP) activity was detected after 3 and 7 days of intervention. Western blotting was performed to detect the expression of Runt-related transcriptional factor 2 (Runx2), Yes-associated protein (YAP) and connective tissue growth factor (CTGF) after the third day of the intervention. A reverse transcription polymerase chain reaction was performed at 7 days to detect the expression of osteopontin (OPN) and collagen 1a and after 3 days to detect the expression of the YAP target gene, CTGF and ankyrin repeating domain 1 (Ankrd1). Results Icariin and CTS at 1000 μ, 2000 μ or 3000 μ could all significantly promote the expression of ALP protein. CTS at 2000 μ was the most effective. The co-treatment with icariin and CTS significantly promoted ALP protein expression compared with icariin or CTS treatment alone. It also significantly promoted the expression of Runx2 and CTGF protein. Icariin or CTS (2000 μ) alone could not promote the expression of YAP protein, but icariin combined with CTS (2000 μ) promoted it significantly. Either icariin or CTS (2000 μ) could significantly promote ALP activity after 3 and 7 days, but icariin combined with CTS had the most obvious effect. Both icariin and CTS (2000 μ) could also significantly promote the expression of the osteogenesis-related genes OPN and collagen 1a, as well as the YAP targeted genes CTGF and Ankrd1. However, the combination of icariin and CTS had the greatest effect in promoting the expression of OPN mRNA, collagen 1a mRNA, CTGF mRNA and ankrd1 mRNA. Conclusion Icariin and CTS co-treatment may promote osteogenic differentiation of ASCs via activating YAP expression. Key words: Icariin; Cyclic tensile strain; Adipose-derived stem cells; Osteogenic differentiation

Integrin α2β1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma.

The Hippo pathway regulates the down-stream target Yes-associated protein (YAP) to maintain organ homeostasis, which is commonly inactivated in many types of cancers. However, how cell adhesion dysregulates the Hippo pathway activating YAP oncogene in hepatocellular carcinoma (HCC) remains unclear. Our findings demonstrate that α2β1 integrin (but not other β1 integrins) expressed in HCC cells, after binding to collagen extracellular matrix, could inhibit MST1 kinase phosphorylation and activate YAP pro-oncogenic activities. Knockdown of integrin α2 gene (ITGA2) suppressed YAP targeted gene expression in vitro. α2β1 and collagen binding resulted in suppressing Hippo signaling of mammalian sterile 20-like kinase 1 (MST1) and Large tumor suppressor homolog 1 (LATS1) with concomitant activation of YAP-mediated connective tissue growth factor (CTGF) gene expression. In vitro kinase assay showed that MST1 is an immediate downstream target of integrin α2 with S1180 residue as the critical phosphorylation site. Clinical correlational analysis using a gene expression dataset of 228 HCC tumors revealed that ITGA2 expression was significantly associated with tumor progression, and co-expression with YAP targeted genes (AXL receptor tyrosine kinase, CTGF, cyclin D1, glypican 3, insulin like growth factor 1 receptor, and SRY-box 4) correlated with survivals of HCC patients. In conclusion, α2β1 integrin activation through cellular adhesion impacts the Hippo pathway in solid tumors and modulates MST1-YAP signaling cascade. Targeting integrin α2 holds promises for treating YAP-positive HCC.

Oestrogens Stimulate Proliferation in Colorectal Cancer via GPER and the Hippo signalling pathway

The concentration of circulating oestrogens is associated with the incidence and outcomes of colorectal cancer (CRC). Both the physiological and pathological effects of oestrogens are mediated by oestrogen receptors. This project aims to investigate the potential role of G protein-coupled oestrogen receptor 1 (GPER) as an oestrogen-induced mediator in CRC proliferation. To achieve this, colorectal adenoma and carcinoma cell lines were examined for protein expression of oestrogen receptors. Immunoblotting did not report ERa and ERb expression, although GPER was detected. Following this, the GPER-associated gene connective tissue growth factor (CTGF) expression was measured after I7β-estradiol (E2) and GPER agonist (G1) treatment. qRT-PCR results showed no significant increase in CTGF expression levels, 24 and 48 hours after treatment. In addition, GPER interaction with the Hippo pathway in CRC was examined by treating cells with E2 and G1 for 0, 15, 30 minutes, 1, and 2 hours. Alterations in P-Y API expression were unclear after treatment in the cell lines examined. However, addition of GPER antagonist, G15, resulted in significant inhibition in YAP1 phosphorylation in HCT116 cells, 15 and 30 minutes of treatment. Consequently, our data supports that oestrogens and G1 treatment leads to increase in YAP phosphorylation and nucleus-cytoplasmic shuttling via GPER stimulation. YAP1 knock-down studies and pharmacological inhibition followed by proliferation assays established that this early metabolic effect translates into increased cellular proliferation. Collectively, our data propose a novel oestrogen-driven pro-proliferative pathway via GPER through Hippo pathway's key downstream effector, YAP1, in CRC. Further studies are required to 'reveal the detailed signalling cascade by which GPER mediates the increased YAP1 phosphorylation.

Per2-Mediated Vascular Dysfunction Is Caused by the Upregulation of the Connective Tissue Growth Factor (CTGF).

Period 2-mutant mice (Per2m/m), which possess a circadian dysfunction, recapitulate the retinal vascular phenotype similar to diabetic retinopathy (DR). The vascular dysfunction in Per2m/m is associated with an increase in connective tissue growth factor (CTGF/CCN2). At the molecular level, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. The nuclear binding of β-catenin to a transcription factor, lymphoid enhancer binding protein (Lef)/ T-cell factor (TCF/LEF), leads to downstream activation of CTGF. For this study, we hypothesized that the silencing of Per2 results in nuclear translocation and subsequent transactivation of the CTGF gene. To test this hypothesis, we performed immunofluorescence labeling for CTGF in retinal sections from wild-type (WT) and Per2m/m mice. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2, and the protein expression of CTGF and β-catenin was evaluated. The TCF/LEF luciferase reporter (TOPflash) assay was performed to validate the involvement of β-catenin in the activation of CTGF. Per2m/m retinas exhibited an increased CTGF immunostaining in ganglion cell layer and retinal endothelium. Silencing of Per2 using siRNA resulted in an upregulation of CTGF and β-catenin. The TOPflash assay revealed an increase in luminescence for HRECs transfected with Per2 siRNA. Our studies show that loss of Per2 results in an activation of CTGF via nuclear entry of β-catenin. Our study provides novel insight into the understanding of microvascular dysfunction in Per2m/m mice.

Short Hairpin RNA Knockdown of Connective Tissue Growth Factor by Ultrasound-Targeted Microbubble Destruction Improves Renal Fibrosis

The purpose of this study was to evaluate whether ultrasound-targeted microbubble destruction transfer of interfering RNA against connective tissue growth factor (CTGF) in the kidney would ameliorate renal fibrosis in vivo. A short hairpin RNA (shRNA) targeting CTGF was cloned into a tool plasmid and loaded onto the surface of a cationic microbubble product. A unilateral ureteral obstruction (UUO) model in mice was used to evaluate the effect of CTGF knockdown. Mice were administered the plasmid-carrying microbubble intravenously, and ultrasound was applied locally to the obstructed kidney. Mice undergoing a sham UUO surgery and untreated UUO mice were used as disease controls, and mice administered plasmid alone, plasmid with ultrasound treatment and microbubbles and plasmid without ultrasound were used as treatment controls. Mice were treated once and then evaluated at day 14. CTGF in the kidney was measured by quantitative reverse transcription polymerase chain reaction and Western blot. Expression of CTGF, transforming growth factor β1, α smooth muscle actin and type I collagen in the obstructed kidney was evaluated by immunohistochemistry. The cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA and protein expression of CTGF (p < 0.01). Furthermore, CTGF gene silencing decreased the interstitial deposition of transforming growth factor β1, α smooth muscle actin and type I collagen as assessed in immunohistochemistry, as well as reduced renal fibrosis in pathologic alterations (p < 0.01). No significant changes in target mRNA, protein expression or disease pathology were observed in the control cohorts. A single treatment of ultrasound-targeted microbubble destruction is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in disease severity. This technique may be a potential therapy for treatment of renal fibrosis.