Ageing is associated with reduction of mechanically-induced activation of Smad2/3P signaling in articular cartilage.

Mechanical signals control key cellular processes in articular cartilage. Previously we have shown that mechanical compression is an important ALK5/Smad2/3P activator in cartilage explants. However, age-related changes in the cartilage are known to affect tissue mechanosensitivity and also ALK5/Smad2/3P signaling. We have investigated whether ageing of cartilage is associated with an altered response to mechanical compression. Articular cartilage explants of two different age groups (young-6-36 months old, aged-6 - 13 years old) were subjected to dynamic mechanical compression with 3 MPa (physiological) or 12 MPa (excessive) load. Subsequently, essential cartilage extracellular matrix (ECM) components and tissue growth factors gene expression was measured in young and aged cartilage by QPCR. Furthermore, the ability of young and aged cartilage, to activate the Smad2/3P signaling in response to compression was analyzed and compared. This was done by immunohistochemical (IH) Smad2P detection and Smad3-responsive gene expression analysis. Aged cartilage showed a highly reduced capacity for mechanically-mediated activation of Smad2/3P signaling when compared to young cartilage. Compression of aged cartilage, induced collagen type II (Col2a1) and fibronectin (Fn1) expression to a far lesser extent than in young cartilage. Additionally, in aged cartilage no mechanically mediated up-regulation of bone morphogenetic protein 2 (Bmp2) and connective tissue growth factor (Ctgf) was observed. We identified age-related changes in cellular responses to mechanical stimulation of articular cartilage. We propose that these changes might be associated with age-related alterations in cartilage functioning and can underlie mechanisms for development of age-related cartilage diseases like osteoarthritis (OA).

Madej W ，van Caam A ，Davidson EN ，Hannink G ，Buma P ，van der Kraan PM ... -  《-》

Physiological and excessive mechanical compression of articular cartilage activates Smad2/3P signaling.

Transforming growth factor beta (TGF-β) in articular cartilage can signal via two routes, the ALK5/Smad2/3P and the ALK1/Smad1/5/8P route, the first being protective and the latter favoring chondrocyte terminal differentiation. Since biomechanical factors are known to play an essential role in osteoarthritis (OA) initiation and progression, we investigated if excessive mechanical compression can alter TGF-β signaling in cartilage shifting it from ALK5/Smad2/3P to ALK1/Smad1/5/8P pathway, favoring terminal differentiation of chondrocytes. Articular cartilage explants were harvested from bovine metacarpophalangeal joints. After equilibration, explants were subjected to unconfined dynamic mechanical compression (1 Hz) with 3 MPa (physiological) or 12 MPa (excessive) stress. After different time intervals samples were frozen and mRNA levels of selected genes were examined using real-time polymerase chain reaction. In articular cartilage compressed with 3 MPa and also 12 MPa stress the expression of Smad2/3P responsive genes bSerpine1, bSmad7 and bAlk5 was up-regulated, whereas the expression of Smad1/5/8P responsive gene bId1 was down-regulated. Furthermore, the expression of bTgfb1 was significantly up-regulated in both compression groups. When ALK5/Smad2/3P pathway was blocked with a selective ALK4/5/7 inhibitor, the effect of excessive mechanical compression on bSmad7 and bAlk5 expression was prevented. Here we show that excessive mechanical compression alone is not able to shift TGF-β signaling toward the ALK1/Smad1/5/8P pathway. In contrast, we show that mechanical compression not only with physiological but also with excessive stress can activate Smad2/3P signaling, which is known to be protective for articular cartilage and to block chondrocyte terminal differentiation.

Madej W ，van Caam A ，Blaney Davidson EN ，van der Kraan PM ，Buma P ... -  《-》

Transforming growth factor-β1 induces type II collagen and aggrecan expression via activation of extracellular signal-regulated kinase 1/2 and Smad2/3 signaling pathways.

Zhu Y ，Tao H ，Jin C ，Liu Y ，Lu X ，Hu X ，Wang X ... -  《-》

TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-β (TGF-β), which in turn stimulates chondrocytes to produce NGF. Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-β1 and/or Interleukin-1 (IL-1)β. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-β pathway inducing NGF. NGF expression was consistently induced in higher levels by TGF-β than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-β-induced NGF whereas it fully blocked IL-1β-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-β-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-β exposure led to a consistent high level of NGF induction. We show for the first time that TGF-β induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Blaney Davidson EN ，van Caam AP ，Vitters EL ，Bennink MB ，Thijssen E ，van den Berg WB ，Koenders MI ，van Lent PL ，van de Loo FA ，van der Kraan PM ... -  《-》

Age-related alterations in TGF beta signaling as a causal factor of cartilage degeneration in osteoarthritis.

van der Kraan PM 《-》