Ubiquitin specific peptidase Usp53 regulates osteoblast versus adipocyte lineage commitment

Abstract

We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (− 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

Expression and Role of Ubiquitin-Specific Peptidases in Osteoblasts

“…The scope of our work covers the characterization of novel molecular mechanisms involved in the anabolic action of parathyroid hormone (1–34) (PTH) in osteoblasts [ 77 , 78 , 79 , 80 ]. Administrating PTH at a low dosage once a day (intermittent PTH, iPTH) promotes bone formation through pleiotropic effects on osteoblasts and osteocytes [ 81 , 82 , 83 ].…”

“…Work from our laboratory led to the identification and characterization of the nascent-polypeptide-associated complex and coregulator alpha (NACA), a transcriptional coregulator, as a target of the iPTH-PKA axis in osteoblasts (reviewed in [ 84 ]). The physiological relevance of this pathway is mediated through the induction of downstream effectors crucial for osteoblast function and bone biology [ 77 , 78 , 79 , 80 ]. Using RNA-sequencing and ChIP-sequencing against NACA, we identified Usp53 as a transcriptional target induced by the iPTH-NACA axis in osteoblasts [ 77 ].…”

“…The physiological relevance of this pathway is mediated through the induction of downstream effectors crucial for osteoblast function and bone biology [ 77 , 78 , 79 , 80 ]. Using RNA-sequencing and ChIP-sequencing against NACA, we identified Usp53 as a transcriptional target induced by the iPTH-NACA axis in osteoblasts [ 77 ]. The RNA-sequencing data of PTH-treated MC3T3-E1 osteoblastic cells has yielded hundreds of differentially regulated genes [ 77 ], some of which belonged to the USP family of deubiquitinases ( Table 1 ).…”

“…Using RNA-sequencing and ChIP-sequencing against NACA, we identified Usp53 as a transcriptional target induced by the iPTH-NACA axis in osteoblasts [ 77 ]. The RNA-sequencing data of PTH-treated MC3T3-E1 osteoblastic cells has yielded hundreds of differentially regulated genes [ 77 ], some of which belonged to the USP family of deubiquitinases ( Table 1 ). An RNA-seq analysis has revealed that Usp9x was predominantly expressed in MC3T3-E1 cells at basal levels, followed by Usp18 , Usp12 , Usp30, and Usp36 that showed moderate levels of expression and Usp53 , Usp35 , Usp2 , and Usp27x that exhibited the lowest levels of expression [ 77 ].…”

“…The RNA-sequencing data of PTH-treated MC3T3-E1 osteoblastic cells has yielded hundreds of differentially regulated genes [ 77 ], some of which belonged to the USP family of deubiquitinases ( Table 1 ). An RNA-seq analysis has revealed that Usp9x was predominantly expressed in MC3T3-E1 cells at basal levels, followed by Usp18 , Usp12 , Usp30, and Usp36 that showed moderate levels of expression and Usp53 , Usp35 , Usp2 , and Usp27x that exhibited the lowest levels of expression [ 77 ]. Interestingly, Usp53 and Usp2 were significantly upregulated by more than two-fold following iPTH treatment ( Table 1 ).…”