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Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Eaton GJ, Zhang QS, Diallo C, Matsuzawa A, Ichijo H, Steinbeck MJ, Freeman TA - Cell Death Dis (2014)

Bottom Line: These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death.However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation.These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

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Altered death signaling in KO MEFs in response to stress. (a) Twenty-fourhours culture with H2O2 caused increased phosphorylation ofJNK and p38 in WT MEFs, both were decreased in ASK1 KO cells. (b) Anincrease in cl-CASP3 and cl-PARP, both apoptotic activators, was also observed inWT MEFs, but not in KO MEFs. (c) Less expression of the apoptotic effectorsBAD, BAX and BID was observed in KO cells, in contrast to their upregulation in WTcells. (d) For KO cells cultured in the presence of Staur, decreasedphosphorylation of JNK and p38 was observed. (e) Decreased cl-CASP3 andcl-PARP were also observed in KO MEF Staur culture. (f) Similarly, BAX, BIDand cytochrome c release were reduced in KO MEFs cultured with Staur. In eachcase, the opposite was seen in WT MEFs. (n=3 replicates;*P<0.05; **P<0.01;#P<0.05 to WT Ctrl;##P<0.01 to WT Ctrl) (g) An MTT viabilityassay showed that WT cells have significantly decreased viability compared withASK1 KO cells after 24 h culture with H2O2 or Staur(n=4 replicates; **P<0.01). (h)Immunohistochemistry revealed decreased cl-CASP3 in the growth plate of KO mice,compared with WT mice. (Scale bar=50 μm)
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fig6: Altered death signaling in KO MEFs in response to stress. (a) Twenty-fourhours culture with H2O2 caused increased phosphorylation ofJNK and p38 in WT MEFs, both were decreased in ASK1 KO cells. (b) Anincrease in cl-CASP3 and cl-PARP, both apoptotic activators, was also observed inWT MEFs, but not in KO MEFs. (c) Less expression of the apoptotic effectorsBAD, BAX and BID was observed in KO cells, in contrast to their upregulation in WTcells. (d) For KO cells cultured in the presence of Staur, decreasedphosphorylation of JNK and p38 was observed. (e) Decreased cl-CASP3 andcl-PARP were also observed in KO MEF Staur culture. (f) Similarly, BAX, BIDand cytochrome c release were reduced in KO MEFs cultured with Staur. In eachcase, the opposite was seen in WT MEFs. (n=3 replicates;*P<0.05; **P<0.01;#P<0.05 to WT Ctrl;##P<0.01 to WT Ctrl) (g) An MTT viabilityassay showed that WT cells have significantly decreased viability compared withASK1 KO cells after 24 h culture with H2O2 or Staur(n=4 replicates; **P<0.01). (h)Immunohistochemistry revealed decreased cl-CASP3 in the growth plate of KO mice,compared with WT mice. (Scale bar=50 μm)

Mentions: During the process of hypertrophy, chondrocytes in the growth plate are exposed tostress followed by apoptotic cell death. To investigate whether loss of ASK1protects chondrocytes from death, MEFs isolated from WT and KO mice were seeded asa micromass and cultured in chondrogenic media containingH2O2 or Staur. WT chondrogenic MEFs treated withH2O2 displayed a significant increase in ASK1 signaling,as evidenced by increased phosphorylation of JNK and p38 (Figure 6a). Conversely, JNK and p38 phosphorylation afterH2O2 stimulation were decreased in KO MEFs. A significantdecrease in cleavage of both CASP3 and Poly (ADP-ribose) polymerase (PARP), twoinitiators of apoptosis, was also observed (Figure6b). Furthermore, decreased expression of the apoptosis effectorsBcl-2-associated death promoter (BAD), Bcl-2-associated X protein (BAX) and BH3interacting-domain death agonist (BID) was observed in the KO MEFs (Figure 6c). Similarly, treatment with Staur showedsignificant decreases in ASK1 signaling in the KO MEFs compared with WT (Figure 6d), concurrent with a decrease in apoptoticproteins cl-CASP3, cl-PARP (Figure 6e), BAX, BID andcytochrome c (Figure 6f).


Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

Eaton GJ, Zhang QS, Diallo C, Matsuzawa A, Ichijo H, Steinbeck MJ, Freeman TA - Cell Death Dis (2014)

Altered death signaling in KO MEFs in response to stress. (a) Twenty-fourhours culture with H2O2 caused increased phosphorylation ofJNK and p38 in WT MEFs, both were decreased in ASK1 KO cells. (b) Anincrease in cl-CASP3 and cl-PARP, both apoptotic activators, was also observed inWT MEFs, but not in KO MEFs. (c) Less expression of the apoptotic effectorsBAD, BAX and BID was observed in KO cells, in contrast to their upregulation in WTcells. (d) For KO cells cultured in the presence of Staur, decreasedphosphorylation of JNK and p38 was observed. (e) Decreased cl-CASP3 andcl-PARP were also observed in KO MEF Staur culture. (f) Similarly, BAX, BIDand cytochrome c release were reduced in KO MEFs cultured with Staur. In eachcase, the opposite was seen in WT MEFs. (n=3 replicates;*P<0.05; **P<0.01;#P<0.05 to WT Ctrl;##P<0.01 to WT Ctrl) (g) An MTT viabilityassay showed that WT cells have significantly decreased viability compared withASK1 KO cells after 24 h culture with H2O2 or Staur(n=4 replicates; **P<0.01). (h)Immunohistochemistry revealed decreased cl-CASP3 in the growth plate of KO mice,compared with WT mice. (Scale bar=50 μm)
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fig6: Altered death signaling in KO MEFs in response to stress. (a) Twenty-fourhours culture with H2O2 caused increased phosphorylation ofJNK and p38 in WT MEFs, both were decreased in ASK1 KO cells. (b) Anincrease in cl-CASP3 and cl-PARP, both apoptotic activators, was also observed inWT MEFs, but not in KO MEFs. (c) Less expression of the apoptotic effectorsBAD, BAX and BID was observed in KO cells, in contrast to their upregulation in WTcells. (d) For KO cells cultured in the presence of Staur, decreasedphosphorylation of JNK and p38 was observed. (e) Decreased cl-CASP3 andcl-PARP were also observed in KO MEF Staur culture. (f) Similarly, BAX, BIDand cytochrome c release were reduced in KO MEFs cultured with Staur. In eachcase, the opposite was seen in WT MEFs. (n=3 replicates;*P<0.05; **P<0.01;#P<0.05 to WT Ctrl;##P<0.01 to WT Ctrl) (g) An MTT viabilityassay showed that WT cells have significantly decreased viability compared withASK1 KO cells after 24 h culture with H2O2 or Staur(n=4 replicates; **P<0.01). (h)Immunohistochemistry revealed decreased cl-CASP3 in the growth plate of KO mice,compared with WT mice. (Scale bar=50 μm)
Mentions: During the process of hypertrophy, chondrocytes in the growth plate are exposed tostress followed by apoptotic cell death. To investigate whether loss of ASK1protects chondrocytes from death, MEFs isolated from WT and KO mice were seeded asa micromass and cultured in chondrogenic media containingH2O2 or Staur. WT chondrogenic MEFs treated withH2O2 displayed a significant increase in ASK1 signaling,as evidenced by increased phosphorylation of JNK and p38 (Figure 6a). Conversely, JNK and p38 phosphorylation afterH2O2 stimulation were decreased in KO MEFs. A significantdecrease in cleavage of both CASP3 and Poly (ADP-ribose) polymerase (PARP), twoinitiators of apoptosis, was also observed (Figure6b). Furthermore, decreased expression of the apoptosis effectorsBcl-2-associated death promoter (BAD), Bcl-2-associated X protein (BAX) and BH3interacting-domain death agonist (BID) was observed in the KO MEFs (Figure 6c). Similarly, treatment with Staur showedsignificant decreases in ASK1 signaling in the KO MEFs compared with WT (Figure 6d), concurrent with a decrease in apoptoticproteins cl-CASP3, cl-PARP (Figure 6e), BAX, BID andcytochrome c (Figure 6f).

Bottom Line: These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death.However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation.These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.

Show MeSH
Related in: MedlinePlus