TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

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-ß2) 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-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 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-ß2 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-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Discovery of calcite as a new pro-inflammatory calcium-containing crystal in human osteoarthritic synovial fluid.

OBJECTIVE: We aimed to characterize calcium-containing crystals present in synovial fluid from patients with knee osteoarthritis (OA) using Raman spectroscopy, and specifically investigate the biological effects of calcite crystals. DESIGN: Thirty-two synovial fluid samples were collected pre-operatively from knee OA patients undergoing total joint arthroplasty. An integrated Raman polarized light microscope was used for identification of crystals in synovial fluid. Human peripheral blood mononuclear cells (PBMC's), human OA articular chondrocytes (HACs) and fibroblast-like synoviocytes (FLSs) were exposed to calcite crystals. Expression of relevant cytokines and inflammatory genes were measured using ELISA and real-time PCR. RESULTS: Various calcium-containing crystals were identified, including calcium pyrophosphate (37.5 %) and basic calcium phosphate (21.8 %), but they were never found simultaneously in the same OA synovial fluid sample. For the first time, we discovered the presence of calcite crystals in 93.8 % of the samples, while dolomite was detected in 25 % of the cases. Characterization of the cellular response to calcite crystal exposure revealed increased production of innate immune-derived cytokines by PBMC's, when co-stimulated with lipopolysaccharide (LPS). Additionally, calcite crystal stimulation of HACs and FLSs resulted in enhanced secretion of pro-inflammatory molecules and alterations in the expression of extracellular matrix remodeling enzymes. CONCLUSIONS: This study highlights the unique role of Raman spectroscopy in OA crystal research and identified calcite as a novel pro-inflammatory crystal type in OA synovial fluid. Understanding the role of specific crystal species in the OA joint may open new avenues for pharmacological interventions and personalized approaches to treating OA.

The BMP7-Derived Peptide p[63-82] Reduces Cartilage Degeneration in the Rat ACLT-pMMx Model for Posttraumatic Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks. DESIGN: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis. RESULTS: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery. CONCLUSIONS: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

Introduction: Ribosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation. Materials: SnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling. Results: As a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5-6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation. Conclusion: The expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

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.

Prediction of the Effect of the Osteoarthritic Joint Microenvironment on Cartilage Repair.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

Ribosome dysfunction in osteoarthritis.

PURPOSE OF REVIEW: Translation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central position of the ribosome in the translation of proteins, and considering the major proteomic changes that occur in the joint during osteoarthritis development and progression, the ribosome has received very limited attention as driver of osteoarthritis pathogenesis. RECENT FINDINGS: We provide an overview of the limited literature regarding this developing topic for the osteoarthritis field. Recent key findings that connect ribosome biogenesis and activity with osteoarthritis include: ribosomal RNA transcription, processing and maturation, ribosomal protein expression, protein translation capacity and preferential translation. SUMMARY: The ribosome as the central cellular protein synthesis hub is largely neglected in osteoarthritis research. Findings included in this review reveal that in osteoarthritis, ribosome aberrations have been found from early-stage ribosome biogenesis, through ribosome build-up and maturation, up to preferential translation. Classically, osteoarthritis has been explained as an imbalance between joint tissue anabolism and catabolism. We postulate that osteoarthritis can be interpreted as an acquired ribosomopathy. This hypothesis fine-tunes the dogmatic anabolism/katabolism point-of-view, and may provide novel molecular opportunities for the development of osteoarthritis disease-modifying treatments.

BMP7 reduces the fibrocartilage chondrocyte phenotype.

The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFß-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA)12-reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA)12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule.

Uncovering pathways regulating chondrogenic differentiation of CHH fibroblasts.

Mutations in the non-coding snoRNA component of mitochondrial RNA processing endoribonuclease (RMRP) are the cause of cartilage-hair hypoplasia (CHH). CHH is a rare form of metaphyseal chondrodysplasia characterized by disproportionate short stature and abnormal growth plate development. The process of chondrogenic differentiation within growth plates of long bones is vital for longitudinal bone growth. However, molecular mechanisms behind impaired skeletal development in CHH patients remain unclear. We employed a transdifferentiation model (FDC) combined with whole transcriptome analysis to investigate the chondrogenic transdifferentiation capacity of CHH fibroblasts and to examine pathway regulation in CHH cells during chondrogenic differentiation. We established that the FDC transdifferentiation model is a relevant in vitro model of chondrogenic differentiation, with an emphasis on the terminal differentiation phase, which is crucial for longitudinal bone growth. We demonstrated that CHH fibroblasts are capable of transdifferentiating into chondrocyte-like cells, and show a reduced commitment to terminal differentiation. We also found a number of key factors of BMP, FGF, and IGF-1 signalling axes to be significantly upregulated in CHH cells during the chondrogenic transdifferentiation. Our results support postulated conclusions that RMRP has pleiotropic functions and profoundly affects multiple aspects of cell fate and signalling. Our findings shed light on the consequences of pathological CHH mutations in snoRNA RMRP during chondrogenic differentiation and the relevance and roles of non-coding RNAs in genetic diseases in general.

Isolation of Nucleus Pulposus and Annulus Fibrosus Cells from the Intervertebral Disc.

Cells isolated from the intervertebral disc are often used for in vitro experimentation. Correctly separating the intervertebral disc tissue in annulus fibrosus and nucleus pulposus is particularly challenging when working with surplus material from surgery or specimens from donors with an advanced age. Moreover, lineage controls are only sparsely reported to verify tissue of origin. Here we describe an approach to intervertebral disc cell isolation from human and bovine origin.

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

OBJECTIVE: Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP). DESIGN: We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature. RESULTS: Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R (CLDN11, TMEFF2, CA12, ANXA2, CD44) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S (COLEC12, LPAR1) and AF-nS (CHIC1). CONCLUSIONS: The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

The antiviral protein viperin regulates chondrogenic differentiation via CXCL10 protein secretion.

Viperin (also known as radical SAM domain-containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor ß (TGF-ß)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin-CXCL10-TGF-ß/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.

Identification of TGFß-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets.

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor ß (TGFß) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFß-related genes are regulated during murine OA and endochondral ossification. METHODS: A list of 580 TGFß-related genes, including TGFß signaling pathway components and TGFß-target genes, was generated. Regulation of these TGFß-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development). RESULTS: A total of 106 TGFß-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients. CONCLUSION: By focusing on TGFß-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.

Interleukin 1 ß-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFß signaling in primary human mesenchymal stem cells.

BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFß) signaling. TGFß binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 ß (IL1ß) on SMAD2/3 signaling dynamics and post-translational modifications. RESULTS: Co-stimulation of MSC with TGFß and IL1ß did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFß and IL1ß compared to TGFß alone. Furthermore, IL1ß stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFß signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFß, IL1ß or TGFß and IL1ß stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1ß-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFß. Co-stimulation with both factors resulted in a similar kinetic profile as TGFß alone. Nevertheless, IL1ß did subtly alter TGFß-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFß target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1ß on TGFß signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1ß-induced SMAD2 linker modifications and CAGA12-luciferase activity. CONCLUSIONS: TGFß and IL1ß signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFß target genes were repressed by IL1ß independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1ß-induced TGFß signal modulation.

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc.

BACKGROUND: Cells in the intervertebral disc have unique phenotypes and marker genes that separate the nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) have been identified. Recently, it was shown that phenotypic marker genes exhibit variable expression in humans. In this study, the bovine tail was used to determine the ability of marker genes to distinguish the outer and inner AF from NP tissue and isolated cells. METHODS: Bovine tail intervertebral discs from 13 donors were dissected and correct isolation of tissue was confirmed. mRNA was isolated directly from tissue or passage 0 monolayer cells and used for gene expression measurements (qPCR). Conventional marker genes (bAcan, bCol1a1, bCol2a1) and novel marker genes (bAdamts17, bBrachyury/T, bCD24, bCol5a1, bCol12a1, bFoxf1, bKrt19, bPax1, bSfrp2) were evaluated. RESULTS: As expected bAcan, bCol2a1 and bCol1a1 distinguished outer AF from NP tissue, while inner AF and NP could not be discriminated. The NP markers bT, bCd24 and bKrt19 were significantly higher expressed in NP than inner and outer AF tissue. bFoxF1 and bPax1 only distinguished IVD tissues from AC. The AF markers bAdamts17, bCol5a1, bCol12a1 and bSfrp2 were higher expressed in the outer AF compared with inner AF and NP tissue. Monolayer culturing strongly decreased bAcan, bCol2a1, bCD24 and bCol5a1 expression, while bCol1a1, bT, bKrt19 and bSfrp2 were not affected. CONCLUSION: The IVD phenotypic marker genes bT, bKrt19, bSfrp2 and bCol12a1 convincingly distinguished NP from outer AF in situ and in vitro.

SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFß-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-ß (TGFß) receptor ALK5 is crucial for chondrogenesis induced by TGFß. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFß-induced chondrogenesis. Activation of TGFß signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFß signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFß-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFß-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFß-induced chondrogenesis of human BMSCs.

Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology.

Osteoclasts are cells specialized in bone resorption. Currently, studies on murine osteoclasts are primarily performed on bone marrow-derived cells with the use of many animals and limited cells available. ER-Hoxb8 cells are conditionally immortalized monocyte/macrophage murine progenitor cells, recently described to be able to differentiate toward functional osteoclasts. Here, we produced an ER-Hoxb8 clonal cell line from C57BL/6 bone marrow cells that strongly resembles phenotype and function of the conventional bone marrow-derived osteoclasts. We then used CRISPR/Cas9 technology to specifically inactivate genes by biallelic mutation. The CRISPR/Cas9 system is an adaptive immune system in Bacteria and Archaea and uses small RNAs and Cas nucleases to degrade foreign nucleic acids. Through specific-guide RNAs, the nuclease Cas9 can be redirected toward any genomic location to genetically modify eukaryotic cells. We genetically modified ER-Hoxb8 cells with success, generating NFATc1-/- and DC-STAMP-/- ER-Hoxb8 cells that lack the ability to differentiate into osteoclasts or to fuse into multinucleated osteoclasts, respectively. In conclusion, this method represents a markedly easy highly specific and efficient system for generating potentially unlimited numbers of genetically modified osteoclast precursors.

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types.

BACKGROUND: Immediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells. METHODS: To further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach. RESULTS: We show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1ß than the NP progenitor cells and aspects of this response were controlled by EGR1. CONCLUSIONS: Our current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells' epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

INTRODUCTION: Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. METHODS: Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor ß (TGFß). RESULTS: The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFß induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. CONCLUSION: We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.

INTRODUCTION: Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. METHODS: Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. RESULTS: A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. CONCLUSIONS: Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.