Stability of mRNA influences osteoporotic bone mass via CNOT3

Abstract

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3′-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3′-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.RNA stability | osteopenia

Adipocyte‐specific disruption of mouse Cnot3 causes lipodystrophy

“…As Cnot3 facilitates CCR4-NOT deadenylase activity [8][9][10], deadenylation-induced mRNA decay of specific targets may contribute to maintenance of adipose homeostasis. As Cnot3 facilitates CCR4-NOT deadenylase activity [8][9][10], deadenylation-induced mRNA decay of specific targets may contribute to maintenance of adipose homeostasis.…”

“…The mammalian CCR4-NOT complex comprises at least 10 subunits, Cnot1-Cnot3, Cnot6, Cnot6l, and Cnot7-Cnot11 [6]. Cnot3, a noncatalytic subunit, is involved in substrate recognition, complex formation, and regulation of deadenylase activity [7][8][9][10]. Cnot3 participates in energy metabolism and senses nutrient states [11,12].…”

Profilin 1 Negatively Regulates Osteoclast Migration in Postnatal Skeletal Growth, Remodeling, and Homeostasis in Mice

“…RAW264.7 cells were maintained in culture with Dulbecco's modified Eagle's medium or alpha‐MEM supplemented with 10% FBS by subconfluent stage. By replating the cells onto individual plates or wells, the osteoclastogenesis assay was conducted using RANKL (100 ng/mL; Oriental Yeast Co., Tokyo, Japan) as described previously . The siRNA transfection was conducted using lipofectamine RNAiMax reagent (Life Technologies; Thermo Fisher Scientific), according to the manufacturer's standard protocol.…”

“…By replating the cells onto individual plates or wells, the osteoclastogenesis assay was conducted using RANKL (100 ng/mL; Oriental Yeast Co., Tokyo, Japan) as described previously. (18,22) The siRNA transfection was conducted using lipofectamine RNAiMax reagent (Life Technologies; Thermo Fisher Scientific), according to the manufacturer's standard protocol. The siRNA for the mouse Pfn1 (Ambion Silencer Select #4390771-s71525, Thermo Fisher Scientific; Sense CAAUGGAUCUUCGUACCAATT, Antisense UUGGUAC-GAAGAUCCAUUGTA) and control siRNA (Ambion Silencer Negative Control No.1 siRNA) was purchased from the vendor (Life Technologies; Thermo Fisher Scientific).…”

CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State

“…Ccr4-Not is the main mRNA deadenylase complex in eukaryotic cells that shortens mRNA poly(A) tails (Collart and Panasenko, 2012, Shirai et al., 2014, Xu et al., 2014). It has been implicated in various physiological and developmental processes, such as spermatogenesis (Berthet et al., 2004), heart development (Neely et al., 2010), energy metabolism (Morita et al., 2011), B cell differentiation (Inoue et al., 2015), osteoporosis (Watanabe et al., 2014), reprogramming (Kamon et al., 2014), and ESC self-renewal (Hu et al., 2009, Zheng et al., 2012). However, its role in early development and the mechanism by which it regulates pluripotency remain to be fully elucidated.…”