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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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ASK1 KO histological examination of bone and cartilage. (a) Alizarin redstaining of femurs from WT, Het and KO at PD14. (b) Analysis confirmed asignificant increase in trabecular length in the KO mouse. (c) Saffranin Ostaining of the growth plates showed an increase in the length of the hypertrophiczone (black brackets), but a decrease in the proliferative zone (white brackets)for the Het and KO mice. (d) The significant increase in the length of thehypertrophic zone of both Het and KO mice expressed as % of growth plate.(e) The overall length of the growth plates is similar. (f) TRAPstaining of growth plate from WT, Het and KO at day 14. (g) Analysis showedthat the number of osteoclasts present was increased in Het and KO mice.(h) The size of osteoclasts was not significantly increased. (i) Theosteoclast surface to bone surface ratio (Oc.S/BS) was not significantlyincreased. (n=10 limbs each genotype; *P<0.05;**P≤0.01; NS, not significant; scale bar,50 μm)
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fig3: ASK1 KO histological examination of bone and cartilage. (a) Alizarin redstaining of femurs from WT, Het and KO at PD14. (b) Analysis confirmed asignificant increase in trabecular length in the KO mouse. (c) Saffranin Ostaining of the growth plates showed an increase in the length of the hypertrophiczone (black brackets), but a decrease in the proliferative zone (white brackets)for the Het and KO mice. (d) The significant increase in the length of thehypertrophic zone of both Het and KO mice expressed as % of growth plate.(e) The overall length of the growth plates is similar. (f) TRAPstaining of growth plate from WT, Het and KO at day 14. (g) Analysis showedthat the number of osteoclasts present was increased in Het and KO mice.(h) The size of osteoclasts was not significantly increased. (i) Theosteoclast surface to bone surface ratio (Oc.S/BS) was not significantlyincreased. (n=10 limbs each genotype; *P<0.05;**P≤0.01; NS, not significant; scale bar,50 μm)

Mentions: To determine the cellular dynamics responsible for the increase in diaphyseallength, WT, Het and KO mouse femurs from PD14 were sectioned and stained withalizarin red (mineralization), alcian blue (proteoglycan of cartilage) andtartrate-resistant acid phosphatase (TRAP, osteoclast marker; n=10per genotype). Results from the alizarin red staining showed that Het and KO micetrabeculae were longer than those in the WT mice (Figures 3aand b, P=0.005), but there was no apparent differencein the total mineral deposition. Comparison of the femoral growth plates showed anincrease in the length of the hypertrophic zone in Het and KO animals (Figure 3c, black brackets, D, P<0.0001,P<0.0001, respectively), but a decrease in the proliferative zone(Figure 3c, white brackets). The oppositedifferences in the length of these two zones ultimately resulted in no overallchange in growth plate length (Figure 3e). Finally,osteoclast parameters, including osteoclast number, area and osteoclast surface onbone surface (Oc.S/BS), were determined on the equivalent femur sections forall three genotypes (Figure 3f). Both the Het and KOmice presented statistically significant increases in the number of osteoclasts(Figure 3g, P=0.007,P<0.0001, respectively), while there was no significant difference ineither the osteoclast area or the Oc.S/BS measurements (Figures 3h and i).


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)

ASK1 KO histological examination of bone and cartilage. (a) Alizarin redstaining of femurs from WT, Het and KO at PD14. (b) Analysis confirmed asignificant increase in trabecular length in the KO mouse. (c) Saffranin Ostaining of the growth plates showed an increase in the length of the hypertrophiczone (black brackets), but a decrease in the proliferative zone (white brackets)for the Het and KO mice. (d) The significant increase in the length of thehypertrophic zone of both Het and KO mice expressed as % of growth plate.(e) The overall length of the growth plates is similar. (f) TRAPstaining of growth plate from WT, Het and KO at day 14. (g) Analysis showedthat the number of osteoclasts present was increased in Het and KO mice.(h) The size of osteoclasts was not significantly increased. (i) Theosteoclast surface to bone surface ratio (Oc.S/BS) was not significantlyincreased. (n=10 limbs each genotype; *P<0.05;**P≤0.01; NS, not significant; scale bar,50 μm)
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fig3: ASK1 KO histological examination of bone and cartilage. (a) Alizarin redstaining of femurs from WT, Het and KO at PD14. (b) Analysis confirmed asignificant increase in trabecular length in the KO mouse. (c) Saffranin Ostaining of the growth plates showed an increase in the length of the hypertrophiczone (black brackets), but a decrease in the proliferative zone (white brackets)for the Het and KO mice. (d) The significant increase in the length of thehypertrophic zone of both Het and KO mice expressed as % of growth plate.(e) The overall length of the growth plates is similar. (f) TRAPstaining of growth plate from WT, Het and KO at day 14. (g) Analysis showedthat the number of osteoclasts present was increased in Het and KO mice.(h) The size of osteoclasts was not significantly increased. (i) Theosteoclast surface to bone surface ratio (Oc.S/BS) was not significantlyincreased. (n=10 limbs each genotype; *P<0.05;**P≤0.01; NS, not significant; scale bar,50 μm)
Mentions: To determine the cellular dynamics responsible for the increase in diaphyseallength, WT, Het and KO mouse femurs from PD14 were sectioned and stained withalizarin red (mineralization), alcian blue (proteoglycan of cartilage) andtartrate-resistant acid phosphatase (TRAP, osteoclast marker; n=10per genotype). Results from the alizarin red staining showed that Het and KO micetrabeculae were longer than those in the WT mice (Figures 3aand b, P=0.005), but there was no apparent differencein the total mineral deposition. Comparison of the femoral growth plates showed anincrease in the length of the hypertrophic zone in Het and KO animals (Figure 3c, black brackets, D, P<0.0001,P<0.0001, respectively), but a decrease in the proliferative zone(Figure 3c, white brackets). The oppositedifferences in the length of these two zones ultimately resulted in no overallchange in growth plate length (Figure 3e). Finally,osteoclast parameters, including osteoclast number, area and osteoclast surface onbone surface (Oc.S/BS), were determined on the equivalent femur sections forall three genotypes (Figure 3f). Both the Het and KOmice presented statistically significant increases in the number of osteoclasts(Figure 3g, P=0.007,P<0.0001, respectively), while there was no significant difference ineither the osteoclast area or the Oc.S/BS measurements (Figures 3h and i).

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