Endothelial cell-derived MMP19 promotes pulmonary fibrosis by inducing E(nd)MT and monocyte infiltration.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in remodeling the extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). MMP19, which is an MMP, was significantly upregulated in hyperplastic alveolar epithelial cells in IPF lung tissues and promoted epithelial-mesenchymal transition (EMT). Recent studies have demonstrated that endothelial-to-mesenchymal transition (E(nd)MT) contributes to pulmonary fibrosis. However, the role of MMP19 in pulmonary vascular injury and repair and E(nd)MT remains unclear. METHODS: To determine the role of MMP19 in E(nd)MT and pulmonary fibrosis. MMP19 expressions were determined in the lung endothelial cells of IPF patients and bleomycin (BLM)-induced mice. The roles of MMP19 in E(nd)MT and endothelial barrier permeability were studied in the MMP19 cDNA-transfected primary human pulmonary microvascular endothelial cells (HPMECs) and MMP19 adenoassociated virus (MMP19-AAV)-infected mice. The regulatory mechanism of MMP19 in pulmonary fibrosis was elucidated by blocking its interacting proteins SDF1 and ET1 with AMD3100 and Bosentan, respectively. RESULTS: In this study, we found that MMP19 expression was significantly increased in the lung endothelial cells of IPF patients and BLM-induced mice compared to the control groups. MMP19 promoted E(nd)MT and the migration and permeability of HPMECs in vitro, stimulated monocyte infiltration into the alveolus, and aggravated BLM-induced pulmonary fibrosis in vivo. SDF1 and Endothelin-1 (ET1) were physically associated with MMP19 in HPMECs and colocalized with MMP19 in endothelial cells in IPF patient lung tissues. AMD3100 and bosentan alleviated the fibrosis induced by MMP19 in the BLM mouse model. CONCLUSION: MMP19 promoted E(nd)MT by interacting with ET1 and stimulated monocyte infiltration into lung tissues via the SDF1/CXCR4 axis, thus aggravating BLM-induced pulmonary fibrosis. Vascular integrity regulated by MMP19 could be a promising therapeutic target for suppressing pulmonary fibrosis. Video abstract.

Prolactin Protects the Structural Integrity of Human Fetal Membranes by Downregulating Inflammation-induced Secretion of Matrix Metalloproteinases.

Prolactin (PRL) is a pleiotropic hormone with a key role in pregnancy. In fetal membranes, PRL can regulate the secretion of pro-inflammatory factors, which induces the activation of matrix metalloproteinases (MMPs). The increase and activation of MMPs deregulate the turnover of the extracellular matrix in the fetal membranes, altering its structure and function, causing premature rupture of the membranes and preterm labor. In this work, we evaluate the effect of PRL upon the secretion of MMP-1, MMP-2, MMP-9, MMP-13, and the tissue inhibitors of metalloproteinases (TIMPs) in human fetal membranes after lipopolysaccharide (LPS) challenge. Nine fetal membranes from healthy non-laboring cesarean deliveries at term were cultured in a 2-independent chamber system and pre-treated with 250, 500, 1000 or 4000 ng/ml of PRL for 24 h, then choriodecidual region was stimulated with 500 ng/ml of LPS plus fresh PRL for 24 h. The MMPs and TIMPs secretion were quantified by ELISA, additionally MMP-2 and MMP-9 gelatinolytic activity was measured by zymography. LPS induced the MMP-9 and MMP-1 secretion, but no MMP-2 or MMP-13 in comparison with basal levels. PRL co-treatment decreased the MMP-2, MMP-9 and MMP-1 secretion induced by LPS. The active forms were present in the tissue extract, showing a response consistent with the secretion profile. TIMP-1 and TIMP-2 secretion was decreased after LPS treatment and the PRL co-treatment reverts this effect. The present results support that PRL may favor the balance between these factors involved in the structural maintenance of fetal membranes in an inflammatory event.

Long noncoding RNA ZEB1-AS1 affects paclitaxel and cisplatin resistance by regulating MMP19 in epithelial ovarian cancer cells.

PURPOSE: The long noncoding RNA (lncRNA) ZEB1-AS1 is reported overexpressed in sensitive ovarian cancer cells A2780 compared with paclitaxel (PTX)-and cisplatin (DDP)- resistant. However, the function and mechanism of ZEB1-AS1 in EOC cells still unknown. METHODS: We used quantitative real-time PCR (qPCR) to detect ZEB1-AS1 expression in A2780 and A2780/R cells. A combination of siRNA, plasmids, CCK8 and flow cytometry was used to detect the effect of ZEB1-AS1 on ovarian cancer cell A2780 PTX and DDP resistance. Transcriptome sequencing, qPCR, and western blot were used for further mechanistic studies. RESULTS: ZEB1-AS1 depletion using siRNA in chemosensitive A2780 cells significantly increased PTX and DDP resistance. In contrast, ZEB1-AS1 overexpression in PTX- and DDP-resistant A2780/resistant (A2780/R) cells reversed the observed drug resistance. Thus, ZEB1-AS1 plays an important role in PTX and DDP resistance in EOC cells. However, quantitative real-time PCR (qPCR) and western blot results suggested that ZEB1-AS1 did not regulate chemoresistance through regulation of ZEB1 protein. We used sequencing to detect mRNA expression changes in A2780 cells after ZEB1-AS1 silencing. The results indicated that MMP19 was the likely downstream factor of ZEB1-AS1. We further examined whether ZEB1-AS1 played an important role in chemoresistance by silencing MMP19 in ZEB1-AS1-overexpressing cells. CCK8 assay results suggested that MMP19 knockdown promoted ZEB1-AS1-induced chemoresistance to PTX and DDP in A2780 cells. CONCLUSION: This study is the first to reveal that ZEB1-AS1 plays a pivotal role in cancer chemoresistance.

The prognostic value and potential mechanism of Matrix Metalloproteinases among Prostate Cancer.

Background: Matrix Metalloproteinases (MMPs) play an indispensable role in the initial alteration and development of PCa. We tried to generate an MMP-related prognostic signature (MMPS) in prostate cancer (PCa). Methods: TCGA-PRAD, MSKCC/GSE21032, GSE116918, GSE70769 cohorts were enrolled to assess the prognostic value of MMPs. The least absolute shrinkage and selection operator (LASSO) Cox regression was employed to generate the MMPS signature. The log-rank test and Kaplan-Meier (K-M) survival curve were applied to show the difference RFS, The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) was plotted to predict the accuracy of signature. CIBERSORT was conducted to analyze the different immune infiltration in MMPS-H and MMPS-L groups. Potential signaling pathways activated in the MMPS-H groups by Metascape. Results: MMP1, MMP7, MMP11, MMP24 and MMP26 were selected by LASSO regression and established the MMPS predict signature. The MMPS showed the high prognostic value in TCGA-PRAD training cohort (AUC=0.714) and validation cohorts (GSE116918: AUC=0.976, GSE70769: AUC=0.738, MSKCC: AUC=0.793). Pid integrin1 pathway, G2M checkpoint, and response to growth factor signaling pathways were activated in MMPS-H group, patients with the high MMPS risk score and low M2 macrophage showed the worst recurrence-free survival (RFS). Conclusion: MMPs involved and played an essential role in the tumorigenesis and biochemical recurrence in PCa patients. The MMPS signature could accurately predict the recurrence of PCa patients and validated in several cohorts.

Microglial activation in an amyotrophic lateral sclerosis-like model caused by Ranbp2 loss and nucleocytoplasmic transport impairment in retinal ganglion neurons.

Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ran-binding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1+-motoneurons develop cardinal ALS-like motor traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This will help to understand the role of nucleocytoplasmic transport in the differential vulnerability of neuronal cell types to ALS and to uncover non-motor endophenotypes with pathognomonic signs of ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1+-RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified CD11b+ and CD45+-microglia, increase of F4\80+-microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80+-microglia intermingled between RGNs of naive mice. Further, there was the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Hence, Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neuronal-type selective substrates. Immune-modulators underpinning RGN-to-microglial signaling are regulated by Ranbp2, and this neuronal-glial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of motoneuron diseases, such as ALS.

Mechanical injury and IL-1ß regulated LOXs and MMP-1, 2, 3 expression in ACL fibroblasts co-cultured with synoviocytes.

OBJECTIVE: Interleukin (IL)-1ß in the joint cavity increases to promote healing after anterior cruciate ligament (ACL) injury. Synovial tissue is a major joint microenvironmental regulator after ACL injury. The purpose of this study was to investigate the effects of synovial cells (SCs) on lysyl oxidase (LOX) and matrix metalloproteinase (MMP) production by ACL fibroblasts (ACLfs) in the presence of IL-1ß. RESULTS: This study sheds light on the regulation of LOX and MMP-1, -2, -3 expression by ACLfs co-cultured with SCs and treated with IL-1ß. LOX and MMP-1, 2, 3 gene/protein expression in IL-1ß/stretch-stimulated ACLfs co-cultured with SCs were measured by real-time quantitative PCR and Western blot. Meanwhile, MMP-2 activity was analyzed by zymogram. The results showed that co-culture with SCs increased LOX and MMP-1, -2, -3 gene and protein expression in the presence of IL-1ß. Next, ACLfs were subjected to 12% mechanical stretch to simulate pathological injury. Under these conditions, SCs inhibited IL-1ß-mediated upregulation of LOXs. However, IL-1ß enhanced the expression of MMP-1, -2, -3 in injured ACLfs. CONCLUSIONS: SCs can either inhibit or increase LOX production in the presence of IL-1ß, while promoting the accumulation of MMP in injured ACLfs. These results may provide crucial insights into the mechanisms underlying ACL poor healing capacity after injury.

Role of Cyclosporine in Gingival Hyperplasia: An In Vitro Study on Gingival Fibroblasts.

BACKGROUND: Gingival hyperplasia could occur after the administration of cyclosporine A. Up to 90% of the patients submitted to immunosuppressant drugs have been reported to suffer from this side effect. The role of fibroblasts in gingival hyperplasia has been widely discussed by literature, showing contrasting results. In order to demonstrate the effect of cyclosporine A on the extracellular matrix component of fibroblasts, we investigated the gene expression profile of human fibroblasts after cyclosporine A administration. MATERIALS AND METHODS: Primary gingival fibroblasts were stimulated with 1000 ng/mL cyclosporine A solution for 16 h. Gene expression levels of 57 genes belonging to the "Extracellular Matrix and Adhesion Molecules" pathway were analyzed using real-time PCR in treated cells, compared to untreated cells used as control. RESULTS: Expression levels of different genes were significantly de-regulated. The gene CDH1, which codes for the cell adhesion protein E-cadherin, showed up-regulation. Almost all the extracellular matrix metalloproteases showed down-regulation (MMP8, MMP11, MMP15, MMP16, MMP24, MMP26). The administration of cyclosporine A was followed by down-regulation of other genes: COL7A1, the transmembrane receptors ITGB2 and ITGB4, and the basement membrane constituents LAMA2 and LAMB1. CONCLUSION: Data collected demonstrate that cyclosporine inhibits the secretion of matrix proteases, contributing to the accumulation of extracellular matrix components in the gingival connective tissue, causing gingival overgrowth. Patients affected by gingival overgrowth caused by cyclosporine A need to be further investigated in order to determine the role of this drug on fibroblasts.

[Surgical treatment of internal carotid artery kinking following fibromuscular dysplasia]. / Rezul'taty khirurgicheskogo lecheniya patologicheskoi izvitosti vnutrennei sonnoi arterii na fone fibrozno-myshechnoi displazii.

OBJECTIVE: To evaluate the results of surgical treatment of internal carotid artery kinking following fibromuscular dysplasia. MATERIAL AND METHODS: There were 32 patients who underwent surgical treatment of internal carotid artery kinking following fibromuscular dysplasia. Structural changes of carotid artery wall were analyzed using immunohistochemical survey. Considering destructive changes revealed, we divided all patients into 2 groups in order to assess long-term postoperative outcomes: 1 - ICA resection followed by anastomosis in end-to-end fashion; 2 - ICA replacement. Postoperative analysis included incidence of stroke, thrombosis and deformities of anastomosis zone, regression of cerebrovascular insufficiency. RESULTS: The main «phenotype¼ of arterial wall in patients with ICA kinking following fibromuscular dysplasia is a large number of smooth muscle cells releasing matrix matelloproteinases-2 and -9 and low level of their tissue inhibitor type 1. Postoperative deformities are more common within a year after surgery. Maximum incidence is observed after 12 months. Both ICA resection and replacement are followed by similar incidence of deformity later. No severe deformities were diagnosed. Resection of ICA kinking on the background of fibromuscular dysplasia is followed by comparable results with ICA replacement regarding the incidence stroke, thrombosis and regression of cerebrovascular insufficiency. CONCLUSION: Despite degradation of extracellular matrix, destruction of elastic fibers and their fragmentation, no significant deformities are observed in long-term postoperative period in patients with ICA kinking and fibromuscular dysplasia.

The immunophilin FKBP12 inhibits hepcidin expression by binding the BMP type I receptor ALK2 in hepatocytes.

The expression of the key regulator of iron homeostasis hepcidin is activated by the BMP-SMAD pathway in response to iron and inflammation and among drugs, by rapamycin, which inhibits mTOR in complex with the immunophilin FKBP12. FKBP12 interacts with BMP type I receptors to avoid uncontrolled signaling. By pharmacologic and genetic studies, we identify FKBP12 as a novel hepcidin regulator. Sequestration of FKBP12 by rapamycin or tacrolimus activates hepcidin both in vitro and in murine hepatocytes. Acute tacrolimus treatment transiently increases hepcidin in wild-type mice. FKBP12 preferentially targets the BMP receptor ALK2. ALK2 mutants defective in binding FKBP12 increase hepcidin expression in a ligand-independent manner, through BMP-SMAD signaling. ALK2 free of FKBP12 becomes responsive to the noncanonical inflammatory ligand Activin A. Our results identify a novel hepcidin regulator and a potential therapeutic target to increase defective BMP signaling in disorders of low hepcidin.

Possible Association between Cathepsin V and the Development of Placenta Accreta Spectrum Disorders.

BACKGROUND/AIMS: The study aimed to evaluate molecular changes related to trophoblast adhesion in placenta accreta spectrum (PAS) disorders. METHODS: A retrospective analysis of 10 PAS cases in which both the trophoblast adherent site and the non-adherent site were identified was performed in April 2010 and March 2013. Microarray analysis and reverse transcription polymerase chain reaction (RT-PCR) analyses were performed to extract upregulated genes in the adherent site. Gene expression changes were examined by immunohistochemistry. RESULTS: Microarray analysis showed that 157 transcripts were > 3-fold upregulated, including the following: a disintegrin and metalloproteinase-28 (ADAM28), 3.10-fold; cathepsin V (CTSV), 3.73-fold; cathepsin S (CTSS), 3.46-fold; and matrix metalloproteinase-19 (MMP19), 3.41-fold. RT-PCR showed relatively high mRNA expressions. On immunohistochemistry, extravillous trophoblast (EVT) at the non-adherent site showed weak or no CTSV expression, whereas EVT that invaded myometrium at the adherent site showed strong expression (histological score, median [min-max], 115.6 [37.6-153.6] vs. 184.8 [56.4-222.8], p < 0.05). MMP19 showed moderate staining, with no difference between the adherent and non-adherent sites. ADAM28 and CTSS showed weak or no staining. DISCUSSION: This limited study suggests that CTSV may be involved in the pathogenesis of PAS.

Upregulation and Nuclear Location of MMP28 in Alveolar Epithelium of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive aging-associated disease of unknown etiology. A growing body of evidence indicates that aberrant activated alveolar epithelial cells induce the expansion and activation of the fibroblast population, leading to the destruction of the lung architecture. Some matrix metalloproteinases (MMPs) are upregulated in IPF, indicating that they may be important in the pathogenesis and/or progression of IPF. In the present study, we examined the expression of MMP28 in this disease and evaluated its functional effects in two alveolar epithelial cell lines and in human primary bronchial epithelial cells. We found that the enzyme is expressed in bronchial (apical and cytoplasmic localization) and alveolar (cytoplasmic and nuclear localization) epithelial cells in two different groups of patients with IPF. In vitro MMP28 epithelial silencing decreased the proliferation rate and delayed wound closing, whereas overexpression showed opposite effects, protecting from apoptosis and enhanced epithelial-mesenchymal transition. Our findings demonstrate that MMP28 is upregulated in epithelial cells from IPF lungs, where it may play a role in increasing the proliferative and migratory phenotype in a catalysis-dependent manner.

Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases.

Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP-10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP-9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP-10 expression in UMSCC12 cells (p = 0.0001), and MMP-3 (p = 0.0005) and -9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP-2 (p = 0.0408) and MMP-9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP-2 (p = 0.0023) and -9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion.

Micro-RNAs as critical regulators of matrix metalloproteinases in cancer.

Metastasis is known to be one of the important factors associated with cancer-related deaths worldwide. Several cellular and molecular targets are involved in the metastasis process. Among these targets, matrix metalloproteinases (MMPs) play central roles in promoting cancer metastasis. MMPs could contribute toward tumor growth, angiogenesis, migration, and invasion via degradation of the extracellular matrix and activation of pre-pro-growth factors. Therefore, identification of various cellular and molecular pathways that affect MMPs could contribute toward a better understanding of the metastatic pathways involved in various tumors. Micro-RNAs are important targets that could affect MMPs. Multiple lines of evidence have indicated that deregulation of various micro-RNAs, including miR-9, Let-7, miR-10b, and miR-15b, affects metastasis of tumor cells via targeting MMPs.

Gremlin1 Delivered by Mesenchymal Stromal Cells Promoted Epithelial-Mesenchymal Transition in Human Esophageal Squamous Cell Carcinoma.

Backgroud/Aims: Mesenchymal stromal cells (MSCs) are a major component of the tumor microenvironment (TME). Several studies focusing on tumor-derived MSCs have demonstrated that they exhibit a strong ability to promote the tumor epithelial-mesenchymal transition (EMT). However, the factors mediating these effects are poorly understood. METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry assays were used to detect the expression of Gremlin1 (GREM1) in human esophageal squamous cell carcinoma (ESCC) tissues. ShRNA silencing, flow cytometry, cell counting kit (CCK8) assay, invasion assay, western blot were used to detect the effect of GREM1 in ECa109, TE-1 cell lines and xenograft tumor models. RESULTS: In the current study, we found that the GREM1 was overexpressed in human ESCC tissues. The conditioned medium from mesenchymal stromal cells (MSCs-CM) enhanced the malignancy of xenograft esophageal tumors in vivo, as well as the cell proliferation, viability and invasion of the esophageal carcinoma cell lines ECa109 and TE-1 in vitro. Furthermore, the shRNA silencing of GREM1 in MSCs (shGREM1-MSCs) reversed the increased malignancy of the esophageal tumor in vivo, while the conditioned medium from shGREM1-MSCs (shGREM1-MSCs-CM) affected the cell cycle and cell invasion in vitro. These processes were accompanied by the EMT in the ECa109 and TE-1 cell lines with an alteration in the expression levels of mesenchymal and epithelial markers. Furthermore, the TGF-ß/BMP (transforming growth factor-beta/bone morphogenetic protein) signaling pathway participated in the shGREM1-MSCs-CM-induced anti-tumor effect on enhanced esophageal malignancy induced by MSCs-CM treatment. CONCLUSIONS: Taken together, our study suggested that GREM1 delivered by MSCs promoted EMT in ESCC in vitro and in vivo, which is partly through TGF-ß/BMP signaling pathway. The results provide experimental evidence to a potential therapeutic target in the treatment of esophageal cancer.

The oral administration of clarithromycin prevents the progression and rupture of aortic aneurysm.

OBJECTIVE: The pathogenesis of aortic aneurysm (AA) is associated with chronic inflammation in the aortic wall with increased levels of matrix metalloproteinases (MMPs). Clarithromycin (CAM) has been reported to suppresses MMP activity. In this study, we investigated whether CAM could prevent the formation and rupture of AA. METHODS: Male apolipoprotein E-deficient mice (28-30 weeks of age) were infused with angiotensin II for 28 days. CAM (100 mg/kg/d) or saline (as a control) was administered orally to the mice every day (CAM group, n = 13; control group, n = 13). After the administration period, the aortic diameter, elastin content, macrophage infiltration, MMP levels, and levels of inflammatory cytokines, including nuclear factor κB (NF-κB), were measured. RESULTS: The aortic diameter was significantly suppressed in the CAM group (P < .001). No rupture death was observed in the CAM group in contrast to five deaths (38%) in the control group (P < .01). CAM significantly suppressed the degradation of aortic elastin (56.3% vs 16.5%; P < .001) and decreased the infiltration of inflammatory macrophages (0.05 vs 0.16; P < .01). Compared with the controls, the enzymatic activity of MMP-2 and MMP-9 was significantly reduced in the CAM group (MMP-2, 0.15 vs 0.56 [P < .01]; MMP-9, 0.12 vs 0.60 [P < .01]), and the levels of interleukin 1ß (346.6 vs 1066.0; P < .05), interleukin 6 (128.4 vs 346.2; P < .05), and phosphorylation of NF-κB were also decreased (0.3 vs 2.0; P < .01). CONCLUSIONS: CAM suppressed the progression and rupture of AA through the suppression of inflammatory macrophage infiltration, a reduction in MMP-2 and MMP-9 activity, and the inhibition of elastin degradation associated with the suppression of NF-κB phosphorylation.

Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 µg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.

Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha.

The signal transducers of the transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP), the Smads, promote the expression of a subset of miRNAs by facilitating the cleavage reaction by Drosha. The mechanism that limits Smad-mediated processing to a selective group of miRNAs remained hitherto unexplored. In this study, we expand the number of TGFbeta/BMP-regulated miRNAs (T/B-miRs) to 20. Of interest, a majority of T/B-miRs contain a consensus sequence (R-SBE) within the stem region of the primary transcripts of T/B-miRs (pri-T/B-miRs). Here, we demonstrate that Smads directly bind the R-SBE. Mutation of the R-SBE abrogates TGFbeta/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs and impairs their processing, whereas introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and to allow regulation by TGFbeta/BMP. Thus, Smads are multifunctional proteins that modulate gene expression transcriptionally through DNA binding and posttranscriptionally through pri-miRNA binding and regulation of miRNA processing.

G-protein-coupled receptor kinase-2 is a critical regulator of TNFα signaling in colon epithelial cells.

G-protein-coupled receptor kinase-2 (GRK2) belongs to the GRK family of serine/threonine protein kinases critical in the regulation of G-protein-coupled receptors. Apart from this canonical role, GRK2 is also involved in several signaling pathways via distinct intracellular interactomes. In the present study, we examined the role of GRK2 in TNFα signaling in colon epithelial cell-biological processes including wound healing, proliferation, apoptosis, and gene expression. Knockdown of GRK2 in the SW480 human colonic cells significantly enhanced TNFα-induced epithelial cell wound healing without any effect on apoptosis/proliferation. Consistent with wound-healing effects, GRK2 knockdown augmented TNFα-induced matrix metalloproteinases (MMPs) 7 and 9, as well as urokinase plasminogen activator (uPA; factors involved in cell migration and wound healing). To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα-induced MAPK and NF-κB pathways. Our results demonstrate that while GRK2 knockdown inhibited TNFα-induced IκBα phosphorylation, activation of ERK was significantly enhanced in GRK2 knockdown cells. Our results further demonstrate that GRK2 inhibits TNFα-induced ERK activation by inhibiting generation of reactive oxygen species (ROS). Together, these data suggest that GRK2 plays a critical role in TNFα-induced wound healing by modulating MMP7 and 9 and uPA levels via the ROS-ERK pathway. Consistent with in vitro findings, GRK2 heterozygous mice exhibited enhanced intestinal wound healing. Together, our results identify a novel role for GRK2 in TNFα signaling in intestinal epithelial cells.

Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System.

Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.

Profile of Expression of Genes Encoding Matrix Metallopeptidase 9 (MMP9), Matrix Metallopeptidase 28 (MMP28) and TIMP Metallopeptidase Inhibitor 1 (TIMP1) in Colorectal Cancer: Assessment of the Role in Diagnosis and Prognostication.

BACKGROUND Studies on the pathomechanism of colorectal cancer (CRC) expansion indicate a significant role of metalloproteinases and their inhibitors in the extracellular matrix. The results of the analysis of a profile of transcriptional activity of genes encoding metalloproteinases were the basis of the hypothesis indicating changes in the expression of genes encoding MMP9, MMP28, and TIMP1 as an additional diagnostic and prognostic marker of CRC. MATERIAL AND METHODS The material consisted of samples obtained from resected tumors and healthy tissue samples from 15 CRC patients (aged 46-72 years) at clinical stages (CSs) I and II-IV. Gene expression analysis was done using microarrays. Microarray data analysis was done using the GeneSpring 11.5 platform. The results were validated using the qRT-PCR technique. RESULTS We found high levels of expression of MMP9 at each CS, as well as in the tissues at the early stage of CRC. Additionally, we observed high levels of expression of TIMP1 and low levels of MMP28 genes in CS II-IV. No statistically significant differences based on the stage of CRC were observed. CONCLUSIONS MMP9 gene profile may be a complementary diagnostic marker in CRC. The results suggest a crucial role of MMP9 at the early stage of carcinogenesis in the large intestine. The increase in MMP9 and TIMP1 mRNA concentration and the decrease in MMP28 in the large intestinal tissue may be a confirmation of cancer, but it may not indicate the advance of CRC.