M. Kawai, N. Douris , C. Green , B. Lecka-Czernik, C. Ackert-Bicknell, C. Rosen

Rosiglitazone (ROZ) treatment of mesenchymal cells (MSCs) suppresses  osteoblastogenic genes and increases adipogenic genes. We performed microarray analyses to identify genes whose expression was altered in response to ROZ in MSCs. One such transcript was nocturnin (Noc), a gene originally cloned from Xenopus retinae. Noc is a peripheral clock gene encoding a deadenylase expressed in bone, fat, liver and stem cells. Hepatic Noc mRNA exhibits a circadian rhythm, being highest early at night.  RT-PCR from MSCs showed increased Noc expression during adipogenesis but reduced expression during osteoblastogenesis. Thus, we hypothesized that Noc was involved in cell specification and was an important determinant of bone mass. To test this, we analyzed the phenotypes of Noc knockout mice (Noc -/-). Noc -/- mice showed increased trabecular bone volume and increased cortical thickness by microCT, but reduced number of marrow adipocytes. In vitro, calvarial osteoblasts from Noc -/- mice showed enhanced osteoblastogenesis and impaired adipogenesis. Over-expression of Noc in MC3T3-E1 cells suppressed mineralization, while over-expression of Noc in 3T3-L1 cells enhanced adipogenesis. These data indicate that Noc regulates MSC cell fate by favoring adipogenesis and suppressing osteoblastogenesis. Interestingly, DXA analysis showed that with age Noc-/- mice had progressively enhanced % body fat. At 16 weeks, ovarian fat volume was increased in Noc-/- mice with fewer but larger adipocytes, suggesting that cell hypertrophy rather than number was responsible for increased body adiposity. Next, we focused on downstream targets of Noc. We previously reported that ROZ suppressed IGF-1 expression in MSCs although the mechanism was not known. Calvarial osteoblasts from Noc-/- mice showed increased IGF-1 expression, whereas MC3T3-E1 cells overexpressing Noc (MC3T3-Noc) showed reduced Igf1 mRNA after treatment with actinomycin D. Thus, we hypothesized that Igf1mRNA was a target of Noc de-adenylation. To test this, we isolated the 3’UTR of the Igf1 gene and inserted it into a Luciferase (LUC) vector. When  the 6.4 kb full-length 3’UTR was inserted, MC3T3-Noc showed reduced LUC activity, while Noc did not have any effect on the 170 bp short-form 3’UTR. Thus, Noc recognizes a 6.4 kb full-length 3’UTR and degrades IGF-1 mRNA. In summary, Noc has an important role in cell specification of MSCs, and may be regulating bone mass by modulating IGF-1 expression within the skeletal environment.

Disclosures: None