Background/Purpose:   Mechanisms associated with intrinsic mRNA regulation are important players in development and disease processes. The terminal uridylyl transferases (TUT) are a family of enzymes involved in the regulation of mRNA turnover and apoptosis-mediated global mRNA decay. However, their role in skeletal homeostasis has not been explored. In this study, we investigated the role of ZCCHC6/ TUT7 in osteogenesis and bone homeostasis in mice.

Methods: RNA and protein expression were analyzed using SYBR green-based qPCR and Western blotting respectively. Osteoblasts (OB) and osteoclasts (OC) were generated following our published protocols. TUT7KO mice were generated by gene targeting and their skeletal phenotype was characterized using DXA, mCT, histology and histochemistry. Osteocalcin (OCN), OPG and RANKL were quantified by ELISA. Promoter activity was assessed by Luciferase assays.

Results:   TUT7 showed an age-dependent differential pattern of expression in osseous tissues with expression being high in calvaria and long bones of newborn mice which declined with aging. Next, we generated TUT7KO mice and characterized their skeletal phenotype. Deletion of TUT7 was not lethal and neonatal TUT7KO mice developed normal similiar to wildtype (WT) littermates. However, at 8 weeks of age TUT7KO mice showed significantly increased bone mass compared to WT as determined by DEXA, mCT, and histomorphometric analyses. Serum analyses revealed an increase in OCN and OPG levels as well as a decrease in RANKL suggesting an overall increase in bone mass. Ex vivo analysis of bone marrow derived osteoprogenitor cells and primary calvarial OBs from TUT7KO mice demonstrated increased matrix mineralization as shown by von Kossa staining. These data suggest that TUT7 plays an important role in OB function. Interestingly, osteoclasts derived from TUT7KO mice showed no significant difference in differentiation and function as determined by TRAP staining and osteoclast activity assay. Co-culture of osteoblast and osteoclast precursors from WT and TUT7KO mice revealed that TUT7KO osteoblasts inhibited the generation of TRAP positive osteoclasts. This suggests that the role of TUT7 in bone homeostasis is predominantly mediated by the osteoblasts. Expression analyses of genes known to be important in osteogenesis showed that constitutive expression of master transcription factor osterix (Osx) was significantly upregulated in OB derived from TUT7KO mice compared to OB derived from WT littermates. Importantly genes known to be regulated by Osx were also expressed at significantly higher levels in OB derived from TUT7 KO mice. Regulation of Osx activity by TUT7 was demonstrated using an Osx reporter activity assay and inhibition of the promoter activity by overexpression of TUT7 in OB derived from TUT7 KO mice.

Conclusion:   Overall, our data demonstrate that TUT7 is a negative regulator of bone formation through suppression of Osx activity in OB and influencing osteoblast-mediated regulation of osteoclastogenesis. This study is the first to demonstrate the role of TUT7 in bone formation and identifies a potential target for therapeutic interventions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/zcchc6tut7-negatively-regulates-osterix-activity-and-influences-osteoblast-mediated-regulation-of-osteoclastogenesis/