BACKGROUNDProtein arginine methyltransferases 1 (PRMT1) is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC) migration in vitro.

METHODSIn this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2) were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT) (E-cadherin, N-cadherin), focal adhesion kinase (FAK), Src, AKT, and their corresponding phosphorylated states were detected by Western blot.

RESULTSCell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.

CONCLUSIONSPRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; enzymology ; genetics ; Cell Line ; Cell Movement ; genetics ; physiology ; Epithelial-Mesenchymal Transition ; genetics ; physiology ; Extracellular Matrix Proteins ; metabolism ; Humans ; Protein-Arginine N-Methyltransferases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; physiology

OBJECTIVEDiabetic nephropathy (DN) is the major cause of end-stage renal disease worldwide and its prevalence continues to increase. Currently, therapies for DN provide only partial renoprotection; hence new targets for therapeutic intervention need to be identified. In this review, we summarized the new target, sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway, explored its potential therapeutic role in the prevention and treatment of DN.

DATA SOURCESMost relevant articles were mainly identified by searching PubMed in English.

STUDY SELECTIONMainly original articles and critical review articles by major pioneer investigators in this field were selected to be reviewed.

RESULTSSphK1/S1P pathway can be activated by hyperglycemia, advanced glycation end products, and many pro-inflammatory cytokines, which leads to fibronectin, transforming growth factor-β1 up-regulation and AP-1 activation. And then it could promote glomerular mesangial cells proliferation and extracellular matrix accumulation, mediating the initiation and progression of diabetic renal fibrosis.

CONCLUSIONSSphK1/S1P pathway is closely correlated with the pathogenesis of DN. The results suggest that SphK1/S1P pathway as a new target for clinically improving DN in future is of great prospect.

Diabetic Nephropathies ; enzymology ; metabolism ; Extracellular Matrix ; metabolism ; Humans ; Lysophospholipids ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Signal Transduction ; Sphingosine ; analogs & derivatives ; metabolism

Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10-4 to 10-6 M. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.
Antigens, CD/metabolism ; Caspases/metabolism ; Cell Differentiation/*drug effects ; Cell Survival/drug effects ; Collagen Type I/metabolism ; Diphosphonates/*pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Matrix Metalloproteinase 2/metabolism ; NF-kappa B/metabolism ; Osteoblasts/*cytology/*drug effects/enzymology ; Protein C/*pharmacology ; Receptors, Cell Surface/metabolism ; Up-Regulation/drug effects

OBJECTIVEThe imbalance between extracellular matrix (ECM) synthesis and degradation induces the excessive ECM deposition and thus renal fibrosis. The decoction (A&A) which is a combination of two Chinese herbs, Astragalus membranaceus var. mongholicus and Angelica sinensis, has been shown to alleviate ECM production in animal models of chronic kidney diseases. In this paper, the effect of A&A on ECM degradation was investigated with interstitial fibrosis in rats.

METHODMale Wistar rats were randomly divided into sham, unilateral ureteral obstruction (UUO) and UAA (UUO plus A&A administration) groups. After administration of A&A (14 g x kg(-1) x d(-1)) by gavage for 3, 7 and 10 days, morphological changes were evaluated by HE, PAS and Sirius red staining technique. The expression of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA), the activity of PAI-1 and t-PA were determined by ELISA. The activity of matrix metalloproteinases (MMP-9, MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were evaluated by gelatin zymography or reverse gelatin zymography, respectively.

RESULTMorphological analysis showed severe interstitial mononuclear cells infiltration, tubular atrophy, renal fibrosis and collagen expression in kidneys of UUO group, which was reduced by A&A administration (P < 0.05, UAA vs UUO group). Compared with the sham group, the expression of PAI-1 was significantly increased in UUO group by 63%, 91% and 112% at day 3, 7 and 10 respectively; and there were a remarkable decrease in UAA group by 44%, 43% and 52% at day 3, 7 and 10. The expression of active PAI-1 was strikingly increased in UUO group at day 3 [(30.5 +/- 23.8) ng x g(-1) vs. (0.0 +/- 0.0) ng x g(-1), P < 0.05)], day 7 [(36.5 +/- 11.2) ng x g(-1) vs. (0.0 +/- 0.0) ng x g(-1), P < 0.05)], and day 10 [(54.5 +/- 14.2) ng x g(-1) vs. (0.5 +/- 0.5) ng x g(-1), P < 0.05)]. The active PAI-1 was decreased in UAA group at day 7 [(14.9 +/- 0.5) ng x g(-1) vs. (36.5 +/- 11.2) ng x g(-1), P < 0.05] and day 10 [(15.4 +/- 4.8) ng x g(-1) vs. (54.5 +/- 14.2) ng x g(-1), P < 0.05]. The expression of t-PA was increased in UUO group only at day 3 [(58.1 +/- 16.5) microg x g(-1) vs. (30.1 +/- 17.3) microg x g(-1)], P < 0.05), meanwhile decreased in UAA group [(26.3 +/- 8.7) microg x g(-1) vs. (58.1 +/- 16.5) microg x g(-1), P < 0.05)]. But the expression of active t-PA was shown no significantly difference among the three groups. For MMP-2 and MMP-9 activity, they were significantly higher compared with the sham group in UUO group, but no significantly change after A&A treatment. The TIMP-1 activity was significantly increased in UUO group by 28% and 63% at day 7 and 10 respectively, significantly decreased in UAA group by 40% and 39% at the same time point.

CONCLUSIONThe anti-fibrosis effects of A&A might be associated with modulating the imbalance of PAs/PAIs system as well as MMPs/TIMPs system, thereby alleviate ECM accumulation and interstitial fibrosis.

Angelica sinensis ; chemistry ; Animals ; Astragalus Plant ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; Extracellular Matrix ; metabolism ; Fibrosis ; Humans ; Kidney ; enzymology ; metabolism ; pathology ; Kidney Diseases ; drug therapy ; enzymology ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Plasminogen Activator Inhibitor 1 ; metabolism ; Rats ; Rats, Wistar ; Tissue Plasminogen Activator ; metabolism

We investigate the role of β-catenin signaling in the response of macrophage to lipopolysaccharide (LPS) using RAW264.7 cells. LPS rapidly stimulated cytosolic β-catenin accumulation. β-catenin-mediated transcription was showed to be required for LPS induced gene expression and cell migration. Mechanically, ERK activation-primed GSK3β inactivation by Akt was demonstrated to mediate the LPS induced β-catenin accumulation. Overall, our findings suggest that suppression of GSK3β by ERK stimulates β-catenin signaling therefore contributes to LPS induced cell migration in macrophage activation.
Animals ; Cell Line ; Cell Movement ; drug effects ; Enzyme Activation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; enzymology ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Mice ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Transcription, Genetic ; drug effects ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate the in vitro effects and the primary mechanisms of Changweiqing (, CWQ) on antimetastasis and antiinvasion of hypoxic colon carcinoma cells. In addition, to provide experimental evidence for the Chinese medicinal theory of "strengthening the body's resistance to eliminate pathogenic factors" in the treatment of colorectal cancer, including its invasion and metastasis.

METHODSFirst, CWQ sera were prepared with serum-pharmacology methods. Then, the modified hypoxic chamber was designed and flushed with 5% CO(2) and 95% N(2) at 37 °C to induce a hypoxic environment. The effect of CWQ serum on the viability of LoVo cells was tested with MTT cytotoxicity assay. The wound model and chamber model were established to estimate the effects of CWQ serum on migration and invasion of LoVo cells. The model for cell adhesion was established to evaluate the effect of CWQ serum on LoVo cells' adhesion. The gelatin zymography model was performed to determine the effects of CWQ serum on the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The effects of CWQ serum on the hypoxia-inducible factor 1 α (HIF-1α) nuclear translocation and the mRNA level of vascular endothelial growth factor (VEGF) in LoVo cells were determined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively.

RESULTSCWQ inhibited LoVo cells' migration based on wound healing assay. The inhibitive effect could reach about 68.00% under hypoxic culture and about 29.87% under normoxic culture when cells were treated with 10% CWQ serum for 24 h. The results from both cell invasion and adhesion assays showed that CWQ serum could dose-dependently repress the invasion of LoVo cells and inhibit cells from adhering to extra cellular matrix (ECM). Under the hypoxic culture condition, RT-PCR analysis showed that 10% CWQ serum had down-regulated the expression of VEGF by 45.87%, and the result of Western blot analysis provided further evidence. The HIF-1α amount in the nucleus of the LoVo cells was also diminished in a dose-dependent manner, as shown by the Western blot. Gel zymogram assay revealed that CWQ serum could suppress the activities of MMP-2 and MMP-9.

CONCLUSIONSCWQ could effectively inhibit tumor metastasis in vitro The antimetastatic effects of CWQ were associated with the inhibition of cell motility, which was evidenced by inhibition of cell invasion and adhesion. The molecular mechanisms of the inhibition of tumor invasion by CWQ were due to the reduced expression of both HIF-1α and VEGF and the suppression of MMP-2 and MMP-9 expression.

Animals ; Cell Adhesion ; drug effects ; Cell Hypoxia ; drug effects ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Colorectal Neoplasms ; enzymology ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Matrix ; drug effects ; metabolism ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Protein Transport ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.
Animals ; Bone Morphogenetic Proteins/metabolism ; Cartilage/*cytology/metabolism ; *Cell Differentiation ; Chondrocytes/cytology/enzymology ; Extracellular Matrix Proteins/deficiency/*metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Integrin alpha5/metabolism ; Mesenchymal Stem Cells/cytology/metabolism ; Mice ; Neoplasm Proteins/deficiency/*metabolism ; Osteogenesis ; Protein Binding ; Signal Transduction ; Smad Proteins/metabolism

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4).
ADAM Proteins ; antagonists & inhibitors ; chemistry ; genetics ; physiology ; Aggrecans ; metabolism ; Alternative Splicing ; Arthritis ; enzymology ; genetics ; Cartilage ; enzymology ; Endopeptidases ; genetics ; physiology ; Extracellular Matrix ; enzymology ; Humans ; Protein Structure, Tertiary

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; metabolism ; Gene Expression Regulation, Enzymologic ; Hepatic Stellate Cells ; drug effects ; enzymology ; Leptin ; administration & dosage ; genetics ; pharmacology ; Liver Cirrhosis ; etiology ; pathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

Antigens, Bacterial ; metabolism ; Bacterial Adhesion ; physiology ; Biofilms ; growth & development ; Dental Caries ; microbiology ; Extracellular Matrix ; physiology ; Glucosyltransferases ; metabolism ; Humans ; Mouth ; microbiology ; Polysaccharides, Bacterial ; physiology ; Streptococcus mutans ; enzymology ; pathogenicity ; physiology ; Virulence ; Virulence Factors ; physiology