Summary Objective Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly ...systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. Design Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. Results 3D cultures specifically expressed chondrogenic mRNAs collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. Conclusions For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.
BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. ...Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Patients with mantle-cell lymphoma who have a relapse after chemotherapy and anti-CD20 and BTK inhibitor therapy have a poor prognosis. An injection of CD19-directed CAR T cells induced a complete ...response in 59% of patients; 78% with a complete response were in remission at 1 year. About two thirds of patients had serious adverse events, a finding consistent with previous data.
Summary Objective Bone morphogenic protein (BMP)-2 and BMP-7 are clinically approved and their recombinant proteins are used for bone tissue regenerative purposes and widely evaluated for cartilage ...regeneration. Previous comparison of the in vitro chondrogenic characteristics of BMP-2 vs BMP-7 did not address hypertrophic differentiation and characterizing their chondrogenic properties with a focus in on chondrocyte hypertrophy was topic of investigation in this study. Design Equimolar concentrations of BMP-2 or BMP-7 were added to chondrogenic differentiating ATDC5, human bone marrow stem cells or rabbit periosteal explants. Expression of Col2a1, Sox9, Acan, Col10a1, Runx2, ALP, Mmp13, Mef2c and Bapx1/Nkx3.2 was determined by reverse transcription-quantitative PCR (RT-qPCR) and immunoblotting. Glycosaminoglycan content, cell proliferation capacity and ALP activity were analysed by colourimetric analyses. Expression of Bapx1/Nkx3.2 and Sox9 was targeted by transfection of target specific siRNA duplexes. Results BMP-2 dose-dependently increased chondrocyte hypertrophy during chondrogenic differentiation of progenitor cells, whereas BMP-7 acted hypertrophy-suppressive and chondro-promotive. Both BMPs did not influence cell proliferation, but they did increase total glycosaminoglycan content. In a candidate approach Bapx1/Nkx3.2 was found to be involved in the BMP-7 mediated suppression of chondrocyte hypertrophy in ATDC5 cells. Conclusions BMP-2 and BMP-7 display opposing actions on the chondrogenic outcome of differentiating progenitor cells: BMP-2 acts a specific inducer of chondrocyte hypertrophy, while BMP-7 appears to increase or maintain chondrogenic potential and prevent chondrocyte hypertrophy. Our results pave the way for an application-dependent differential use of BMP-2 or BMP-7.
Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is ...unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis.
Sequencing-based rRNA 2′-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC–MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation.
We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2′-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2′-O-me levels. The 2′-O-me status of 5.8S-U14 (one of identified differential 2′-O-me sites; decreased by 7.7%, 95% CI 0.9–14.5%) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI 33–64%). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI 2.2–4.1) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI 1.3–2.0).
Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.
Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the ...first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods.
Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors.
Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression.
BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.
Mutations in the RMRP-gene, encoding the lncRNA component of the RNase MRP complex, are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by ...impaired skeletal development. However, it is not clear why mutations in RMRP RNA lead to skeletal dysplasia. Since chondrogenic differentiation of the growth plate is required for development of long bones, we hypothesized that RMRP RNA plays a pivotal role in chondrogenic differentiation. Expression of Rmrp RNA and RNase MRP protein subunits was detected in the murine growth plate and during the course of chondrogenic differentiation of ATDC5 cultures, where Rmrp RNA expression was found to be correlated with chondrocyte hypertrophy. Genetic interference with Rmrp RNA expression in ATDC5 cultures caused a deregulation of chondrogenic differentiation, with a prominent impact on hypertrophy and changes in pre-rRNA processing and rRNA levels. Promoter reporter studies showed that Rmrp RNA expression responds to chondrogenic morphogens. Chondrogenic trans-differentiation of cartilage-hair hypoplasia fibroblasts was impaired with a pronounced impact on hypertrophic differentiation. Together, our data show that RMRP RNA expression is regulated during different stages of chondrogenic differentiation and indicate that RMRP RNA may play a pivotal role in chondrocyte hypertrophy, with potential consequences for CHH pathobiology.
Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for ...the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.
In order to explain the cognitive and cerebral mechanisms responsible for the visuospatial peak in autism, and to document its specificity to this condition, a group of eight high-functioning ...individuals with autism and a visuospatial peak (HFA-P) performed a modified block-design task (BDT; subtest from Wechsler scales) at various levels of perceptual cohesiveness, as well as tasks tapping visuomotor speed, global perception, visual memory, visual search and speed of visual encoding. Their performance was compared with that of 8 autistics without a visuospatial peak (HFA-NP), 10 typically developing individuals (TD) and 8 gifted comparison participants with a visuospatial peak (TD-P). Both HFA-P and HFA-NP groups presented with diminished detrimental influence of increasing perceptual coherence compared with their BDT-matched comparison groups. Neither autistic group displayed a deficit in construction of global representations. The HFA-P group showed no differences in performance level or profile in comparison with the gifted BDT-matched i.e. higher full-scale IQ (FSIQ) group, apart from locally oriented perception. Diminished detrimental influence of perceptual coherence on BDT performance is both sensitive and specific to autism, and superior low-level processing interacts with locally oriented bias to produce outstanding BDT performance in a subgroup of autistic individuals. Locally oriented processing, enhanced performance in multiple tasks relying on detection of simple visual material and enhanced discrimination of first-order gratings converge towards an enhanced functioning and role of the primary visual cortex (V1) in autism. In contrast, superior or typical performance of autistics in tasks requiring global processing is inconsistent with the global-deficit-driven Weak Central Coherence hypothesis and its neurobiological magnocellular deficit counterpart.
The NEWS-G collaboration uses spherical proportional counters (SPCs) to search for weakly interacting massive particles (WIMPs). In this paper, we report the first measurements of the nuclear ...quenching factor in neon gas at 2 bar using an SPC deployed in a neutron beam at the TUNL facility. The energy-dependence of the nuclear quenching factor is modeled using a simple power law: α E nrβ ; we determine its parameters by simultaneously fitting the data collected with the detector over a range of energies. We measured the following parameters in Ne:CH4 at 2 bar: α=0.2801±0.0050 (fit) ±0.0045 (sys) and β=0.0867±0.020 (fit) ±0.006 (sys). Our measurements do not agree with expected values from SRIM or Lindhard theory. We demonstrated the feasibility of performing quenching factor measurements at sub-keV energies in gases using SPCs and a neutron beam.
PDF
