Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia.

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

Mechanical stress-induced connective tissue growth factor plays a critical role in intestinal fibrosis in Crohn's-like colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of profibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMCs). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends toward normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.NEW & NOTEWORTHY We found that CTGF expression increased significantly not only in the inflammation site but in the distended segment proximal to inflammation in a rodent model of CD-like colitis. Release of mechanical distention prevented CTGF expression in CD rats, whereas direct stretch induced CTGF expression. Treatment with anti-CTGF antibody reduced fibrosis and collagen contents in CD rats. Thus, mechanical stress, via upregulating profibrotic mediators, i.e., CTGF, may play a critical role in fibrosis in CD.

Ccn2a is an injury-induced matricellular factor that promotes cardiac regeneration in zebrafish.

The ability of zebrafish to heal their heart after injury makes them an attractive model for investigating the mechanisms governing the regenerative process. In this study, we show that the gene cellular communication network factor 2a (ccn2a), previously known as ctgfa, is induced in endocardial cells in the injured tissue and regulates CM proliferation and repopulation of the damaged tissue. We find that, whereas in wild-type animals, CMs track along the newly formed blood vessels that revascularize the injured tissue, in ccn2a mutants CM proliferation and repopulation are disrupted, despite apparently unaffected revascularization. In addition, we find that ccn2a overexpression enhances CM proliferation and improves the resolution of transient collagen deposition. Through loss- and gain-of-function as well as pharmacological approaches, we provide evidence that Ccn2a is necessary for and promotes heart regeneration by enhancing the expression of pro-regenerative extracellular matrix genes, and by inhibiting the chemokine receptor gene cxcr3.1 through a mechanism involving Tgfß/pSmad3 signaling. Thus, Ccn2a positively modulates the innate regenerative response of the adult zebrafish heart.

CTGF regulates mineralization in human mature chondrocyte by controlling Pit-1 and modulating ANK via the BMP/Smad signalling.

OBJECTIVE: Connective tissue growth factor (CTGF) exhibits potent proliferative, differentiated, and mineralizing effects, and is believed to be contribute to cartilage mineralization in Osteoarthritis (OA). However, the underlying mechanism of chondrocyte mineralization induced by CTGF remains obscure. As a key regulator of mineral responses, type III phosphate transporter 1 (Pit-1) has been associated with the pathogenesis of articular mineralization. Therefore, the primary objective of this study was to investigate whether CTGF influences the development of mature chondrocyte mineralization and the underlying mechanisms governing such mineralization. METHODS: The effect of Connective tissue growth factor (CTGF) on human C-28/I2 chondrocytes were investigated. The chondrocytes were treated with CTGF or related inhibitors, and transfected with Overexpression and siRNA transfection of Type III Phosphate Transporter 1(Pit-1). Subsequently, the cells were subjected to Alizarin red S staining, PiPer Phosphate Assay Kit, Alkaline Phosphatase Diethanolamine Activity Kit, ELISA, RT-PCR or Western blot analysis. RESULTS: Stimulation with Connective tissue growth factor (CTGF) significantly upregulated the expression of the Type III Phosphate Transporter 1(Pit-1) and mineralization levels in chondrocytes through activation of α5ß1 integrin and BMP/Samd1/5/8 signaling pathways. Furthermore, treatment with overexpressed Pit-1 markedly increased the expression of Multipass Transmembrane Ankylosis (ANK) transporter in the cells. The inhibitory effect of CTGF receptor blockade using α5ß1 Integrin blocking antibody was demonstrated by significantly suppressed the expression of Pit-1 and ANK transporter, as well as chondrocyte mineralization. CONCLUSIONS: Our data indicate that Connective tissue growth factor (CTGF) plays a critical role inchondrocyte mineralization, which is dependent on the expression of the Type III Phosphate Transporter 1(Pit-1) and Multipass Transmembrane Ankylosis (ANK) transporter. Consequently, inhibition of CTGF activity may represent a novel therapeutic approach for the management of Osteoarthritis (OA).

Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.

The sustained release of profibrotic cytokines, mainly transforming growth factor-ß (TGF-ß), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-ß for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.

Transcriptional regulation of CYR61 and CTGF by LM98: a synthetic YAP-TEAD inhibitor that targets in-vitro vasculogenic mimicry in glioblastoma cells.

Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1 , TEAD1 , AXL , NF2 , CTGF , and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF , or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61 / CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.

Down-regulation of CYTL1 attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting M2 macrophage polarization via the TGF-ß/CCN2 axis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by chronic inflammation, lung tissue fibrotic changes and impaired lung function. Pulmonary fibrosis 's pathological process is thought to be influenced by macrophage-associated phenotypes. IPF treatment requires specific targets that target macrophage polarization. Cytokine-like 1(CYTL1) is a secreted protein with multiple biological functions first discovered in CD34+ haematopoietic cells. However, its possible effects on IPF progression remain unclear. This study investigated the role of CYTL1 in IPF progression in a bleomycin-induced lung injury and fibrosis model. In bleomycin-induced mice, CYTL1 is highly expressed. Moreover, CYTL1 ablation alleviates lung injury and fibrosis in vivo. Further, downregulating CYTL1 reduces macrophage M2 polarization. Mechanically, CYTL1 regulates transforming growth factor ß (TGF-ß)/connective tissue growth factor (CCN2) axis and inhibition of TGF-ß pathway alleviates bleomycin-induced lung injury and fibrosis. In conclusion, highly expressed CYTL1 inhibits macrophage M2 polarization by regulating TGF-ß/CCN2 expression, alleviating bleomycin-induced lung injury and fibrosis. CYTL1 could, therefore, serve as a promising IPF target.

Neutralization of CX3CL1 Attenuates TGF-ß-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-ß1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Context-dependent effects of CCN2 on ß-cell mass expansion and indicators of cell stress in the setting of acute and chronic stress.

Stimulation of functional ß-cell mass expansion can be beneficial for the treatment of type 2 diabetes. Our group has previously demonstrated that the matricellular protein CCN2 can induce ß-cell mass expansion during embryogenesis, and postnatally during pregnancy and after 50% ß-cell injury. The mechanism by which CCN2 stimulates ß-cell mass expansion is unknown. However, CCN2 does not induce ß-cell proliferation in the setting of euglycemic and optimal functional ß-cell mass. We thus hypothesized that ß-cell stress is required for responsiveness to CCN2 treatment. In this study, a doxycycline-inducible ß-cell-specific CCN2 transgenic mouse model was utilized to evaluate the effects of CCN2 on ß-cell stress in the setting of acute (thapsigargin treatment ex vivo) or chronic [high-fat diet or leptin receptor haploinsufficiency (db/+) in vivo] cellular stress. CCN2 induction during 1 wk or 10 wk of high-fat diet or in db/+ mice had no effect on markers of ß-cell stress. However, CCN2 induction did result in a significant increase in ß-cell mass over high-fat diet alone when animals were fed high-fat diet for 10 wk, a duration known to induce insulin resistance. CCN2 induction in isolated islets treated with thapsigargin ex vivo resulted in upregulation of the gene encoding the Nrf2 transcription factor, a master regulator of antioxidant genes, suggesting that CCN2 further activates this pathway in the presence of cell stress. These studies indicate that the potential of CCN2 to induce ß-cell mass expansion is context-dependent and that the presence of ß-cell stress does not ensure ß-cell proliferation in response to CCN2.NEW & NOTEWORTHY CCN2 promotes ß-cell mass expansion in settings of suboptimal ß-cell mass. Here, we demonstrate that the ability of CCN2 to induce ß-cell mass expansion in the setting of ß-cell stress is context-dependent. Our results suggest that ß-cell stress is necessary but insufficient for CCN2 to increase ß-cell proliferation and mass. Furthermore, we found that CCN2 promotes upregulation of a key antioxidant transcription factor, suggesting that modulation of ß-cell oxidative stress contributes to the actions of CCN2.

YAP, CTGF and Cyr61 are overexpressed in tamoxifen-resistant breast cancer and induce transcriptional repression of ERα.

About 70% of breast cancers overexpress estrogen receptor α (ERα, encoded by ESR1). Tamoxifen, a competitive inhibitor of estrogen that binds to ER, has been widely used as a treatment for ER-positive breast cancer. However, 20-30% of breast cancer is resistant to tamoxifen treatment. The mechanisms underlying tamoxifen resistance remain elusive. We found that Yes-associated protein (YAP; also known as YAP1), connective tissue growth factor (CTGF; also known as CCN2) and cysteine-rich angiogenic inducer 61 (Cyr61; also known as CCN1) are overexpressed, while ERα is downregulated in tamoxifen-resistant breast cancer. Inhibition of YAP, CTGF and Cyr61 restored ERα expression and increased sensitivity to tamoxifen. Overexpression of YAP, CTGF, and Cyr61 led to downregulation of ERα and conferred resistance to tamoxifen in ER-positive breast cancer cells. Mechanistically, CTGF and Cyr61 downregulated ERα expression at the transcriptional level by directly binding to the regulatory regions of the ERα-encoding gene, leading to increased tamoxifen resistance. Also, CTGF induced Glut3 (also known as SLC2A3) expression, leading to increased glycolysis, which enhanced cell proliferation and migration in tamoxifen-resistant cells. Together, these results demonstrate a novel role of YAP, CTGF and Cyr61 in tamoxifen resistance and provide a molecular basis for their function in tamoxifen-resistant breast cancer.

METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner†.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy, and it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

The role of YAP1 target gene CTGF in the anoikis resistance of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). METHODS: Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. RESULTS: Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and ß1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. CONCLUSION: The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention.

Hsa-miR-379 down-regulates Rac1/MLK3/JNK/AP-1/Collagen I cascade reaction by targeting connective tissue growth factor in human alveolar basal epithelial A549 cells.

OBJECTIVE: This study was aimed to screen and identify miRNAs that could regulate human CTGF gene and downstream cascade reaction Rac1/MLK3/JNK/AP-1/Collagen I by bioinformatics and experimental means. METHODS: TargetScan and Tarbase were used to predict miRNAs that may have regulatory effects on human CTGF gene. The dual-luciferase reporter gene assay was employed to verify the results obtained in bioinformatics. Human alveolar basal epithelial A549 cells were exposed to silica (SiO2) culture medium for 24 h to establish an in vitro model of pulmonary fibrosis, and bleomycin (BLM) of 100 ng/mL was used as a positive control. The miRNA and mRNA expression levels were determined by RT-qPCR, and the protein levels were measured by western blot in hsa-miR-379-3p overexpression group or not. RESULTS: A total of 9 differentially expressed miRNAs that might regulate the human CTGF gene were predicted. Hsa-miR-379-3p and hsa-miR-411-3p were selected for the subsequent experiments. The results of the dual-luciferase reporter assay showed that hsa-miR-379-3p could bind to CTGF, but hsa-miR-411-3p could not. Compared with the control group, SiO2 exposure (25 and 50 µg/mL) could significantly reduce the expression level of hsa-miR-379-3p in A549 cells. SiO2 exposure (50 µg/mL) could significantly increase the mRNA expression levels of CTGF, Collagen I, Rac1, MLK3, JNK, AP1, and VIM in A549 cells, while CDH1 level was significantly decreased. Compared with SiO2 + NC group, the mRNA expression levels of CTGF, Collagen I, Rac1, MLK3, JNK, AP1, and VIM were significantly decreased, and CDH1 level was significantly higher when hsa-miR-379-3p was overexpressed. At the same time, overexpression of hsa-miR-379-3p improved the protein levels of CTGF, Collagen I, c-Jun and phospho-c-Jun, JNK1 and phospho-JNK1 significantly compared with SiO2 + NC group. CONCLUSION: Hsa-miR-379-3p was demonstrated for the first time that could directly target and down-regulate human CTGF gene, and further affect the expression levels of key genes and proteins in Rac1/MLK3/JNK/AP-1/Collagen I cascade reaction.

EGF promotes human trophoblast cell invasion by downregulating CTGF expression via PI3K/AKT signaling.

In brief: Although the pro-invasive role of epidermal growth factor (EGF) has been reported in human trophoblast cells, the underlying mechanism remains largely unexplored. This work reveals that EGF-induced downregulation of connective tissue growth factor (CTGF) mediates the EGF-stimulated human trophoblast cell invasion. Abstract: During the development of the placenta, trophoblast cell invasion must be carefully regulated. Although EGF has been shown to promote trophoblast cell invasion, the underlying mechanism remains largely undetermined. Our previous study using RNA-sequencing (RNA-seq) has identified that kisspeptin-1 is a downstream target of EGF in a human trophoblast cell line, HTR-8/SVneo, and mediates EGF-stimulated cell invasion. In the present study, after re-analysis of our previous RNA-seq data, we found that the CTGF was also downregulated in response to the EGF treatment. The inhibitory effects of EGF on CTGF mRNA and protein levels were confirmed in HTR-8/SVneo cells by reverse transcription quantitative real-time PCR and western blot, respectively. Treatment with EGF activated both PI3K/AKT and ERK1/2 signaling pathways. Using pharmacological inhibitors, our results showed that EGFR-mediated activation of PI3K/AKT signaling was required for the EGF-downregulated CTGF mRNA and protein levels. Matrigel-coated transwell invasion assays demonstrated that EGF treatment stimulated cell invasion. In addition, the invasiveness of HTR-8/SVneo cells was suppressed by treatment with recombinant human CTGF. By contrast, siRNA-mediated knockdown of CTGF increased cell invasion. Notably, the EGF-promoted HTR-8/SVneo cell invasion was attenuated by co-treatment with CTGF. This study provides important insights into the molecular mechanisms mediating EGF-stimulated human trophoblast cell invasion and increases the understanding of the biological functions of CTGF in the human placenta.

CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s.

BACKGROUND: Functional alveolar regeneration is essential for the restoration of normal lung homeostasis after acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Lung is a relatively quiescent organ and a variety of stem cells are recruited to participate in lung repair and regeneration after lung tissue injury. However, there is still no effective method for promoting the proliferation of endogenous lung stem cells to promote repair and regeneration. METHODS: Using protein mass spectrometry analysis, we analyzed the microenvironment after acute lung injury. RNA sequencing and image cytometry were used in the alveolar epithelial type 2 cells (AEC2s) subgroup identification. Then we used Sftpc+AEC2 lineage tracking mice and purified AEC2s to further elucidate the molecular mechanism by which CTGF regulates AEC2s proliferation both in vitro and in vivo. Bronchoalveolar lavage fluid (BALF) from thirty ARDS patients who underwent bronchoalveolar lavage was collected for the analysis of the correlation between the expressing of Krt5 in BALF and patients' prognosis. RESULTS: Here, we elucidate that AEC2s are the main facultative stem cells of the distal lung after ALI and ARDS. The increase of connective tissue growth factor (CTGF) in the microenvironment after ALI promoted the proliferation of AEC2s subpopulations. Proliferated AEC2s rapidly expanded and differentiated into alveolar epithelial type 1 cells (AEC1s) in the regeneration after ALI. CTGF initiates the phosphorylation of LRP6 by promoting the interaction between Krt5 and LRP6 of AEC2s, thus activating the Wnt signaling pathway, which is the molecular mechanism of CTGF promoting the proliferation of AEC2s subpopulation. CONCLUSIONS: Our study verifies that CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of AEC2s subpopulation.

Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation in renal fibroblast.

Kinesin family member 26b (Kif26b) is essential for kidney development, and its deletion in mice leads to kidney agenesis. However, the roles of this gene in adult settings remain elusive. Thus, this study aims to investigate the role of Kif26b in the progression of renal fibrosis. A renal fibrosis model with adenine administration using Kif26b heterozygous mice and wild-type mice was established. Renal fibrosis and the underlying mechanism were investigated. The underlying pathways and functions of Kif26b were evaluated in an in vitro model using primary renal fibroblasts. Kif26b heterozygous mice were protected from renal fibrosis with adenine administration. Renal expressions of connective tissue growth factor (CTGF) and myofibroblast accumulation were reduced in Kif26b heterozygous mice. The expression of nonmuscle myosin heavy chain II (NMHCII), which binds to the C-terminus of Kif26b protein, was also suppressed in Kif26b heterozygous mice. The in vitro study revealed reduced expressions of CTGF, α-smooth muscle actin, and myosin heavy chain 9 (Myh9) via transfection with siRNAs targeting Kif26b in renal fibroblasts (RFB). RFBs, which were transfected by the expression vector of Kif26b, demonstrated higher expressions of these genes than non-transfected cells. Finally, Kif26b suppression and NMHCII blockage led to reduced abilities of migration and collagen gel contraction in renal fibroblasts. Taken together, Kif26b contributes to the progression of interstitial fibrosis via migration and myofibroblast differentiation through Myh9 in the renal fibrosis model. Blockage of this pathway at appropriate timing might be a therapeutic approach for renal fibrosis.