- Email: [email protected]
Transthyretin deposition accelerates the development of experimental osteoarthritis in mice
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Results: Chondrocytes lacking either TRPC6 or CXCR2 expressed lower mRNA levels of the chondrocyte differentiation markers SOX9 and type II collagen in comparison to controls, whilst the culture of chondrocytes in micromass resulted in significantly less sulfated proteoglycan production by TRPC6-/- chondrocytes compared to wild type. Activation of CXCR2 in articular chondrocytes resulted in increased intracellular calcium mobilization. AKT phosphorylation was increased in wild type chondrocytes following CXCL6 treatment, but this was not observed in TRPC6-/- cells. In vitro activation of TRPC6 using hyp9 led to increased AKT phosphorylation in wild type chondrocytes, whereas chelation of intracellular calcium inhibited CXCL6-induced phosphorylation of AKT. Finally, activation of TRPC6 resulted in a significant increase in SOX9 and type II collagen mRNA expression, together with a decrease in type X collagen mRNA expression and was demonstrated to rescue the decreased SOX9 expression previously observed in CXCR2-/- chondrocytes. Conclusions: TRPC6 calcium channel activity is required for chondrocyte phenotypic stability. In vitro TRPC6 activation is sufficient to increase AKT phosphorylation and the expression of key chondrocyte phenotypic markers in murine chondrocytes. This data suggests that TRPC6 may play a role within CXCR2-mediated chondrocyte phenotypic stability and may provide an ideal therapeutic target for the modulation of this pathway. 250 TRANSTHYRETIN DEPOSITION ACCELERATES THE DEVELOPMENT OF EXPERIMENTAL OSTEOARTHRITIS IN MICE T. Matsuzaki y, O. Alvarez-Garcia y, Y. Akasaki z, N. Reixach y, J. Buxbaum y, M.K. Lotz y. y The Scripps Res. Inst., La Jolla, CA, USA; z Dept. of Orthopaedic Surgery, Graduate Sch. of Med. Sci., Kyushu Univ., Fukuoka, Japan Purpose: Deposition of amyloid is a common aging-associated phenomenon and a key factor in the pathogenesis of several aging-related diseases. Osteoarthritis (OA) is the most prevalent joint disease and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein. We previously reported that amyloid and TTR deposition were increased in human OA cartilage with aging and OA grade. In addition, amyloidogenic TTR affected chondrocyte survival and induced the OA related genes such as ADAMTS4, interleukin 6 and inducible nitric oxide synthase. Here we investigated the role of TTR in vivo. Transgenic mice for wild-type human TTR (hTTR TG mice) were analyzed using an experimental OA model. Methods: TTR in cartilage and chondrocytes was analyzed by immunohistochemistry and Western blotting. OA was surgically induced by destabilizing the medial meniscus of the hTTR TG mice (n ¼ 26) and control mice (n ¼ 22). Mice were sacrificed 10 weeks after surgery. To investigate the effects of TTR on cartilage, 6-month-old mice were sacrificed and examined by immunohistochemistry, real-time PCR and Western blotting. In addition, quantitative analysis of cell number in cartilage was examined in 6-, 12-month-old mice, and surgical model. OA-related tissue changes were evaluated using the Glasson’s semiquantitative cartilage scoring system and Krenn's synovitis score. Results: TTR protein was detected cartilage in hTTR TG mice, but chondrocytes did not express TTR mRNA, the transgene was highly overexpressed in the liver. ADAMTS4 and MMP13 mRNA were significantly elevated in cartilage in 6 month-old hTTR TG mice compared with control mice. Immunohistochemical and Western blotting analysis showed increased MMP13 expression in the hTTR TG mice 10 weeks after surgery compared with control mice. In addition, nuclear factor-kB (NF-kB) p65 and Phospho-NF-kB p65 was elevated in hTTR TG mice. In the surgical model, both histological OA score and synovitis score were significantly increased in hTTR TG mice. Additionally, cellularity was significantly lower in the 6-month-old hTTR TG mice and hTTR TG mice with surgical OA compared with control mice. Conclusions: These findings are the first to show that TTR deposition accelerated the development of OA in the surgically-induced murine OA model. TTR amyloid formation represents a novel mechanism that contributes to aging as a risk factor for OA and is a new target for OA prevention and treatment. 251 PANNEXIN 3, BUT NOT PANNEXIN 1 IS AN IMPORTANT PROCATABOLIC MEDIATOR IN OSTEOARTHRITIS. P. Moon, S. Penuela, D.W. Laird, F. Beier. Univ. of Western Ontario, London, ON, Canada
S153
Purpose: Osteoarthritis (OA) is a disabling degenerative joint disease associated with high levels of individual suffering and a tremendous societal burden. Although much progress has been made identifying novel regulators of cartilage metabolism in OA, to date, no disease modifying therapies exist. This highlights the need for a deeper understanding of the underlying molecular mechanisms driving OA initiation and progression. Recently, we have identified Pannexin 3 (Panx3) as a novel pro-catabolic factor in OA and showed that both cartilage specific and global Panx3 deletion confers protection against surgically induced OA development. In follow up to this data, we have investigated the effects of a variety of signalling mediators on primary chondrocyte cultures isolated from wild-type (WT) and Panx3 KO mice to identify pathways upstream of Panx3 in chondrocytes. Additionally, we have investigated the effects of Panx1 deletion on surgically induced OA progression due to its homology to Panx3 and its overlapping expression pattern. Methods: In vitro studies utilized primary immature murine articular chondrocytes (IMAC) isolated from tibiae and femurs of 5 day-old mice. Cultures were treated with TNF, IL1, ATP and TNF plus the Pannexin inhibitor probenecid. Following 24 or 48 hours of treatment, cells were harvested and RNA or protein extracted. qPCR is used to analyze gene expression and western blotting to quantify protein levels. To examine the effects of Panx1 deletion on OA progression, destabilization of the medial meniscus was performed on the left knees of WT and Panx1 KO mice. Eight weeks post surgery, mice were euthanized and joints harvested, paraffinized and sectioned. Safranin-O/Fast Green stained sections were scored according to the OARSI histopathology scoring system. Immunohistochemical analysis of Panx3 was also performed on serial sections. Results: WT and Panx3 KO chondrocytes showed similar responses to all cytokines as measured through QPCR, indicating that the effect of Panx3 deletion observed in our in vivo studies is not mediated by alterations in pro-inflammatory responses. Treatment with probenecid had no effect on either WT or Panx3 KO chondrocytes. Interestingly however, in chondrocytes isolated from Rac1 KO mice, Panx3 expression was significantly elevated, indicating interplay between these two pathways in the onset of chondrocyte hypertrophy, possibly in the chondrocyte mechano-response. In contrast to the chondroprotective effects observed in the Panx3 KO mice, Panx1 KO’s were not protected from the effects of surgical joint destabilization. Similar grade OA was observed in the KO DMM mice as in the WT mice, suggesting that Panx3 is of greater importance in this process. Additionally, we did not see increased Panx3 expression in Panx1 KO mice above what is typically seen in OA cartilage. Conclusions: Although no differences were observed in the responsiveness of Panx3 KO chondrocytes to various pro-inflammatory cytokines, the potential interplay between Rac1 and Panx3 provides us with an exciting new pathway, one we are currently investigating. Observing no differences in OA progression between Panx1 KO and WT DMM operated mice increases our confidence in the central role of Panx3 in the cartilage, and we are further investigating its role in aging associated OA in mice. Ultimately, these results provide novel insights into the specific molecular role of Panx3 in cartilage homeostasis and OA. 252 THE ROLE OF NUCLEAR RECEPTORS LIVER X RECEPTOR AND RETINOID X RECEPTOR IN MURINE CARTILAGE M.-G. Sun, A. Ratneswaran, H. Dupuis, F. Beier. Univ. of Western Ontario, London, ON, Canada Purpose: Osteoarthritis (OA) is a disabling disease characterized by cartilage degradation of the joint. A better understanding of the genetic regulators of cartilage breakdown is needed to understand OA pathogenesis and develop novel therapeutic strategies. Previously, studies have shown activation of a nuclear receptor, the Liver X Receptor (LXR), to possess protective effects against cartilage degradation. Our recent studies similarly show LXR activation to suppress chondrocyte hypertrophy. However, LXR regulates transcription by forming obligate heterodimers with the Retinoid X Receptor (RXR) - a common heterodimeric partner of different nuclear receptors. In order to elucidate the underlying mechanisms of LXR’s protective effect in cartilage, we investigate and compare the effects of LXR and RXR activation in the maintenance of cartilage health. Methods: The specific LXR agonist, GW3965, as well as the specific RXR agonists, SR11237 and Bexarotene, were used to examine the effect of
Results: Chondrocytes lacking either TRPC6 or CXCR2 expressed lower mRNA levels of the chondrocyte differentiation markers SOX9 and type II collagen in comparison to controls, whilst the culture of chondrocytes in micromass resulted in significantly less sulfated proteoglycan production by TRPC6-/- chondrocytes compared to wild type. Activation of CXCR2 in articular chondrocytes resulted in increased intracellular calcium mobilization. AKT phosphorylation was increased in wild type chondrocytes following CXCL6 treatment, but this was not observed in TRPC6-/- cells. In vitro activation of TRPC6 using hyp9 led to increased AKT phosphorylation in wild type chondrocytes, whereas chelation of intracellular calcium inhibited CXCL6-induced phosphorylation of AKT. Finally, activation of TRPC6 resulted in a significant increase in SOX9 and type II collagen mRNA expression, together with a decrease in type X collagen mRNA expression and was demonstrated to rescue the decreased SOX9 expression previously observed in CXCR2-/- chondrocytes. Conclusions: TRPC6 calcium channel activity is required for chondrocyte phenotypic stability. In vitro TRPC6 activation is sufficient to increase AKT phosphorylation and the expression of key chondrocyte phenotypic markers in murine chondrocytes. This data suggests that TRPC6 may play a role within CXCR2-mediated chondrocyte phenotypic stability and may provide an ideal therapeutic target for the modulation of this pathway. 250 TRANSTHYRETIN DEPOSITION ACCELERATES THE DEVELOPMENT OF EXPERIMENTAL OSTEOARTHRITIS IN MICE T. Matsuzaki y, O. Alvarez-Garcia y, Y. Akasaki z, N. Reixach y, J. Buxbaum y, M.K. Lotz y. y The Scripps Res. Inst., La Jolla, CA, USA; z Dept. of Orthopaedic Surgery, Graduate Sch. of Med. Sci., Kyushu Univ., Fukuoka, Japan Purpose: Deposition of amyloid is a common aging-associated phenomenon and a key factor in the pathogenesis of several aging-related diseases. Osteoarthritis (OA) is the most prevalent joint disease and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein. We previously reported that amyloid and TTR deposition were increased in human OA cartilage with aging and OA grade. In addition, amyloidogenic TTR affected chondrocyte survival and induced the OA related genes such as ADAMTS4, interleukin 6 and inducible nitric oxide synthase. Here we investigated the role of TTR in vivo. Transgenic mice for wild-type human TTR (hTTR TG mice) were analyzed using an experimental OA model. Methods: TTR in cartilage and chondrocytes was analyzed by immunohistochemistry and Western blotting. OA was surgically induced by destabilizing the medial meniscus of the hTTR TG mice (n ¼ 26) and control mice (n ¼ 22). Mice were sacrificed 10 weeks after surgery. To investigate the effects of TTR on cartilage, 6-month-old mice were sacrificed and examined by immunohistochemistry, real-time PCR and Western blotting. In addition, quantitative analysis of cell number in cartilage was examined in 6-, 12-month-old mice, and surgical model. OA-related tissue changes were evaluated using the Glasson’s semiquantitative cartilage scoring system and Krenn's synovitis score. Results: TTR protein was detected cartilage in hTTR TG mice, but chondrocytes did not express TTR mRNA, the transgene was highly overexpressed in the liver. ADAMTS4 and MMP13 mRNA were significantly elevated in cartilage in 6 month-old hTTR TG mice compared with control mice. Immunohistochemical and Western blotting analysis showed increased MMP13 expression in the hTTR TG mice 10 weeks after surgery compared with control mice. In addition, nuclear factor-kB (NF-kB) p65 and Phospho-NF-kB p65 was elevated in hTTR TG mice. In the surgical model, both histological OA score and synovitis score were significantly increased in hTTR TG mice. Additionally, cellularity was significantly lower in the 6-month-old hTTR TG mice and hTTR TG mice with surgical OA compared with control mice. Conclusions: These findings are the first to show that TTR deposition accelerated the development of OA in the surgically-induced murine OA model. TTR amyloid formation represents a novel mechanism that contributes to aging as a risk factor for OA and is a new target for OA prevention and treatment. 251 PANNEXIN 3, BUT NOT PANNEXIN 1 IS AN IMPORTANT PROCATABOLIC MEDIATOR IN OSTEOARTHRITIS. P. Moon, S. Penuela, D.W. Laird, F. Beier. Univ. of Western Ontario, London, ON, Canada
S153
Purpose: Osteoarthritis (OA) is a disabling degenerative joint disease associated with high levels of individual suffering and a tremendous societal burden. Although much progress has been made identifying novel regulators of cartilage metabolism in OA, to date, no disease modifying therapies exist. This highlights the need for a deeper understanding of the underlying molecular mechanisms driving OA initiation and progression. Recently, we have identified Pannexin 3 (Panx3) as a novel pro-catabolic factor in OA and showed that both cartilage specific and global Panx3 deletion confers protection against surgically induced OA development. In follow up to this data, we have investigated the effects of a variety of signalling mediators on primary chondrocyte cultures isolated from wild-type (WT) and Panx3 KO mice to identify pathways upstream of Panx3 in chondrocytes. Additionally, we have investigated the effects of Panx1 deletion on surgically induced OA progression due to its homology to Panx3 and its overlapping expression pattern. Methods: In vitro studies utilized primary immature murine articular chondrocytes (IMAC) isolated from tibiae and femurs of 5 day-old mice. Cultures were treated with TNF, IL1, ATP and TNF plus the Pannexin inhibitor probenecid. Following 24 or 48 hours of treatment, cells were harvested and RNA or protein extracted. qPCR is used to analyze gene expression and western blotting to quantify protein levels. To examine the effects of Panx1 deletion on OA progression, destabilization of the medial meniscus was performed on the left knees of WT and Panx1 KO mice. Eight weeks post surgery, mice were euthanized and joints harvested, paraffinized and sectioned. Safranin-O/Fast Green stained sections were scored according to the OARSI histopathology scoring system. Immunohistochemical analysis of Panx3 was also performed on serial sections. Results: WT and Panx3 KO chondrocytes showed similar responses to all cytokines as measured through QPCR, indicating that the effect of Panx3 deletion observed in our in vivo studies is not mediated by alterations in pro-inflammatory responses. Treatment with probenecid had no effect on either WT or Panx3 KO chondrocytes. Interestingly however, in chondrocytes isolated from Rac1 KO mice, Panx3 expression was significantly elevated, indicating interplay between these two pathways in the onset of chondrocyte hypertrophy, possibly in the chondrocyte mechano-response. In contrast to the chondroprotective effects observed in the Panx3 KO mice, Panx1 KO’s were not protected from the effects of surgical joint destabilization. Similar grade OA was observed in the KO DMM mice as in the WT mice, suggesting that Panx3 is of greater importance in this process. Additionally, we did not see increased Panx3 expression in Panx1 KO mice above what is typically seen in OA cartilage. Conclusions: Although no differences were observed in the responsiveness of Panx3 KO chondrocytes to various pro-inflammatory cytokines, the potential interplay between Rac1 and Panx3 provides us with an exciting new pathway, one we are currently investigating. Observing no differences in OA progression between Panx1 KO and WT DMM operated mice increases our confidence in the central role of Panx3 in the cartilage, and we are further investigating its role in aging associated OA in mice. Ultimately, these results provide novel insights into the specific molecular role of Panx3 in cartilage homeostasis and OA. 252 THE ROLE OF NUCLEAR RECEPTORS LIVER X RECEPTOR AND RETINOID X RECEPTOR IN MURINE CARTILAGE M.-G. Sun, A. Ratneswaran, H. Dupuis, F. Beier. Univ. of Western Ontario, London, ON, Canada Purpose: Osteoarthritis (OA) is a disabling disease characterized by cartilage degradation of the joint. A better understanding of the genetic regulators of cartilage breakdown is needed to understand OA pathogenesis and develop novel therapeutic strategies. Previously, studies have shown activation of a nuclear receptor, the Liver X Receptor (LXR), to possess protective effects against cartilage degradation. Our recent studies similarly show LXR activation to suppress chondrocyte hypertrophy. However, LXR regulates transcription by forming obligate heterodimers with the Retinoid X Receptor (RXR) - a common heterodimeric partner of different nuclear receptors. In order to elucidate the underlying mechanisms of LXR’s protective effect in cartilage, we investigate and compare the effects of LXR and RXR activation in the maintenance of cartilage health. Methods: The specific LXR agonist, GW3965, as well as the specific RXR agonists, SR11237 and Bexarotene, were used to examine the effect of