Skp2 inhibits osteogenesis by promoting ubiquitin–proteasome degradation of Runx2

Abstract

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

CDK2 destabilizes tumor suppressor C/EBPα expression through ubiquitin‐mediated proteasome degradation in acute myeloid leukemia

“…Western blot analysis was performed as earlier described . In brief, cells were lysed either in RIPA lysis buffer (1% (w/w) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.15 M NaCl, 5 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol, phosphatase and protease inhibitors) or in Urea lysis buffer.…”

“…Western blot analysis was performed as earlier described. 16,26,27 In brief, cells were lysed either in RIPA lysis buffer (1% (w/w) NP40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.15 M NaCl, 5 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol, phosphatase and protease inhibitors) or in Urea lysis buffer. Equal amount of protein was loaded on 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred on polyvinylidene fluoride membrane, and probed with 1:1000 dilution of primary antibody (1:500 for endogenous proteins) and 1:10 000 for HRP-conjugated secondary antibody.…”

Nano‐LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells

“…Antibodies used for Enolase (sc‐15343), anti‐GST (sc‐459), ER alpha (sc‐543x) were purchased from Santa Cruz Biotechnology Inc; anti‐HA (2367S) from Cell Signalling Technology, E6AP (E8655) and anti β‐actin (A3854) from Sigma Aldrich. Chemiluminescence was detected with ECL western blotting detection reagents (Millipore) using LAS 4010 (GE Healthcare) as described previously …”

“…MDA‐MB‐231 cells were plated in chamber slide one day before staining. Next day, cells were processed for immunofluorescence microscopy as previously described . Alexa Fluor‐594 donkey antirabbit (A21207; Invitrogen) and Alexa‐Fluor 488 goat antimouse (A11055; Invitrogen) secondary antibodies and 4′, 6‐diamino‐2‐phenylindole staining were procured from Invitrogen and Sigma Aldrich, respectively.…”

Retenção prolongada de dentes decíduos: possíveis fatores etiológicos locais e sistêmicos

“…Enquanto os osteoblastos expressam o RANKL como um fator associado à membrana, os préosteoclastos, que expressam RANK, reconhecem o RANKL pela interação célulacélula e diferenciam-se em osteoclastos 17 . A partir deste fato, tem importante papel então o RUNX2, fator essencial para induzir a diferenciação osteoblástica 17,52,[96][97][98][99][100] . O processo completo de diferenciação de células tronco mesenquimais indiferenciadas em osteoblastos ocorre em diferentes fases, sendo que cada uma delas é caracterizada por um padrão particular de expressão de genes marcadores de osteoblastos.…”