Shift in Chondrocyte TGF-βReceptor Usage, ALK5 to ALK1 … · 2020. 9. 23. · Paper No. 122 •...
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Paper No. 122 • 54th Annual Meeting of the Orthopaedic Research Society Shift in Chondrocyte TGF-β Receptor Usage, ALK5 to ALK1 Signaling, as a potential Cause for Osteoarthritis Esmeralda N. Blaney Davidson 1 , Dennis F. Remst 1 , Elly L. Vitters 1 , Henk B. van Beuningen 1 , Arjen B. Blom 1 , Marie-Jose Goumans 2 , Wim B. van den Berg 1 , Peter M. van der Kraan 1 1 Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; 2 University Medical Centre Utrecht, Utrecht, Netherlands [email protected] Introduction: Ageing is the primary risk factor for osteoarthritis (OA). During OA, chondrocytes show deviant behavior resembling terminal differentiation of growth-plate chondrocytes, characterized by elevated MMP-13 expression. Previously we demonstrated a reduction in TGF-β signaling via the ALK5 recep- tor in aged mice and in experimental OA. ALK5 activates the Smad2/3 route, which is known to suppress terminal dif- ferentiation. Recently, it has been shown that in certain cell types TGF-β is also able to sig- nal via the alternative TGF-β receptor ALK1, thereby activating the Smad1/5/8 route instead of the Smad2/3 route. The Smad1/5/8 route has been shown to induce chondrocyte terminal differentiation. Therefore, we investigated whether during ageing and OA, TGF-β signaling switched from ALK5 to ALK1, favoring progression of chondrocyte differentiation. Materials and Methods: First we investigated whether TGF-β was able to sig- nal via both ALK5 and ALK1 in chondrocytes. Therefore we analyzed phospho- rylation of Smad2 (Smad2P) and Smad1/5/8 (Smad1/5/8P) upon TGF-β stimu- lation by Western Blotting. Furthermore, we assessed whether ALK5 and ALK1 signaled specifically via Smad2 or Smad1/5/8 respectively. To assay the latter we transfected chondrocytes with constitutive active TGF-β receptors, Ad-caALK5 or Ad-caALK1, after which a Western Blot was performed staining for Smad2P and Smad1/5/8P. RNA was isolated to evaluate the response to ALK5 or ALK1 sig- naling on expression of aggrecan, collagen type II and MMP-13 mRNA. In addition, knee joints were isolated from naive C57Bl/6 mice aged 1 or 2 years, OA prone STR/ort mice (aged 3, 6 and 9 months and 1 year) and 3 months old C57Bl/6 mice in which OA was induced by destabilizing the medial meniscus (DMM model) and prepared for histology in paraffin sections. Immunohistochemistry for ALK5, ALK1, PAI1 (marker Smad2/3 route) and Id1 (marker Smad1/5/8 route) was performed. The number of cells staining positive for each receptor in tibial cartilage was measured with a computerized imaging sys- tem and corrected for the cell number in HE-stained sections. Results: In vitro, chondrocytes displayed both Smad2P and Smad1/5/8P upon stimulation with TGF-β. Thus both routes can be activated by TGF-β. Transfection of chondrocytes with caALK5 specifically led to Smad2P, whereas caALK1 specifically led to Smad1/5/8P. Chondrocytes that overexpressed caALK5 showed enhanced aggrecan expression and slightly reduced collagen type II expres- sion. Chondrocytes overexpressing caALK1 had elevated expression of aggrecan, collagen type II and MMP-13, thereby displaying an OA-associated phenotype. From 1 year old to 2 year old mice ALK5 decreased 88% in medial tibial car- tilage and 76% in lateral tibial cartilage. ALK1 also was reduced, but only 51% in medial and and 33% in lateral tibial cartilage. PAI1 expression had been drastical- ly reduced in old mice by 77% on the medial side and 61% on the lateral side of the tibia. Id1 expression had also decreased, but by 58% and 56% respectively. Thus, with age there was a stronger reduction in ALK5 than ALK1 expression and sig- naling via Smad2/3 (PAI1) appears to be more reduced than signaling via Smad1/5/8 (Id1). In the DMM model no change was observed on the lateral side, but a reduc- tion of 91% ALK5 positive cells compared to 75% ALK1 positive cells in the OA developing medial side. PAI and Id1 only showed a reduction in staining in the cartilage on the medial side with 79% and 70% respectively. This again shows a stronger reduction in ALK5 signaling than signaling via ALK1. STR/ort mice develop the most severe OA in the medial tibia. The number of cells staining positive for ALK5 was already reduced to 7% in the medial tibial car- tilage by 3 months of age. This declined to less than 1 % by 9 months. (Figure 1) On the lateral side there was still abundant ALK5 expression at 3 months of age. This rapidly decreased in time to less than 10% by 9 months and 1 year of age. PAI expression was already low on the medial side of the tibia by 3 months of age and rapidly declined to less than 1% by 9 months of age. On the lateral side PAI expression was abundantly present at 3 months of age and declined by 26% by 6 months of age. Thereafter there was a drastic reduction to 3% and 1% by 9 months and 1 year of age respectively. The percentage of cells positive for ALK1 was much higher than those for ALK5 on the medial side of the tibia. (Figure 1) This was 33% in 3-month-old mice and declined to 13% by 6 months of age. Thereafter the number of cells increased again to 22% and 27% by 9 months and 1 year respec- tively. On the lateral tibia the expression levels of ALK5 and ALK1 were compa- rable at the age of 3 months. Thereafter, the ratio changed clearly favoring the ALK1 side. Overall, with progressing age and OA development a significant increase in the ALK1/ALK5 ratio was observed. The dominance of Smad1/5/8 signaling (ALK1) compared to Smad2/3 signaling (ALK5) was also reflected by the increased Id1/PAI ratio. Discussion: Our data show a strong decrease in ALK5 expression in cartilage of ageing mice, whereas ALK1 expression only slightly diminished. In murine models of OA we showed a pronounced decrease of ALK5 expression in progres- sive OA and a less pronounced decrease of ALK1. In addition, PAI1 expression decreased more rapidly than Id1 expression, indicating that the change in receptor expression ratio results in an altered downstream signaling and protein expression. Thus, with age and OA progression, the balance between ALK1 and ALK5 expression is in favour of the ALK1 side. We have shown in vitro that ALK1 sig- naling in chondrocytes induces OA-like changes indicating a role for ALK1 sig- naling in OA development. Overall our data suggest a role for decreased ALK5 and increased ALK1 sig- naling in deviant chondrocyte behavior in ageing and OA development.
Transcript of Shift in Chondrocyte TGF-βReceptor Usage, ALK5 to ALK1 … · 2020. 9. 23. · Paper No. 122 •...
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Paper No. 122 • 54th Annual Meeting of the Orthopaedic Research Society
Shift in Chondrocyte TGF-β Receptor Usage, ALK5 to ALK1 Signaling, as a potential Cause for Osteoarthritis
Esmeralda N. Blaney Davidson1, Dennis F. Remst1, Elly L. Vitters1, Henk B. van Beuningen1, Arjen B. Blom1, Marie-Jose Goumans2, Wim B. vanden Berg1, Peter M. van der Kraan1
1Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; 2University Medical Centre Utrecht, Utrecht, [email protected]
Introduction: Ageing is the primary risk factor for osteoarthritis (OA). DuringOA, chondrocytes show deviant behavior resembling terminal differentiation ofgrowth-plate chondrocytes, characterized by elevated MMP-13 expression.Previously we demonstrated a reduction in TGF-β signaling via the ALK5 recep-tor in aged mice and in experimental OA.
ALK5 activates the Smad2/3 route, which is known to suppress terminal dif-ferentiation.
Recently, it has been shown that in certain cell types TGF-β is also able to sig-nal via the alternative TGF-β receptor ALK1, thereby activating the Smad1/5/8route instead of the Smad2/3 route. The Smad1/5/8 route has been shown toinduce chondrocyte terminal differentiation. Therefore, we investigated whetherduring ageing and OA, TGF-β signaling switched from ALK5 to ALK1, favoringprogression of chondrocyte differentiation.
Materials and Methods: First we investigated whether TGF-β was able to sig-nal via both ALK5 and ALK1 in chondrocytes. Therefore we analyzed phospho-rylation of Smad2 (Smad2P) and Smad1/5/8 (Smad1/5/8P) upon TGF-β stimu-lation by Western Blotting. Furthermore, we assessed whether ALK5 and ALK1signaled specifically via Smad2 or Smad1/5/8 respectively. To assay the latter wetransfected chondrocytes with constitutive active TGF-β receptors, Ad-caALK5 orAd-caALK1, after which a Western Blot was performed staining for Smad2P andSmad1/5/8P. RNA was isolated to evaluate the response to ALK5 or ALK1 sig-naling on expression of aggrecan, collagen type II and MMP-13 mRNA.
In addition, knee joints were isolated from naive C57Bl/6 mice aged 1 or 2years, OA prone STR/ort mice (aged 3, 6 and 9 months and 1 year) and 3 monthsold C57Bl/6 mice in which OA was induced by destabilizing the medial meniscus(DMM model) and prepared for histology in paraffin sections.Immunohistochemistry for ALK5, ALK1, PAI1 (marker Smad2/3 route) and Id1(marker Smad1/5/8 route) was performed. The number of cells staining positivefor each receptor in tibial cartilage was measured with a computerized imaging sys-tem and corrected for the cell number in HE-stained sections.
Results: In vitro, chondrocytes displayed both Smad2P and Smad1/5/8P uponstimulation with TGF-β. Thus both routes can be activated by TGF-β.Transfection of chondrocytes with caALK5 specifically led to Smad2P, whereascaALK1 specifically led to Smad1/5/8P. Chondrocytes that overexpressed caALK5showed enhanced aggrecan expression and slightly reduced collagen type II expres-sion. Chondrocytes overexpressing caALK1 had elevated expression of aggrecan,collagen type II and MMP-13, thereby displaying an OA-associated phenotype.
From 1 year old to 2 year old mice ALK5 decreased 88% in medial tibial car-tilage and 76% in lateral tibial cartilage. ALK1 also was reduced, but only 51% inmedial and and 33% in lateral tibial cartilage. PAI1 expression had been drastical-ly reduced in old mice by 77% on the medial side and 61% on the lateral side of thetibia. Id1 expression had also decreased, but by 58% and 56% respectively. Thus,with age there was a stronger reduction in ALK5 than ALK1 expression and sig-naling via Smad2/3 (PAI1) appears to be more reduced than signaling viaSmad1/5/8 (Id1).
In the DMM model no change was observed on the lateral side, but a reduc-tion of 91% ALK5 positive cells compared to 75% ALK1 positive cells in the OAdeveloping medial side. PAI and Id1 only showed a reduction in staining in thecartilage on the medial side with 79% and 70% respectively. This again shows astronger reduction in ALK5 signaling than signaling via ALK1.
STR/ort mice develop the most severe OA in the medial tibia. The number ofcells staining positive for ALK5 was already reduced to 7% in the medial tibial car-tilage by 3 months of age. This declined to less than 1 % by 9 months. (Figure 1)
On the lateral side there was still abundant ALK5 expression at 3 months ofage. This rapidly decreased in time to less than 10% by 9 months and 1 year of age.PAI expression was already low on the medial side of the tibia by 3 months of ageand rapidly declined to less than 1% by 9 months of age. On the lateral side PAIexpression was abundantly present at 3 months of age and declined by 26% by 6months of age. Thereafter there was a drastic reduction to 3% and 1% by 9 monthsand 1 year of age respectively. The percentage of cells positive for ALK1 was muchhigher than those for ALK5 on the medial side of the tibia. (Figure 1) This was33% in 3-month-old mice and declined to 13% by 6 months of age. Thereafter thenumber of cells increased again to 22% and 27% by 9 months and 1 year respec-tively. On the lateral tibia the expression levels of ALK5 and ALK1 were compa-rable at the age of 3 months. Thereafter, the ratio changed clearly favoring theALK1 side.
Overall, with progressing age and OA development a significant increase in theALK1/ALK5 ratio was observed. The dominance of Smad1/5/8 signaling (ALK1)compared to Smad2/3 signaling (ALK5) was also reflected by the increasedId1/PAI ratio.
Discussion: Our data show a strong decrease in ALK5 expression in cartilageof ageing mice, whereas ALK1 expression only slightly diminished. In murinemodels of OA we showed a pronounced decrease of ALK5 expression in progres-sive OA and a less pronounced decrease of ALK1. In addition, PAI1 expressiondecreased more rapidly than Id1 expression, indicating that the change in receptorexpression ratio results in an altered downstream signaling and protein expression.Thus, with age and OA progression, the balance between ALK1 and ALK5expression is in favour of the ALK1 side. We have shown in vitro that ALK1 sig-naling in chondrocytes induces OA-like changes indicating a role for ALK1 sig-naling in OA development.
Overall our data suggest a role for decreased ALK5 and increased ALK1 sig-naling in deviant chondrocyte behavior in ageing and OA development.
