Distinct effects of loss of classical estrogen receptor signaling versus complete deletion of estrogen receptor alpha on bone

Abstract

Estrogen receptor (ER) α is a major regulator of bone metabolism which can modulate gene expression via a "classical" pathway involving direct DNA binding to estrogen-response elements (EREs) or via "non-classical" pathways involving protein-protein interactions. While the skeletal consequences of loss of ERE binding by ERα have been described, a significant unresolved question is how loss of ERE binding differs from complete loss of ERα. Thus, we compared the skeletal phenotype of wild-type (ERα(+/+)) and ERα knock out (ERα(-/-)) mice with that of mice in which the only ERα present had a knock-in mutation abolishing ERE binding (non-classical ERα knock-in [NERKI], ERα(-/NERKI)). All three groups were in the same genetic background (C57BL/6). As compared to both ERα(+/+) and ERα(-/-) mice, ERα(-/NERKI) mice had significantly reduced cortical volumetric bone mineral density and thickness at the tibial diaphysis; this was accompanied by significant decreases in periosteal and endocortical mineral apposition rates. Colony forming unit (CFU)-fibroblast, CFU-alkaline phosphatase, and CFU-osteoblast numbers were all increased in ERα(-/-) compared to ERα(+/+) mice, but reduced in ERα(-/NERKI) mice compared to the two other groups. Thus, using mice in identical genetic backgrounds, our data indicate that the presence of an ERα that cannot bind DNA but can function through protein-protein interactions may have more deleterious skeletal effects than complete loss of ERα. These findings suggest that shifting the balance of classical versus non-classical ERα signaling triggers pathways that impair bone formation. Further studies defining these pathways may lead to novel approaches to selectively modulate ER signaling for beneficial skeletal effects.

Figure 1

ER signaling pathways. The classical pathway of ERα action (left) requires direct binding to EREs found in the control regions of estradiol (E₂)-regulated genes. The non-classical pathway can involve either nuclear (middle) or membrane (right) signaling. In non-classical nuclear signaling, the ER interacts with other transcription factors, such as the jun/fos complex which binds to AP-1 sites, whereas in the membrane pathway, ER signaling is via alterations in kinase activity.

Figure 2

(A) Body weight (n = 48–49 per group) and (B) percent body fat by DXA in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 14–25 per group). **P < 0.01 and ***P < 0.001 versus ERα^+/+; ^†††P < 0.001 versus ERα^−/−.

Figure 3

(A) Total body aBMD, (B) spine aBMD, and (C) femur aBMD by DXA in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 14–25 per group). ***P < 0.001 versus ERα^+/+; ^†††P < 0.001 versus ERα^−/−.

Figure 4

(A) Total vBMD, (B) trabecular vBMD, and (C) cortical vBMD at the tibial metaphysis by QCT in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 18–31 per group). ***P < 0.001 versus ERα^+/+; ^†††P < 0.001 versus ERα^−/−.

Figure 5

(A) Cortical vBMD, (B) cortical thickness, (C) periosteal circumference, and (D) endosteal circumference at the tibial diaphysis in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 18–31 per group). ^aP = 0.051, *P < 0.05, **P < 0.01 and ***P < 0.001 versus ERα^+/+; ^††P < 0.01 and ^†††P < 0.001 versus ERα^−/−.

Figure 6

(A) Representative µCT images of the tibial metaphysis and (B) representative histological sections of the lumbar spine in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice.

Figure 7

(A) CFU-F, (B) CFU-AP, and (C) CFU-OB colonies (per 10⁷ cells plated) in ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 4 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 versus ERα^+/+; ^†††P < 0.001 versus ERα^−/−.

Figure 8

Oil-red O+ (adipocyte) cells in bone marrow cultures from ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 5–10 per group). *P < 0.05 versus ERα^+/+ mice.

Figure 9

(A) Osteoblast colonies and (B) Oil-red O+ (adipocyte) cells (open bars, vehicle control; solid bars, 10⁻⁸ M E₂) in bone marrow cultures from ERα^+/+, ERα^−/−, and ERα^−/NERKI mice (n = 5–10 per group). *P < 0.05 and **P < 0.01 versus the vehicle control.