Esmeralda N. Blaney Davidson

A role for age-related changes in TGFβ signaling in aberrant chondrocyte differentiation and osteoarthritis

Transforming growth factor beta (TGFβ) is a growth factor with many faces. In our osteoarthritis (OA) research we have found that TGFβ can be protective as well as deleterious for articular cartilage. We postulate that the dual effects of TGFβ on chondrocytes can be explained by the fact that TGFβ can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFβ not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFβ on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFβ signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFβ signaling in OA development.

TGF β-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.

Canonical Wnt signaling skews TGF-β signaling in chondrocytes towards signaling via ALK1 and Smad 1/5/8

BACKGROUNDnBoth Wnt signaling and TGF-β signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-β signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-β signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-β signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway.nnnRESULTSnStimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-β-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-β signaling via Smad 2/3, measured by the functional CAGA12-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation.nnnCONCLUSIONSnOur data show that canonical Wnt signaling is able to skew TGF-β signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.

Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.

Unravelling osteoarthritis-related synovial fibrosis: a step closer to solving joint stiffness

Synovial fibrosis is often found in OA, contributing heavily to joint pain and joint stiffness, the main symptoms of OA. At this moment the underlying mechanism of OA-related synovial fibrosis is not known and there is no cure available. In this review we discuss factors that have been reported to be involved in synovial fibrosis. The aim of the study was to gain insight into how these factors contribute to the fibrotic process and to determine the best targets for therapy in synovial fibrosis. In this regard, the following factors are discussed: TGF-β, connective tissue growth factor, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2, tissue inhibitor of metalloproteinase 1, A disintegrin and metalloproteinase domain 12, urotensin-II, prostaglandin F2α and hyaluronan.

How to build an inducible cartilage-specific transgenic mouse

Transgenic mice are used to study the roles of specific proteins in an intact living system. Use of transgenic mice to study processes in cartilage, however, poses some challenges. First of all, many factors involved in cartilage homeostasis and disease are also crucial factors in embryogenesis. Therefore, meddling with these factors often leads to death before birth, and mice who do survive cannot be considered normal. The build-up of cartilage in these mice is altered, making it nearly impossible to truly interpret the role of a protein in adult cartilage function. An elegant way to overcome these limitations is to make transgenic mice time- and tissue-specific, thereby omitting side-effects in tissues other than cartilage and during embryology. This review discusses the potential building blocks for making an inducible cartilage-specific transgenic mouse. We review which promoters can be used to gain chondrocyte-specificity - all chondrocytes or a specific subset thereof - as well as different systems that can be used to enable inducibility of a transgene.

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

BackgroundCells 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.MethodsBovine 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.ResultsAs 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.ConclusionThe IVD phenotypic marker genes bT, bKrt19, bSfrp2 and bCol12a1 convincingly distinguished NP from outer AF in situ and in vitro.

Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation

The claimed beneficial effect of milk on bone is still a matter for debate. Recently extracellular vesicles (EVs) that contain proteins and RNA were discovered in milk, but their effect on bone formation has not yet been determined. We demonstrated previously that bovine milk-derived EVs (BMEVs) have immunoregulatory properties. Our aim was to evaluate the effect of BMEVs on osteogenesis by mice and human mesenchymal stem cells (hMSCs). Oral delivery of two concentrations of BMEVs to female DBA/1J mice during 7weeks did not alter the tibia trabecular bone area; however, the osteocytes number increased. In addition, the highest dose of BMEVs markedly increased the woven bone tissue, which is more brittle. The exposure of hMSCs to BMEVs during 21days resulted in less mineralization but higher cell proliferation. Interestingly BMEVs reduced the collagen production, but enhanced the expression of genes characteristic for immature osteoblasts. A kinetic study showed that BMEVs up-regulated many osteogenic genes within the first 4days. However, the production of type I collagen and expression of its genes (COL1A1 and COL1A2) were markedly reduced at days 21 and 28. At day 28, BMEVs again lead to higher proliferation, but mineralization was significantly increased. This was associated with increased expression of sclerostin, a marker for osteocytes, and reduced osteonectin, which is associated to bone matrix formation. Our study adds BMEVs to the list of milk components that can affect bone formation and may shed new light on the contradictory claims of milk on bone formation.

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

BACKGROUNDnChondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ 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 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications.nnnRESULTSnCo-stimulation of MSC with TGFβ and IL1β 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 TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ 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 TGFβ, IL1β or TGFβ and IL1β 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 IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ 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 IL1β-induced SMAD2 linker modifications and CAGA12-luciferase activity.nnnCONCLUSIONSnTGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β 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 IL1β-induced TGFβ signal modulation.

TGFβ1-induced SMAD2/3 and SMAD1/5 phosphorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity

BackgroundDysregulated transforming growth factor β (TGFβ) signaling is implicated in osteoarthritis development, making normalizing TGFβ signaling a possible therapy. Theoretically, this can be achieved with small molecule inhibitors specifically targeting the various TGFβ receptors and downstream mediators. In this study we explore in primary chondrocytes the use of small molecule inhibitors to target TGFβ-induced pSmad1/5/9-, pSmad2/3- and TGFβ-activated kinase 1 (TAK1)-dependent signaling.MethodPrimary bovine chondrocytes and explants were isolated from metacarpophalangeal joints. To modulate TGFβ signaling the activin receptor-like kinase (ALK)1/2/3/6 inhibitor LDN-193189, the ALK4/5/7 inhibitor SB-505124 and the TAK1 inhibitor (5Z)-7-Oxozeaenol were used. pSmad1/5 and pSmad2 were measured using western blot analysis and TGFβ1-induced gene expression was measured using quantitative real time PCR (qPCR).ResultsIn chondrocytes, TGFβ1 strongly induced both pSmad1/5 and pSmad2. Remarkably, LDN-193189 did not inhibit TGFβ-induced pSmad1/5. In contrast, SB-505124 did inhibit both TGFβ-induced Smad2 and Smad1/5 phosphorylation. Furthermore, (5Z)-7-Oxozeaenol also profoundly inhibited TGFβ-induced pSmad2 and pSmad1/5. Importantly, both SB-505124 and (5Z)-7-Oxozeaenol did not significantly inhibit constitutively active ALK1, making an off-target effect unlikely. Additionally, LDN-193189 was able to potently inhibit BMP2/7/9-induced pSmad1/5, showing its functionality. On gene expression, LDN-193189 did not affect TGFβ1-induced regulation, whereas both SB-505124 and (5Z)-7-Oxozeaenol did. Similar results were obtained in cartilage explants, although pSmad1/5 was not strongly induced by addition of TGFβ1.ConclusionOur data suggest that ALK5 kinase activity plays a central role in both TGFβ-induced Smad1/5 and Smad2/3 phosphorylation, making it difficult to separate both pathways with the use of currently available small molecule inhibitors. Furthermore, our data regarding (5Z)-7-Oxozeaenol suggest that TAK1 facilitates Smad-dependent signaling.