MicroRNA‐214 exerts a Cardio‐protective effect by inhibition of fibrosis

The miRNAs play important roles in regulating myocardial fibrosis. The purpose of this study was to determine the potential roles of microRNA‐214 (miR‐214) in cardiac fibrosis in vitro and in vivo. In vitro experiment, Ang II‐induced cardiac fibroblasts (CFBs) are transfected with pre‐miR‐214, anti‐miR‐214 and their oligo controls. Gene expression was checked by Quantitative realtime‐PCR (qRT‐PCR) and western blotting. In the present experiment, compared with controls, expressions of collagen type I (COL I), collagen type III (COL III), transforming growth factor (TGF)‐β1, and tissue inhibitors of metalloproteinase (TIMP)‐1 were all increased, but matrix metalloproteinase (MMP)‐1 was reduced in CFB by Ang II treatment at both mRNA and protein levels, and these alterations were found reversed by miR‐214 transfection. In vivo, an anterior transmural acute myocardial infarction (AMI) was created by occlusion of the left anterior descending coronary artery after Ad‐pre‐miR‐214, Ad‐anti‐miR‐214 or Ad‐GFP was delivered separately. Four weeks after AMI, protein contents of COL I, COL III and TGF‐β1 in tissue from border area were found increased after AMI, but impaired by overexpression of miR‐214. While the expression of MMP‐1 was increased by miR‐214 stimulation but decreased by miR‐214 inhibition. These results suggested that miR‐214 exerts cardio‐protective effects by inhibition of fibrosis and the inhibitory effect involves TGF‐β1 suppression and MMP‐1/TIMP‐1 regulation. Anat Rec, 299:1348–1357, 2016. © 2016 Wiley Periodicals, Inc.     

Co‐expression of TLR‐9 and MMP‐13 is associated with the degree of tumour differentiation in prostate cancer

In vitro experiments demonstrated that stimulation of Toll‐like receptor 9 (TLR‐9) by synthetic TLR‐9 ligands induces the invasion of TLR‐9‐expressing prostate cancer cells through matrix metalloproteinase 13 (MMP‐13). However, the clinical value of TLR‐9 and MMP‐13 co‐expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR‐9 and MMP‐13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high‐grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR‐9 and MMP‐13 markers. Subsequently, the correlation between the TLR‐9 and MMP‐13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR‐9 and MMP‐13 were found in PCa and high‐grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP‐13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR‐9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR‐9 and MMP‐13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR‐9^high/MMP‐13^high and TLR‐9^low/MMP‐13^low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR‐9 and MMP‐13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa.     

Plasma levels of matrix metalloproteinase‐9 in chronic urticaria patients correlate with disease severity and C‐reactive protein but not with circulating histamine‐releasing factors

Background Matrix metalloproteinase‐9 (MMP‐9) is an endopeptidase produced by many inflammatory cells that has been found in increased amounts in plasma from patients with chronic urticaria (CU). Objective To evaluate plasma levels of MMP‐9 and its tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in CU patients in relation with disease severity, C‐reactive protein (CRP) and circulating histamine‐releasing factors. Methods Fifty‐two consecutive CU patients were included in the study and disease activity was graded from 0 to 3. Plasma MMP‐9, TIMP‐1 and CRP levels were measured by enzyme immunoassays. Circulating histamine‐releasing factors were assessed using in vivo (autologous serum skin test) and in vitro (basophil histamine release) tests. Seven CU patients were studied both during active disease and during remission. Thirty healthy subjects were used as normal controls. Results Plasma levels of MMP‐9, TIMP‐1 and CRP were significantly higher in CU patients than in healthy controls (P=0.0001, 0.003 and 0.005, respectively) and a trend towards a higher MMP‐9/TIMP‐1 molar ratio was found (P=0.051). A significant correlation was found between plasma MMP‐9 levels and urticaria severity score (r=0.48, P<0.0001). CRP levels correlated with MMP‐9 levels (r=0.37, P=0.008) and CU severity score (r=0.52, P=0.0001), but not with TIMP‐1 (r=0.13) concentrations. MMP‐9, TIMP‐1 and CRP plasma levels and MMP‐9/TIMP‐1 molar ratio did not differ in patients either with or without an evidence of circulating histamine‐releasing factors. Seven patients evaluated during remission showed a significant reduction of MMP‐9 and CRP plasma levels. Conclusion Plasma levels of MMP‐9 and its inhibitor TIMP‐1 are increased in CU patients. MMP‐9 levels are associated with disease severity and CRP levels, but not with skin reactivity to autologous serum and with circulating histamine‐releasing factors. These findings suggest that in CU there is an ongoing inflammatory process independent of the presence of circulating histamine‐releasing factors.     

Over‐expression of prothymosin‐α antagonizes TGFβ signalling to promote the development of emphysema

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor‐β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up‐regulation of histone acetylation in the promoters of pro‐inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non‐histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ–Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down‐regulates TGFβ–Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R‐Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3–Smad4 complex to the TIMP‐3 promoter, resulting in reduced TIMP‐3 expression. These effects were detected in ProT‐over‐expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP‐3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)‐induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Chronic exposure of interleukin‐13 suppress the induction of matrix metalloproteinase‐1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt

Peripheral blood mononuclear cells taken from patients with scleroderma express increased levels of interleukin (IL)‐13. Moreover, the expression of matrix metalloproteinase‐1 (MMP‐1) from involved scleroderma skin fibroblasts is refractory to stimulation by tumour necrosis factor (TNF)‐α. To elucidate the mechanism(s) involved, we examined the effect of IL‐13 on TNF‐α‐induced MMP‐1 expression in normal and scleroderma human dermal fibroblast lines and studied the involvement of serine/threonine kinase B/protein kinase B (Akt) in this response. Dermal fibroblast lines were stimulated with TNF‐α in the presence of varying concentrations of IL‐13. Total Akt and pAkt were quantitated using Western blot analyses. Fibroblasts were treated with or without Akt inhibitor VIII in the presence of IL‐13 followed by TNF‐α stimulation. MMP‐1 expression was analysed by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (anova ) or Student's t‐test. Upon TNF‐α stimulation, normal dermal fibroblasts secrete more MMP‐1 than systemic sclerosis (SSc) fibroblasts. This increase in MMP‐1 is lost when fibroblasts are co‐incubated with IL‐13 and TNF‐α. IL‐13 induced a significant increase in levels of pAkt in dermal fibroblasts, while Akt inhibitor VIII reversed the suppressive effects of IL‐13 on the response of cultured fibroblasts to TNF‐α, increasing their expression of MMP‐1. We show that IL‐13 suppresses MMP‐1 in TNF‐α‐stimulated normal and scleroderma dermal fibroblast. Akt inhibitor VIII is able to reverse the suppressive effect of IL‐13 on MMP‐1 expression and protein synthesis. Our data suggest that IL‐13 regulates MMP‐1 expression in response to TNF‐α through an Akt‐mediated pathway and may play a role in fibrotic diseases such as scleroderma.     

miR‐29a inhibition normalizes HuR over‐expression and aberrant AU‐rich mRNA stability in invasive cancer

The activities of RNA‐binding proteins are perturbed in several pathological conditions, including cancer. These proteins include tristetraprolin (TTP, ZFP36) and HuR (ELAVL1), which respectively promote the decay or stability of adenylate‐uridylate‐rich (AU‐rich) mRNAs. Here, we demonstrated that increased stabilization and subsequent over‐expression of HuR mRNA were coupled to TTP deficiency. These findings were observed in breast cancer cell lines with an invasive phenotype and were further confirmed in ZFP36‐knockout mouse fibroblasts. We show that TTP–HuR imbalance correlated with increased expression of AU‐rich element (ARE) mRNAs that code for cancer invasion genes. The microRNA miR‐29a was abundant in invasive breast cancer cells when compared to non‐tumourigenic cell types. When normal breast cells were treated with miR‐29a, HuR mRNA and protein expression were up‐regulated. MiR‐29a recognized a seed target in the TTP 3′ UTR and a cell‐permeable miR‐29a inhibitor increased TTP activity towards HuR 3′ UTR. This led to HuR mRNA destabilization and restoration of the aberrant TTP–HuR axis. Subsequently, the cancer invasion factors uPA, MMP‐1 and MMP‐13, and cell invasiveness, were decreased. The TTP:HuR mRNA ratios were also perturbed in samples from invasive breast cancer patients when compared with normal tissues, and were associated with invasion gene expression. This study demonstrates that an aberrant ARE‐mediated pathway in invasive cancer can be normalized by targeting the aberrant and functionally coupled TTP–HuR axis, indicating a potential therapeutic approach. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Influence of LPS, TNF, TGF-ß1 and IL-4 on the expression of MMPs, TIMPs and selected cytokines in rat synovial membranes incubated in vitro

Synovial membranes are formed by four main types of cells, i.e. fibroblasts, macrophages, epitheliocytes and adipocytes. To study the combined effect of various factors on these cell populations, synovial membranes dissected from rat knee joints were incubated in control medium or medium with lipopolysaccharide (LPS), TNF, TGF-?1 or IL-4 for 12 h. LPS stimulated TNF secretion and both agents stimulated secretion of IL-6. TGF-?1 slightly increased IL-6 secretion. LPS increased the mRNA levels of IL-6, IL-1?, TGF-?1, MMP1a, MMP1b, MMP3, MMP9, MMP13, MMP14, TIMP1 and TIMP3 while the mRNA levels of MMP2, TIMP2 and TIMP4 were significantly decreased. Expression of IL-1?, MMP1a, MMP1b, MMP3, MMP9, MMP13 and TIMP1 increased after TNF treatment, while mRNA levels of MMP2, MMP14, TIMP2, TIMP3 and TIMP4 were decreased. TGF-?1 decreased the mRNA levels of IL-1?, all MMPs, TIMP1, TIMP2, TIMP4 and increased mRNA levels of itself and TIMP3. IL-4 decreased mRNA levels of IL-1?, TGF-?1, MMP2, MMP9, MMP13 and all TIMPs. Only LPS decreased the amount and activity of MMP2. The effect of LPS and cytokines on most of the MMPs and TIMPs produced by whole synovial membrane was in good agreement with previous studies on their action on similar types of cells as those present in synovial membranes, but originating from other tissues. All tested agents decreased MMP2 mRNA expression levels and in the case of LPS also the protein level and its activity determined by zymography, contrary to previous observations on isolated cell populations. This indicates that the response of the organized tissue is an interplay of all components and cannot be deduced from the individual reactions.     

Bone Morphogenetic Protein‐2 (BMP‐2) Increases Gene Expression of FSH Receptor and Aromatase and Decreases Gene Expression of LH Receptor and StAR in Human Granulosa Cells

Citation Shi J, Yoshino O, Osuga Y, Koga K, Hirota Y, Nose E, Nishii O, Yano T, Taketani Y. Bone morphogenetic protein‐2 (BMP‐2) increases gene expression of FSH receptor and aromatase and decreases gene expression of LH receptor and stAR in human granulosa Cells. Am J Reprod Immunol 2011; 65: 421–427 Problem A growing body of evidence indicates that bone morphogenetic protein (BMP) cytokines play a key role in female fertility in mammals. BMP‐2 is known to be expressed in the ovary of many species. In the present study, we examined the expression and function of BMP‐2 in the human ovary. Method of Study BMP‐2 mRNA expression in the human ovary was evaluated by in situ hybridization. Human granulosa cells were obtained from in vitro fertilization patients. Human granulosa cells were cultured with recombinant BMP‐2 or human chorionic gonadotrophin (HCG), followed by RNA extraction. Results BMP‐2 expression was detected in granulosa cells of antral follicles but not of corpus luteum. The in vitro study showed that BMP‐2 induced follicular stimulating hormone (FSH) receptor and aromatase expression, while decreasing luteinizing hormone (LH) receptor and steroidogenic acute regulatory protein expression in human granulosa cells. HCG decreased gene expression of BMP‐2 and increased BMP and activin membrane‐bound inhibitor (BAMBI), an antagonist of BMP‐2. Conclusion Expression and disappearance of BMP‐2 might contribute to folliculogenesis and luteinization by regulating gonadotropin receptor expression in human granulosa cells. HCG can modulate BMP‐2 function by controlling BMP‐2 and BAMBI expression.     

LAIR‐1 shedding from human fibroblast‐like synoviocytes in rheumatoid arthritis following TNF‐α stimulation

This study examined the expression of the inhibitory receptor, leucocyte‐associated immunoglobulin (Ig)‐like receptor‐1 (LAIR‐1) in fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA) patients to investigate its potential role in the modulation of inflammatory cytokines, matrix metalloproteinases (MMPs) and invasiveness of synoviocytes. LAIR‐1 expression in synovial tissues from RA patients, osteoarthritis patients and healthy donors was analysed by immunohistochemistry. The membrane‐bound form (mLAIR‐1) was detected by flow cytometry. Factors involved in inflammation and MMP activity in FLS were analysed by quantitative polymerase chain reaction (qPCR). LAIR‐1 expression was higher in the synovia of the RA patients than those of the osteoarthritis patients. Co‐immunostaining of vimentin/LAIR‐1 demonstrated that LAIR‐1 was localized mainly in FLS in the RA patients. Surprisingly, primary FLS isolated from the RA patients had low levels of mLAIR‐1 expression, with cytoplasmic distribution. The extracellular domain of LAIR‐1 was shed from the cell surface in response to tumour necrosis factor (TNF)‐α, and this process could be blocked by serine protease inhibitors. Additional experiments indicated that LAIR‐1 over‐expression reduced FLS invasion considerably, which reduced simultaneously the mRNA levels of interleukin (IL)‐6, IL‐8 and MMP‐13 in the presence of TNF‐α. Our study demonstrated that LAIR‐1 is an anti‐inflammatory molecule, and was up‐regulated in FLS in the RA patients; however, cell‐surface LAIR‐1 could be shed from cells in the inflammatory microenvironment in RA. This may weaken the interaction of LAIR‐1 with its ligand, thus reducing the anti‐inflammatory effects of LAIR‐1. These findings suggested that LAIR‐1 may be an important factor involved in the mediation of the progressive joint destruction in RA.     

The differential effects of 1,25‐dihydroxyvitamin D3 on Salmonella‐induced interleukin‐8 and human beta‐defensin‐2 in intestinal epithelial cells

Salmonellosis or Salmonella, one of the most common food‐borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion of Salmonella by interleukin (IL)?8 and human β‐defensin‐2 (hBD‐2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression of Salmonella‐induced IL‐8 but enhancement of hBD‐2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhanced Salmonella‐induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3‐kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 on Salmonella‐induced IL‐8 expression, while knock‐down of NOD2 by siRNA diminished the enhanced hBD‐2 expression. These data suggest differential regulation of 1,25D3 on Salmonella‐induced IL‐8 and hBD‐2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D‐enhanced anti‐microbial peptide in Salmonella‐infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D‐induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasive Salmonella infection.     

The effect of gender and genetic polymorphisms on matrix metalloprotease (MMP) and tissue inhibitor (TIMP) plasma levels in different infectious and non‐infectious conditions

Matrix metalloproteases (MMPs) are increased in different infections due to their role in controlling immune responses and are regulated by tissue inhibitors (TIMPs). Different MMP promoter single nucleotide polymorphisms (SNPs) induce changes in MMP genes, mRNA and protein expression. Gender might also modify MMP plasma levels. In order to determine the weight of these variables on MMP secretion we studied MMP‐1, ‐2, ‐3, ‐8, ‐9, ‐10, ‐13 and TIMP‐1, ‐2, ‐4 plasma levels in 90 patients with severe bacterial sepsis, 102 with anti‐retroviral (ARV)‐treated HIV monoinfection, 111 with ARV‐treated HIV–hepatitis C virus (HCV) co‐infection and 86 non‐infected controls (45 stroke and 41 trauma patients). MMP‐1(‐1607 1G/2G), MMP‐3(‐1612 5A/6A), MMP‐8(‐799C/T), MMP‐9(‐1562 C/T) and MMP‐13(‐77A/G) SNPs were genotyped. MMP‐3 plasma levels were significantly higher in men than in women in each diagnostic group, and MMP‐3 SNP allele 6A carriers also had higher levels than allele 5A carriers, an effect that was magnified by sepsis. Independent predictors of higher MMP‐3 levels were male gender (P = 0·0001), MMP‐3(‐1612 5A/6A) SNP (P = 0·001), higher levels of TIMP‐4 (P = 0·004) and MMP‐8 (P = 0·006) and lower levels of MMP‐1 (P = 0·03) by multivariate analysis. No strong associations with gender or SNPs were observed for other MMPs or TIMPs. In conclusion, male gender and MMP‐3(‐1612 5A/6A) 6A allele carriage increased MMP‐3 plasma levels significantly, especially in patients with severe bacterial sepsis. This confounding gender effect needs to be addressed when evaluating MMP‐3 plasma levels in any infectious or non‐infectious condition.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

The induced RNA‐binding protein,HuR, targets 3′‐UTR region of IL‐6 mRNA and enhances its stabilization in periodontitis

RNA‐binding proteins (RBPs) regulate mRNA stability by binding to the 3′‐untranslated region (UTR) region of mRNA. Human antigen‐R (HuR), one of the RBPs, is involved in the progression of diseases, such as rheumatoid arthritis, diabetes mellitus and some inflammatory diseases. Interleukin (IL)‐6 is a major inflammatory cytokine regulated by HuR binding to mRNA. Periodontal disease (PD) is also an inflammatory disease caused by elevations in IL‐6 following an infection by periodontopathogenic bacteria. The involvement of HuR in the progression of PD was assessed using in‐vitro and in‐vivo experiments. Immunohistochemistry of inflamed periodontal tissue showed strong staining of HuR in the epithelium and connective tissue. HuR mRNA and protein level was increased following stimulation with Porphyromonas gingivalis (Pg), one of the periodontopathogenic bacteria, lipopolysacchride (LPS)‐derived from Pg (PgLPS) and tumour necrosis factor (TNF)‐α in OBA‐9, an immortalized human gingival epithelial cell. The luciferase activity of 3′‐UTR of IL‐6 mRNA was increased by TNF‐α, Pg and PgLPS in OBA‐9. Luciferase activity was also increased in HuR‐over‐expressing OBA‐9 following a bacterial stimulation. Down‐regulation of HuR by siRNA resulted in a decrease in mRNA expression and production of IL‐6. In contrast, the over‐expression of HuR increased IL‐6 mRNA expression and production in OBA‐9. The HuR inhibitor, quercetin, suppressed Pg‐induced HuR mRNA expression and IL‐6 production in OBA‐9. An oral inoculation with quercetin also inhibited bone resorption in ligature‐induced periodontitis model mice as a result of down‐regulation of IL‐6. These results show that HuR modulates inflammatory responses by regulating IL‐6.     

Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression

Striking similarities exist between molecular mechanisms driving embryonic liver development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryonic hepatic development. BMP expression has been observed in neoplasia but the expression and biological role of BMP4 in human HCC are unknown. We found increased BMP4 mRNA and protein in HCC cell lines and tissue samples compared to primary human hepatocytes and corresponding non‐tumourous tissue. Hypoxia further induced BMP4 expression in HCC cells, which was abolished by transfection of a dominant negative form of HIF‐1alpha (dnHIF‐1alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to a putative hypoxia‐response element in the BMP4 promoter. Sequence analysis of the BMP4 promoter revealed two Ets‐1 binding sites, and Ets‐1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF‐1alpha completely abrogated hypoxia‐induced Ets‐1 activity as well as BMP4 expression. Overexpression of Ets‐1 markedly enhanced BMP4 promoter activity, while antisense Ets‐1 almost completely abolished basal as well as hypoxia‐induced BMP4 expression. These data demonstrate that Ets‐1 activity contributes to baseline expression of the BMP4 gene and is the predominant mediator of the HIF‐dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP4 expression, HCC cell lines were treated with antisense BMP4 constructs or siRNA against BMP4. BMP4 suppression resulted in a strong reduction of the migratory and invasive potential and anchorage‐independent growth. Furthermore, tube formation assays indicated that BMP4 expressed by HCC cells promotes vasculogenesis. Our findings demonstrate that BMP4 is increased in HCC and promotes HCC progression. Therefore, BMP4 expression may have clinical relevance, and interfering with BMP4 signalling appears as an attractive therapeutic target for this highly aggressive tumour. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Macrophage‐specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP‐9 pathways

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS‐producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase‐induced emphysema using NOX2‐ and NOX1‐deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2‐positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase‐induced alveolar airspace enlargement and elastin degradation were prevented in NOX2‐deficient mice, but not in NOX1‐deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase‐9 (MMP‐9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage‐restricted functional NOX2 in elastase‐induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68‐positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase‐induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage‐specific NOX2 participates in elastase‐induced emphysema through the involvement of SIRT1/MMP‐9 pathways in mice. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Suppression of oxLDL‐Induced MMP‐9 and EMMPRIN Expression by Berberine via Inhibition of NF‐κB Activation in Human THP‐1 Macrophages

Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF‐κB activation regulates many key inflammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP‐9 and EMMPRIN expression, the role of NF‐κB activation in oxLDL‐stimulated macrophages, and the possible mechanism in which NF‐κB activation is involved were investigated. Berberine inhibited the expression of MMP‐9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IκB‐α and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF‐κB activation was inhibited by berberine in oxLDL‐stimulated macrophages. Overall, berberine suppressed the expression of MMP‐9 and EMMPRIN by at least reducing partly the activity of NF‐κB in oxLDL‐induced macrophages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Canine Oral Mucosal Fibroblasts Differentiate into Osteoblastic Cells in Response to BMP‐2

Several lines of evidence show that transplantation of osteoblastic cells or genetically engineered nonosteogenic cells expressing osteoblast‐related genes into bone defects effectively promotes bone regeneration. To extend this possibility, we investigated whether oral mucosal fibroblasts are capable of differentiating into osteoblastic cells by conducting in vitro and in vivo experiments. We investigated the effects of bone morphogenetic protein‐2 (BMP‐2) on osteoblast differentiation of cultured fibroblasts isolated from canine buccal mucosa. We also transplanted green fluorescence protein (GFP)‐expressing fibroblasts with gelatin/BMP‐2 complexes into the subfascial regions of athymic mice, and investigated the localization of GFP‐positive cells in the ectopically formed bones. The cultured canine buccal mucosal fibroblasts differentiated into osteoblastic cells by increasing their alkaline phosphatase (ALP) activity and Osteocalcin, Runx2, and Osterix mRNA expression levels in response to BMP‐2. Transplantation experiments of GFP‐expressing oral mucosal fibroblasts with gelatin/BMP‐2 complexes revealed that 17.1% of the GFP‐positive fibroblasts differentiated into ALP‐positive cells, and these cells accounted for 6.2% of total ALP‐positive cells in the ectopically formed bone. This study suggests that oral mucosal fibroblasts can differentiate into osteogenic cells in response to BMP‐2. Thus, these cells are potential candidates for cell‐mediated bone regeneration therapy in dentistry. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Extracellular 2′5′‐oligoadenylate synthetase 2 mediates T‐cell receptor CD3‐ζ chain down‐regulation via caspase‐3 activation in oral cancer

Decreased expression of CD3‐ζ chain, an adaptor protein associated with T‐cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3‐ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour‐derived factors promote immune suppression by down‐regulating CD3‐ζ chain expression. 2′5′‐Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3‐ζ chain down‐regulation and OAS2 stimulation. The surrogate situation was established by over‐expressing OAS2 in a HEK293 cell line and cell‐free supernatant was collected. These supernatants when incubated with T cells resulted in down‐regulation of CD3‐ζ chain, which shows that the secreted OAS2 is capable of regulating CD3‐ζ chain expression. Incubation of T cells with cell‐free supernatants of oral tumours or recombinant human OAS2 (rh‐OAS2) induced caspase‐3 activation, which resulted in CD3‐ζ chain down‐regulation. Caspase‐3 inhibition/down‐regulation using pharmacological inhibitor or small interfering RNA restored down‐regulated CD3‐ζ chain expression in T cells induced by cell‐free tumour supernatant or rh‐OAS2. Collectively these results show that OAS2 leads to impairment in CD3‐ζ chain expression, so offering an explanation that might be applicable to the CD3‐ζ chain deficiency observed in cancer and diverse disease conditions.     

MT2‐MMP Expression During Early Avian Morphogenesis

Membrane‐type 2 matrix metalloproteinase (MT2‐MMP; also called MMP15) is a membrane‐bound protease that degrades extracellular matrix and activates proMMPs such as proMMP‐2. MMP‐2 expression in avian embryos is well documented, but it is not clear how proMMP‐2 is activated during avian embryogenesis. Herein, we report that MT2‐MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP‐2. These observations suggest MT2‐MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP‐2. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.