Pin1‐mediated Runx2 modification is critical for skeletal development

Abstract

Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

“…DNA Construction and Site-directed Mutagenesis-Construction of the Pin1 (HA-pcDNA3.1-Pin1) WT and mutant (Y23A, W34A, and C113A) expression vectors has been described previously (25). Ds-Red Pin1 WT and mutant constructs have also been described previously (26).…”

“…Cell Culture and Nuclear-Cytoplasmic FractionationMouse myogenic C2C12, HEK293, preosteoblast MC3T3-E1, multipotent ST2 mesenchymal progenitor, and mouse embryonic fibroblast (MEF) cells were cultured as described previously (25,29). Nuclear-cytoplasmic fractionation was conducted using the NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Fisher Scientific) according to the protocol of the manufacturer.…”

“…Extraction of Total RNAs, RT-PCR, and Real-time PCR (Quantitative PCR)-Total RNA extraction from cultured cells, RT-PCR, quantitative PCR, and the primer sets for real-time PCR have been described previously (25,28).…”

“…GST Pulldown Assay, Immunoprecipitation (IP), and Immunoblot Analyses-Cellular proteins and GST pulldown assays were prepared in lysis buffer as described previously (25). The GST pulldown assay, IP, and immunoblot analysis methods have been described previously (25).…”

“…Pin1-catalyzed, post-phosphorylation conformational regulation often functions as a molecular timer (24). We have found previously that the interaction of Pin1 with Runx2, the major transcription factor for osteoblast differentiation, is critical for bone development (25,26). We showed that Pin1 hetero and KO mice exhibit reduced mineralization at birth.…”