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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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Increased trabecular bone in KO mice. To ascertain whether differences in boneformation persisted as the mice aged, 3-month-, 1–year- and 2-year-old mousefemurs from WT, Het and KO were subjected to microCT analysis. (a)Representative microCT generated longitudinal sections of hind limb femursdemonstrated increased number and penetration into the diaphysis of trabeculae(dotted lines) in the KO animal at 3 months. (b) Increased trabeculaeobserved in 200 16-μm slices in cross-section of the KO mouse.(c) Analysis of trabecular bone (red circle in b) shows asignificant increase in mineralized bone volume/total bone volume (BV/TV),trabecular number (Tb.n.) and connectivity density (Conn.Dens., anassessment of the number of connected structures in the trabecular bone network)in KO mice compared with WT. There was no difference in bone quality (mineraldensity) of trabeculae. (d) Cortical bone BV/TV and mean density showedno significant difference. (e) Trabecular analysis by microCT at 1 year ofage showed equalization of BV/TV, which persisted through 2 years (f).(n=3 for each genotype; **P≤0.01; NS, notsignificant; scale bar, 500 μm)
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fig2: Increased trabecular bone in KO mice. To ascertain whether differences in boneformation persisted as the mice aged, 3-month-, 1–year- and 2-year-old mousefemurs from WT, Het and KO were subjected to microCT analysis. (a)Representative microCT generated longitudinal sections of hind limb femursdemonstrated increased number and penetration into the diaphysis of trabeculae(dotted lines) in the KO animal at 3 months. (b) Increased trabeculaeobserved in 200 16-μm slices in cross-section of the KO mouse.(c) Analysis of trabecular bone (red circle in b) shows asignificant increase in mineralized bone volume/total bone volume (BV/TV),trabecular number (Tb.n.) and connectivity density (Conn.Dens., anassessment of the number of connected structures in the trabecular bone network)in KO mice compared with WT. There was no difference in bone quality (mineraldensity) of trabeculae. (d) Cortical bone BV/TV and mean density showedno significant difference. (e) Trabecular analysis by microCT at 1 year ofage showed equalization of BV/TV, which persisted through 2 years (f).(n=3 for each genotype; **P≤0.01; NS, notsignificant; scale bar, 500 μm)

Mentions: To determine whether enhanced bone formation persisted, 3-month-, 1-year- and2-year-old mouse femurs from all three genotypes were analyzed using microCT.MicroCT longitudinal evaluation revealed Het and KO mice displayed visiblyincreased penetration of the trabeculae into the central diaphysis of the femur at3 months (Figure 2a; n=3 pergenotype). Cross-sectional analysis of 200 slices of the femur (Figure 2b, red circles) indicated a significant increase intrabecular bone volume/total volume (BV/TV) (Figures2c, P=0.004), number of trabeculae(P=0.002) and connectivity density (P=0.002) inthe femurs of Het and KO when compared with WT femurs (Figure2c). No significant differences were observed in the cortical boneBV/TV or the quality of bone (bone mineral density; BMD) between the threegenotypes (Figure 2d). Additionally, no differenceswere observed in trabecular or cortical bone of femurs from 1- and 2-year-old KOand WT mice (Figures 2e and f), indicatingintramembranous bone formation is not affected by the lack of ASK1.


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)

Increased trabecular bone in KO mice. To ascertain whether differences in boneformation persisted as the mice aged, 3-month-, 1–year- and 2-year-old mousefemurs from WT, Het and KO were subjected to microCT analysis. (a)Representative microCT generated longitudinal sections of hind limb femursdemonstrated increased number and penetration into the diaphysis of trabeculae(dotted lines) in the KO animal at 3 months. (b) Increased trabeculaeobserved in 200 16-μm slices in cross-section of the KO mouse.(c) Analysis of trabecular bone (red circle in b) shows asignificant increase in mineralized bone volume/total bone volume (BV/TV),trabecular number (Tb.n.) and connectivity density (Conn.Dens., anassessment of the number of connected structures in the trabecular bone network)in KO mice compared with WT. There was no difference in bone quality (mineraldensity) of trabeculae. (d) Cortical bone BV/TV and mean density showedno significant difference. (e) Trabecular analysis by microCT at 1 year ofage showed equalization of BV/TV, which persisted through 2 years (f).(n=3 for each genotype; **P≤0.01; NS, notsignificant; scale bar, 500 μm)
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fig2: Increased trabecular bone in KO mice. To ascertain whether differences in boneformation persisted as the mice aged, 3-month-, 1–year- and 2-year-old mousefemurs from WT, Het and KO were subjected to microCT analysis. (a)Representative microCT generated longitudinal sections of hind limb femursdemonstrated increased number and penetration into the diaphysis of trabeculae(dotted lines) in the KO animal at 3 months. (b) Increased trabeculaeobserved in 200 16-μm slices in cross-section of the KO mouse.(c) Analysis of trabecular bone (red circle in b) shows asignificant increase in mineralized bone volume/total bone volume (BV/TV),trabecular number (Tb.n.) and connectivity density (Conn.Dens., anassessment of the number of connected structures in the trabecular bone network)in KO mice compared with WT. There was no difference in bone quality (mineraldensity) of trabeculae. (d) Cortical bone BV/TV and mean density showedno significant difference. (e) Trabecular analysis by microCT at 1 year ofage showed equalization of BV/TV, which persisted through 2 years (f).(n=3 for each genotype; **P≤0.01; NS, notsignificant; scale bar, 500 μm)
Mentions: To determine whether enhanced bone formation persisted, 3-month-, 1-year- and2-year-old mouse femurs from all three genotypes were analyzed using microCT.MicroCT longitudinal evaluation revealed Het and KO mice displayed visiblyincreased penetration of the trabeculae into the central diaphysis of the femur at3 months (Figure 2a; n=3 pergenotype). Cross-sectional analysis of 200 slices of the femur (Figure 2b, red circles) indicated a significant increase intrabecular bone volume/total volume (BV/TV) (Figures2c, P=0.004), number of trabeculae(P=0.002) and connectivity density (P=0.002) inthe femurs of Het and KO when compared with WT femurs (Figure2c). No significant differences were observed in the cortical boneBV/TV or the quality of bone (bone mineral density; BMD) between the threegenotypes (Figure 2d). Additionally, no differenceswere observed in trabecular or cortical bone of femurs from 1- and 2-year-old KOand WT mice (Figures 2e and f), indicatingintramembranous bone formation is not affected by the lack of ASK1.

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