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1.
Arthritis Rheumatol ; 72(4): 609-619, 2020 04.
Article in English | MEDLINE | ID: mdl-31682073

ABSTRACT

OBJECTIVE: To identify long noncoding RNAs (lncRNAs) associated with the inflammatory phenotype of synovial fibroblasts from obese patients with osteoarthritis (OA), and to explore the expression and function of these lncRNAs. METHODS: Synovium was collected from normal-weight patients with hip fracture (non-OA; n = 6) and from normal-weight (n = 8) and obese (n = 8) patients with hip OA. Expression of RNA was determined by RNA-sequencing and quantitative reverse transcription-polymerase chain reaction. Knockdown of lncRNA was performed using LNA-based GapmeRs. Synovial fibroblast cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: Synovial fibroblasts from obese patients with OA secreted greater levels of interleukin-6 (IL-6) (mean ± SEM 162 ± 21 pg/ml; P < 0.001) and CXCL8 (262 ± 67 pg/ml; P < 0.05) compared to fibroblasts from normal-weight patients with OA (IL-6, 51 ± 4 pg/ml; CXCL8, 78 ± 11 pg/ml) or non-OA patients (IL-6, 35 ± 3 pg/ml; CXCL8, 56 ± 6 pg/ml) (n = 6 patients per group). RNA-sequencing revealed that fibroblasts from obese OA patients exhibited an inflammatory transcriptome, with increased expression of proinflammatory messenger RNAs (mRNAs) as compared to that in fibroblasts from normal-weight OA or non-OA patients (>2-fold change, P < 0.05; n = 4 patients per group). A total of 19 lncRNAs were differentially expressed between normal-weight OA and non-OA patient fibroblasts, and a further 19 lncRNAs were differentially expressed in fibroblasts from obese OA patients compared to normal-weight OA patients (>2-fold change, P < 0.05 for each), which included the lncRNA for metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). MALAT1 was rapidly induced upon stimulation of OA synovial fibroblasts with proinflammatory cytokines, and was up-regulated in the synovium from obese OA patients as compared to normal-weight OA patients (1.6-fold change, P < 0.001) or non-OA patients (6-fold change, P < 0.001). MALAT1 knockdown in OA synovial fibroblasts (n = 4 patients) decreased the levels of mRNA expression and protein secretion of CXCL8 (>1.5-fold change, P < 0.01), whereas it increased expression of mRNAs for TRIM6 (>2-fold change, P < 0.01), IL7R (<2-fold change, P < 0.01), HIST1H1C (>1.5-fold change, P < 0.001), and MAML3 (>1.5-fold change, P < 0.001). In addition, MALAT1 knockdown inhibited the proliferation of synovial fibroblasts from obese patients with OA. CONCLUSION: Synovial fibroblasts from obese patients with hip OA exhibit an inflammatory phenotype. MALAT1 lncRNA may mediate joint inflammation in obese OA patients.


Subject(s)
Fibroblasts/metabolism , Interleukin-6/metabolism , Obesity/metabolism , Osteoarthritis, Hip/metabolism , RNA, Long Noncoding/metabolism , Synovial Membrane/metabolism , Aged , Cell Proliferation/physiology , Female , Humans , Inflammation/metabolism , Male , Middle Aged , Obesity/complications , Osteoarthritis, Hip/complications
2.
Spine Deform ; 7(4): 533-542, 2019 07.
Article in English | MEDLINE | ID: mdl-31202368

ABSTRACT

STUDY DESIGN: An observational descriptive study based on a single cohort of patients. OBJECTIVE: To determine whether spinal facet osteoblasts at the curve apex display a different phenotype to osteoblasts from outside the curve in adolescent idiopathic scoliosis (AIS) patients. SUMMARY OF BACKGROUND DATA: Intrinsic differences in the phenotype of spinal facet bone tissue and in spinal osteoblasts have been implicated in the pathology of AIS. However, no study has compared the phenotype of facet osteoblasts at the curve apex compared with outside the curve in AIS patients. METHODS: Facet spinal tissue was collected perioperatively from three sites, the concave and convex side at the curve apex and from outside the curve (noncurve) from three AIS female patients aged 13-16 years. Spinal tissue was analyzed by micro-computed tomography to determine bone mineral density (BMD) and trabecular structure. Primary osteoblasts were cultured from concave, convex, and noncurve facet bone chips. The phenotype of osteoblasts was determined by assessment of cellular proliferation, cellular metabolism (alkaline phosphatase and Seahorse Analyzer), bone nodule mineralization (Alizarin red assay), and the mRNA expression of Wnt signaling genes (quantitative reverse transcriptase polymerase chain reaction). RESULTS: Convex facet tissue exhibited greater BMD and trabecular thickness, compared with concave facet tissue. Osteoblasts at the convex side of the curve apex exhibited a significantly higher proliferative and metabolic phenotype and a greater capacity to form mineralized bone nodules, compared with concave osteoblasts. mRNA expression of SKP2 was significantly greater in both concave and convex osteoblasts, compared with noncurve osteoblasts. The expression of SFRP1 was significantly downregulated in convex osteoblasts, compared with either concave or noncurve. CONCLUSIONS: Intrinsic differences that affect osteoblast function are exhibited by spinal facet osteoblasts at the curve apex in AIS patients. LEVEL OF EVIDENCE: Level IV, Prognostic.


Subject(s)
Osteoblasts , Scoliosis/physiopathology , Spine/cytology , Spine/physiopathology , Adolescent , Female , Humans , Osteoblasts/cytology , Osteoblasts/physiology , Phenotype , Scoliosis/diagnostic imaging , Scoliosis/metabolism , Scoliosis/surgery , Spine/diagnostic imaging , Spine/metabolism , Wnt Signaling Pathway/physiology , X-Ray Microtomography
3.
Sci Rep ; 8(1): 12887, 2018 08 27.
Article in English | MEDLINE | ID: mdl-30150656

ABSTRACT

Galectin-9 (Gal9) has been postulated to have anti-inflammatory properties based on the ability of exogenous Gal9 to induce apoptosis in synovial fibroblasts in animal models of rheumatoid arthritis (RA). Here we aimed to assess the potential role of endogenous Galectins, including Gal9, in the inflammatory pathology of the RA synovium in humans. Firstly expression of Galectins 1-9 was determined in synovial fibroblasts (RASF) and dermal fibroblasts (DF) isolated from RA patients, the latter representing a non-inflamed site. We then further challenged the cells with pro-inflammatory TLR agonists and cytokines and assessed Galectin expression. Gal9 was found to be differentially and abundantly expressed in RASF compared to DF. Agonists of TLR3 and TLR4, along with IFNgamma were also found to induce Gal9 expression in RASF. siRNA was then used to knock-down Gal9 expression in RASF and the effects of this on apoptosis and cell viability were assessed. Increased apoptosis was observed in RASF following Gal9 knock-down. We conclude that, unlike exogenous Gal9, endogenous Gal9 is protective against apoptosis and enhances synovial fibroblast viability suggesting that its role in RA is both pathogenic and pro-inflammatory.


Subject(s)
Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/metabolism , Fibroblasts/metabolism , Galectins/genetics , Synovial Membrane/cytology , Arthritis, Rheumatoid/pathology , Biomarkers , Cells, Cultured , Cytokines/metabolism , Fluorescent Antibody Technique , Galectins/metabolism , Gene Expression , Gene Silencing , Humans
4.
Sci Rep ; 7(1): 3451, 2017 06 14.
Article in English | MEDLINE | ID: mdl-28615667

ABSTRACT

Increasing evidence suggests that inflammation plays a central role in driving joint pathology in certain patients with osteoarthritis (OA). Since many patients with OA are obese and increased adiposity is associated with chronic inflammation, we investigated whether obese patients with hip OA exhibited differential pro-inflammatory cytokine signalling and peripheral and local lymphocyte populations, compared to normal weight hip OA patients. No differences in either peripheral blood or local lymphocyte populations were found between obese and normal-weight hip OA patients. However, synovial fibroblasts from obese OA patients were found to secrete greater amounts of the pro-inflammatory cytokine IL-6, compared to those from normal-weight patients (p < 0.05), which reflected the greater levels of IL-6 detected in the synovial fluid of the obese OA patients. Investigation into the inflammatory mechanism demonstrated that IL-6 secretion from synovial fibroblasts was induced by chondrocyte-derived IL-6. Furthermore, this IL-6 inflammatory response, mediated by chondrocyte-synovial fibroblast cross-talk, was enhanced by the obesity-related adipokine leptin. This study suggests that obesity enhances the cross-talk between chondrocytes and synovial fibroblasts via raised levels of the pro-inflammatory adipokine leptin, leading to greater production of IL-6 in OA patients.


Subject(s)
Cell Communication , Chondrocytes/metabolism , Fibroblasts/metabolism , Interleukin-6/biosynthesis , Obesity/complications , Osteoarthritis/complications , Osteoarthritis/metabolism , Aged , Body Mass Index , Female , Humans , Interleukin-8/metabolism , Leptin/metabolism , Male , Middle Aged , Models, Biological , Osteoarthritis/pathology , Synovial Fluid/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
5.
HSS J ; 13(1): 20-27, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28167869

ABSTRACT

BACKGROUND: Precision medicine has been adopted in a range of clinical settings where omics data have led to greater characterisation of disease and stratification of patients into subcategories of phenotypes and pathologies. However, in orthopaedics, precision medicine lags behind other disciplines such as cancer. Joint registries have now amassed a huge body of data pertaining to implant performance which can be broken down into performance statistics for different material types in different cohorts of patients. The National Joint Registry of England, Wales and Northern Ireland (NJR) is now one of the largest datasets available. Other registries such as those from Sweden and Australia however contain longer follow-up. Together, these registries can provide a wealth of informative for the orthopaedics community when considering which implant to give to any particular patient. QUESTIONS/PURPOSES: We aim to explore the benefits of combining multiple large data streams including joint registries, published data on osteoarthritis (OA) pathogenesis and pathology and data concerning performance of each implant material combination in terms of biocompatibility. We believe that this analysis will provide a comprehensive overview of implant performance hopefully aiding surgeons in making more informed choices about which implant should be used in which patient. METHODS: Data from three joint registries were combined with established literature to highlight the heterogeneity of OA disease and the different clinical outcomes following arthroplasty with a range of material types. RESULTS: This review confirms that joint registries are unable to consider differences in arthritis presentation or underlying drivers of pathology. OA is now recognised to present with varying pathology with differing morbidity in different patient populations. Equally, just as OA is a heterogeneous disease, there are disparate responses to wear debris from different material combinations used in joint replacement surgery. This has been highlighted by recent high-profile scrutiny of early failure of metal-on-metal total hip replacement (THR) implants. CONCLUSIONS: Bringing together data from joint registries, biomarker analysis, phenotyping of OA patients and knowledge of how different patients respond to implant debris will lead to a truly personalised approach to treating OA patients, ensuring that the correct implant is given to the correct patient at the correct time.

6.
APL Bioeng ; 1(1): 016104, 2017 Dec.
Article in English | MEDLINE | ID: mdl-31069284

ABSTRACT

Human articular chondrocytes lose their native phenotype when expanded in traditional monolayer cultures. As a consequence, hydrogel encapsulation has been investigated as a means to maintain the natural phenotype. Alginate has been widely used for cartilage engineering as it has been shown to enable the recovery of a native collagen type II expressing chondrocyte phenotype. This study has evaluated whether the capacity of the materials to maintain/revert the phenotype is due to the composition of the material or the physical entrapment provided by the gel. To achieve this, an alginate "fluid gel" (a shear-thinning structured gel system) was produced of identical chemistry to a traditionally gelled alginate structure. Both were seeded with passaged primary human articular chondrocytes. Chondrocytes in quiescent alginate showed the recovery of the native phenotype and a spherical morphology. Chondrocytes in alginate fluid gel were unable to maintain the recovered phenotype despite having a spherical morphology and were shown to have a lower level of entrapment than those in quiescent alginate. These findings indicate that geometric entrapment is essential for the maintenance of a recovered chondrocyte phenotype in alginate.

7.
J Pathol ; 240(4): 472-483, 2016 12.
Article in English | MEDLINE | ID: mdl-27578244

ABSTRACT

Muscle wasting is a common feature of inflammatory myopathies. Glucocorticoids (GCs), although effective at suppressing inflammation and inflammatory muscle loss, also cause myopathy with prolonged administration. 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a bidirectional GC-activating enzyme that is potently upregulated by inflammation within mesenchymal-derived tissues. We assessed the regulation of this enzyme with inflammation in muscle, and examined its functional impact on muscle. The expression of 11ß-HSD1 in response to proinflammatory stimuli was determined in a transgenic murine model of chronic inflammation (TNF-Tg) driven by overexpression of tumour necrosis factor (TNF)-α within tissues, including muscle. The inflammatory regulation and functional consequences of 11ß-HSD1 expression were examined in primary cultures of human and murine myotubes and human and murine muscle biopsies ex vivo. The contributions of 11ß-HSD1 to muscle inflammation and wasting were assessed in vivo with the TNF-Tg mouse on an 11ß-HSD1 null background. 11ß-HSD1 was significantly upregulated within the tibialis anterior and quadriceps muscles from TNF-Tg mice. In human and murine primary myotubes, 11ß-HSD1 expression and activity were significantly increased in response to the proinflammatory cytokine TNF-α (mRNA, 7.6-fold, p < 0.005; activity, 4.1-fold, p < 0.005). Physiologically relevant levels of endogenous GCs activated by 11ß-HSD1 suppressed proinflammatory cytokine output (interkeukin-6, TNF-α, and interferon-γ), but had little impact on markers of muscle wasting in human myotube cultures. TNF-Tg mice on an 11ß-11ß-HSD1 knockout background developed greater muscle wasting than their TNF-Tg counterparts (27.4% less; p < 0.005), with smaller compacted muscle fibres and increased proinflammatory gene expression relative to TNF-Tg mice with normal 11ß-HSD1 activity. This study demonstrates that inflammatory stimuli upregulate 11ß-HSD1 expression and GC activation within muscle. Although concerns have been raised that excess levels of GCs may be detrimental to muscle, in this inflammatory TNF-α-driven model, local endogenous GC activation appears to be an important anti-inflammatory response that protects against inflammatory muscle wasting in vivo. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/physiology , Myositis/complications , Sarcopenia/etiology , 11-beta-Hydroxysteroid Dehydrogenase Type 1/biosynthesis , 11-beta-Hydroxysteroid Dehydrogenase Type 1/deficiency , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , Aged , Animals , Biopsy , Cells, Cultured , Chronic Disease , Cytokines/biosynthesis , Disease Models, Animal , Gene Expression Regulation, Enzymologic , Glucocorticoids/physiology , Humans , Hydrocortisone/biosynthesis , Mice, Transgenic , Middle Aged , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myositis/enzymology , Myositis/pathology , Sarcopenia/enzymology , Sarcopenia/pathology , Sarcopenia/prevention & control , Species Specificity , Tumor Necrosis Factor-alpha/genetics , Up-Regulation/immunology
9.
Arthritis Rheumatol ; 68(4): 845-56, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27023358

ABSTRACT

OBJECTIVE: To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. METHODS: OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non-OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase-polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. RESULTS: RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin-1ß (IL-1ß) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50-associated cyclooxygenase 2-extragenic RNA (PACER) and 2 novel chondrocyte inflammation-associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non-OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL-1-stimulated secretion of proinflammatory cytokines. CONCLUSION: The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation-driven cartilage degeneration in OA.


Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Osteoarthritis, Hip/metabolism , Osteoarthritis, Knee/metabolism , RNA, Long Noncoding/metabolism , Aged , Arthroplasty, Replacement, Hip , Arthroplasty, Replacement, Knee , Case-Control Studies , Chondrocytes/immunology , Cytokines/immunology , Female , Femoral Neck Fractures/immunology , Femoral Neck Fractures/metabolism , Femoral Neck Fractures/surgery , Gene Expression Profiling , Humans , In Vitro Techniques , Inflammation , Interleukin-1 , Male , Osteoarthritis, Hip/immunology , Osteoarthritis, Hip/surgery , Osteoarthritis, Knee/immunology , Osteoarthritis, Knee/surgery , RNA, Long Noncoding/immunology , Reverse Transcriptase Polymerase Chain Reaction
10.
Arthritis Rheumatol ; 67(12): 3135-45, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26245941

ABSTRACT

OBJECTIVE: In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA-protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. METHODS: Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. RESULTS: Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. CONCLUSION: Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.


Subject(s)
Arthritis, Rheumatoid/immunology , Autoantigens/immunology , Citrulline/metabolism , DNA/analysis , Hydrolases/immunology , Neutrophils/immunology , Adult , Aged , Arthritis, Psoriatic/immunology , Autoantigens/metabolism , Blotting, Western , Case-Control Studies , Cell Death/immunology , Extracellular Traps , Female , Fluorescent Antibody Technique , Humans , Hydrolases/metabolism , Male , Mass Spectrometry , Middle Aged , Neutrophils/cytology , Osteoarthritis, Knee/immunology , Protein-Arginine Deiminase Type 2 , Protein-Arginine Deiminases , Synovial Fluid/cytology , Synovial Fluid/immunology
11.
Biomaterials ; 67: 232-9, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26231914

ABSTRACT

Cobalt-chromium-molybdenum (CoCrMo) alloy-based metal-on-metal prostheses have been the implant of choice for total hip replacement in younger patients. However 6.2% of patients require revision of their CoCrMo total hip replacement (THR) implant within five years of surgery and their use was restricted in 2013. We aimed to determine if there were individual differences in the immune response to wear debris that might indicate a poor outcome with a CoCrMo prosthesis. Blood from 22 donors was incubated with CoCrMo particles (>99.9% less than 10 µm diameter) generated by a wear simulator for 24 h. T cell phenotype was assessed by immunostaining and secretion of 8 different pro- and anti-inflammatory cytokines was measured using multiplex technology. Clear differences were seen between individuals in the induction of Th17 and Th1 responses, with some donors showing pro-inflammatory responses (increased IL17 or IFNγ) and others showing anti-inflammatory responses (decreased IL17 or IFNγ). The only differences seen for gender and age related to increased IL-10 expression from T cells in females (p = 0.008) and a trend towards decreased IL-6 expression systemically for older donors (p = 0.058). We conclude that individuals show differential responses to CoCrMo wear debris and that these responses could give early indications of the suitability of the patient for a metal-on-metal prosthesis.


Subject(s)
Cytokines/blood , Prosthesis Failure/adverse effects , T-Lymphocytes/immunology , Vitallium/adverse effects , Adult , Aged , Demography , Female , Flow Cytometry , Humans , Inflammation Mediators/metabolism , Male , Microscopy, Electron, Scanning , Middle Aged , Particle Size , Tissue Donors , Young Adult
12.
PLoS One ; 10(3): e0120917, 2015.
Article in English | MEDLINE | ID: mdl-25807374

ABSTRACT

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.


Subject(s)
Arthritis, Rheumatoid/pathology , Fibroblasts/metabolism , Osteoarthritis/pathology , Actin Cytoskeleton/metabolism , Arthritis, Rheumatoid/metabolism , Bone Marrow Cells/cytology , Cells, Cultured , Databases, Factual , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Integrin beta3/pharmacology , Oligonucleotide Array Sequence Analysis , Osteoarthritis/metabolism , Principal Component Analysis , Signal Transduction/drug effects , Skin/cytology , Somatomedins/pharmacology , Synovial Membrane/cytology , Transcriptome
13.
Biochem Biophys Res Commun ; 426(4): 539-43, 2012 Oct 05.
Article in English | MEDLINE | ID: mdl-22982313

ABSTRACT

3' Untranslated regions (3'UTRs) of messenger RNAs have important roles in post-transcriptional regulation of gene expression and this is partly achieved through binding of specific proteins to sequences or structures within these regions. Previously, replacement of a native luciferase 3'UTR with the human albumin 3'UTR has been found to lead to a 10-fold increase in luciferase reporter activity. In this work we investigated protein binding to the human albumin 3'UTR. Electrophoretic mobility shift and UV cross-linking assays indicate that a ∼50kDa protein from Chinese Hamster Ovary (CHO) cells binds to the albumin 3'UTR, and affinity experiments followed by proteomics identified this protein as CUG binding protein 1 (CUG-BP1, also known as CELF1). Deletion analysis of the albumin 3'UTR showed that nucleotides 1-50 and nucleotides 101-150 are not required for binding but that removal of nucleotides 51-100 caused a loss in binding. The results suggest that CUG-BP1 binds to nucleotides 51-100 of the human albumin 3'UTR. In human cells CUG-BP1 binding may thus play a role in regulation of albumin expression and, additionally, it may have a function in post-transcriptional control in CHO cells.


Subject(s)
3' Untranslated Regions , Albumins/genetics , Gene Expression Regulation , RNA-Binding Proteins/metabolism , Animals , CELF1 Protein , CHO Cells , Cricetinae , Electrophoretic Mobility Shift Assay , Humans
14.
Biotechnol J ; 7(11): 1405-11, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22623341

ABSTRACT

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3'untranslated regions (3'UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3'UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2-3-fold by replacing the endogenous 3'UTR with the albumin 3'UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3'UTR. Replacement of the native 3'UTR with the albumin 3'UTR led to a 10-fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3'UTR showed that loss of nucleotides 1-50, which removed an AU-rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3'UTR sequences.


Subject(s)
3' Untranslated Regions , Albumins/genetics , Biotechnology/methods , Recombinant Proteins/genetics , Animals , Base Sequence , CHO Cells , Cloning, Molecular/methods , Cricetinae , Cricetulus , Humans , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , Recombinant Proteins/metabolism
15.
Microcirculation ; 11(3): 295-306, 2004.
Article in English | MEDLINE | ID: mdl-15280083

ABSTRACT

OBJECTIVE: To quantitatively evaluate the role of fibrinogen (Fb) as a determinant of leukocyte (WBC) margination in postcapillary venules in light of its ability to induce red blood cell (RBC) aggregation with reductions in shear rate (gamma) and increase adhesiveness of WBCs to endothelium (EC). METHODS: Red cell aggregation (RCA), WBC margination (flux at the EC), rolling velocity, and adhesion to the EC were measured in rat mesenteric postcapillary venules upon reducing gamma, prior to and following systemic infusion of Fb. Proximal occlusion of feeding microvessels with a blunted probe facilitated reductions in gamma from 600 to 50 s(-1). An index of aggregation (G) was derived from light-scattering properties of RBCs, where G was proportional to the number of RBCs per aggregate. WBC margination was measured as the percentage of total luminal WBC flux that rolled on the EC, F*(WBC). RESULTS: For normal levels of Fb (0.07 g%), reductions in gamma resulted in a 4-fold rise in F*(WBC) and no change in G as gamma was reduced to 50 s(-1). Infusion of Fb to achieve a plasma concentration to 0.7 g% caused a modest 20% increase in G and a 2.5-fold increase in F*(WBC) at gamma = 50 s(-1). WBC-EC adhesion appeared to increase significantly, but much less than with infusion of high molecular weight dextran (Dx). With Dx, G increased 3-fold, with reductions in gamma, but F*(WBC) increased only half the amount incurred with Fb at low shear. The greater margination in the presence of Fb results from RBC rouleaux that promote radial migration of WBCs. In contrast, clumps of RBCs resulting from high molecular weight Dx entrain WBCs within plasma gaps along the vessel centerline. CONCLUSIONS: In the presence of Fb, margination of WBCs increases dramatically at low shear due to rouleaux formation, which enhances radial migration of WBCs. This effect is much greater than with Dx because disruption of the much weaker Fb induced rouleaux precludes reductions in H(MICRO), whereas clumping aggregates induced by Dx form plasma gaps. Thus, modest levels of RCA caused by increased Fb may greatly enhance margination and with an enhancement of adhesiveness synergistically promote firm WBC-EC adhesion in the low flow state.


Subject(s)
Fibrinogen/pharmacology , Leukocytes/physiology , Venules/cytology , Animals , Cell Adhesion/drug effects , Dextrans/administration & dosage , Dextrans/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Erythrocyte Aggregation/drug effects , Fibrinogen/administration & dosage , Hemorheology , Leukocyte Rolling/drug effects , Leukocytes/drug effects , Male , Rats , Rats, Sprague-Dawley , Splanchnic Circulation , Stress, Mechanical , Swine , Venules/physiology
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