Background/Purpose:

Autophagy is an essential, homeostatic process by which cells digest unnecessary or damaged organelles. Atg7 is essentially required for formation of the autophagosome. The DMARD chloroquine (CQ) has been identified to arrest autophagy function by blocking the fusion between autophagosomes and lysosomes. Accumulating evidence demonstrates that autophagy is involved in the pathology of varies diseases including infections, cancer and neurodegeneration and recent reports also linked autophagy to osteoclastogenesis and rheumatoid arthritis.

Methods:

Systemic bone loss was induced by ovariectomy (OVX). Autophagy was modulated by genetic as well as pharmacologic approaches. For genetic inhibition of autophagy, we crossbred Atg7^fl/fl mice with LysM Cre⁺ mice to generate Atg7^fl/fl x LysM Cre⁺ mice with selective inactivation of autophagy function in the monocyte lineage. For pharmacological inhibition, eight-week-old mice underwent daily intraperitoneal injection of 20mg kg^-1 CQ for a total of 50 days. We applied Microcomputed tomography to analyze bone density in vivo. TRAP staining and bone histomorphometry were used to confirm osteoclast number in tissue. To assess osteoclast formation and activity in vitro, Atg7^fl/fl x LysM Cre⁺ BMCs and CQ treated BMCs were cultured on bone slices and analysed by the TRAP staining and Toluidine blue staining (n=6, for each group). To assess the autophagy effect on osteoblast activity in vitro, primary osteoblasts were treated with Cre Adenovirus and CQ to analyse ALP staining and measure osteoblast markers by RT-PCR.

Results:

To investigate the effect of CQ on OVX-induced systemic bone loss, we analyzed the bone density (bone volume/trabecular volume; Bv/Tv) and the trabecular number (Tb. N.) at the proximal tibias. Bv/Tv and Tb. N. were strongly reduced in sham-treated OVX mice. The decreases in Bv/Tv and in Tb. N. were reduced by 76 % and 60% in CQ treated OVX mice (n=6, p<0.05 for both). Inhibition of autophagy by selective knockdown of Atg7 in monocytic cells (Atg7^fl/fl x LysM Cre-mice) also prevented OVX-induced osteoporosis and prevented OVX-induced decreases in Bv/Tv and in Tb. N.. Histomorphometric analyses demonstrated decreased osteoclast counts in CQ-treated mice and Atg7^fl/fl x LysM Cre-mice, respectively, compared to controls. Consistent with these in vivo results, inhibition of autophagy, either by treatment with CQ or by knockdown of Atg7, also prevented osteoclast differentiation and osteoclast mediated bone resorption in vitro. In contrast to the effects on osteoclasts, inhibition of autophagy by genetic or pharmacologic approaches did not inhibit osteoblast activity.   

Conclusion:  

We demonstrate that inhibition of autophagy by genetic or by pharmacological approaches significantly reduced OVX-induced osteoporosis in mice. These findings identify the autophagy machinery as a potential target for the treatment of osteoporoses. Considering the potent anti-osteoporotic effects and the availability of approved inhibitors such as CQ, these findings may have direct translational implications.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-autophagy-prevents-ovariectomy-induced-bone-loss/