Stress to endoplasmic reticulum of mouse osteoblasts induces apoptosis and transcriptional activation for bone remodeling

Abstract

ATF4 is an essential regulator in osteogenesis as well as in stress responses to the endoplasmic reticulum (ER). We addressed a question: Does ER stress to osteoblasts upregulate ATF4 expression? If so, do they exhibit ATF4-mediated bone remodeling or apoptosis? ER stress, induced by Thapsigargin and tunicamycin, elevated a phosphorylated form of eIF2alpha and ATF4, but the cellular fate depended on treatment duration. The treatment for 1h, for instance, activated Runx2, and type I collagen, while the treatment for 24h induced apoptosis. Our observations suggest that there is a threshold for ER stress and osteoblasts present a bi-phasic pattern of their fate.

Figure 1

Protein expression of ATF4 and eIF2α-p in response to Thapsigargin in MC3T3 cells. (A) Responses at 0.01 to 100 nM for 1 and 3 h. (B) Responses at 100 nM to 1.5 μM for 1 and 3 h. (C) Responses at 10 nM and 1.5 μM for 1 to 24 h.

Figure 2

Apoptotic cell death for continuous Thapsigargin treatments in MC3T3 cells. (A) Cellular images. Black bar = 100 μm. (B) Ratio of cell numbers 24 h after a continuous treatment. (C) Ratio of the number of dead cells with respect to the total number of cells. (D) CHOP, pro-caspase 3, and cleaved caspase 3 proteins in response to 10 nM Thapsigargin.

Figure 3

Responses to short exposure to Thapsigargin in MC3T3 cells. (A) ATF4 and eIF2α-p proteins in response to T _5m, T _15m , T _30m, and T _1h. (B) Levels of ATF4, Runx2, Osterix, Osteocalcin (OCN), and type I collagen (Col I) mRNA in response to T _1h.

Figure 4

Messenger RNA levels of ATF4, ATF3, Runx2, Osterix, CHOP, Rankl, Osteocalcin (OCN), and type I collagen (Col I) for the C4 clone in response to Thapsigargin (T_1h, T_2h, and T_3h).

Figure 5

Protein and mRNA expression for the C4 clone in response to 10 nM Thapsigargin (T_1h). (A) Expression of eIF2α-p, ATF4, ATF3, and CHOP proteins. (B) Temporal fold changes of ATF4 protein, eIF2α-p protein, and ATF4 mRNA.

Figure 6

Response to tunicamycin in the C4 clone. (A) Responses to tunicamycin at 0–1 μg/ml, and at 1 μg/ml (T_1h, T_2h, and T_3h). (B) Expression of pro- and cleaved caspase 3 for T _24h, and cell motility ratio in response to 1 μg/ml tunicamycin. (C) Messenger RNA levels of ATF4, ATF3, Runx2, Osterix, CHOP, Osteocalcin (OCN), and type I collagen (Col I) for the C4 clone in response to tunicamycin (T_1h, and T_3h).