Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

Abstract

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.

“Genetic scissors” CRISPR/Cas9 genome editing cutting-edge biocarrier technology for bone and cartilage repair

“… Used the CRISPR/Cas9 system to screen for deubiquitinating enzymes (DUBs) that regulate MSX1 protein. Kaushal et al, [ 95 ] 2022 Wnt16 The Wnt pathway plays an important role in the osteogenic differentiation of BMSCs. Wnt16 is a ligand that affects stem cell proliferation, differentiation and migration through the Wnt pathway.…”

“…MSX1 balances the level of protein degradation during normal osteogenesis. In the present study, Kaushal et al used the CRISPR/Cas9 system to screen for deubiquitinating enzymes (DUBs) that regulate MSX1 protein and identified the Ubiquitin carboxyl-terminal hydrolase 11 (USP11) as a regulator of MSX1 [ 95 ]. USP11 overexpression enhances the expression of osteogenic factors in BMSCs.…”

Ubiquitin‐specific peptidases: Players in bone metabolism

“…Msh isoform 1 (MSX1) is also an upstream and downstream regulator of the BMP signalling pathway, and a balanced MSX1 protein level is crucial to normal osteogenesis 90 . USP11 prevents MSX1 protein degradation via its DUB activity, making it a positive regulator of BMSC osteogenic differentiation 27 …”

“…90 USP11 prevents MSX1 protein degradation via its DUB activity, making it a positive regulator of BMSC osteogenic differentiation. 27 Wnt/β-catenin is a crucial regulatory signalling pathway involved in the osteogenic differentiation of BMSCs. 91 As a member of the FBP family, FBXO31 interacts with the SCF complex to form the functional E3 ubiquitin ligase SCF-FBXO31, which regulates the ubiquitination of target proteins.…”

“…9 In the past decade, USPs have attracted increasing attention as potential therapeutic targets for different diseases. 10 In terms of bone metabolism, USPs have been found to be involved in the regulation of bone metabolism by promoting the osteogenesis of mesenchymal cells or Mouse USP2 PTHR PTH Osteoblast Proliferation (+) [ 16] Human USP4 DVL WNT/β-catenin Osteoblast Differentiation (À) [ 17] Mouse USP4 SIRT1 Sost Osteocyte Sclerostin transcription (À) [ 18] Mouse USP6 BMP Preosteoblast Differentiation and maturation (À) [19] Human USP7 SOX2 and NANOG Mesenchymal stem cell Self-renewal capacity (+) [ 20] Mouse USP7 KDM6B BMP Osteoblast Differentiation and autophagy (+) [21] Human USP7 RUNX2 BMP Mesenchymal stem cell Osteogenic differentiation (+) [ 22] Human USP7 Adipose-derived stem cells Osteogenic differentiation (+) [ 23] Mouse USP7 Axin WNT/β-catenin Osteoblast Differentiation (À) [ 24] Mouse USP8 FZD5 WNT/β-catenin Osteoblast Differentiation (+) [ 25] Mouse USP10 p53 Oestrogen Osteocytes and osteoblast Senescence (+) [ 26] Human USP11 MSX1 BMP Mesenchymal stem cell Osteogenic differentiation (+) [ 27] Mouse USP13 PTEN NF-κB Osteoclast Differentiation (À) [ 28] Mouse USP14 NLRC5 NF-κB Osteoclast Differentiation (À) [ 29] Mouse USP14 p53 p21 Osteoblast Senescence (+) [ 30] Mouse USP15 IκBα NF-κB Osteoclast Differentiation (À) [ 31] Mouse USP15 ALK3 BMP Myoblast progenitor Osteogenic differentiation (+) [ 32] Mouse USP15 β-catenin WNT/β-catenin Osteoblast Differentiation (+) [ 33] Human USP18 TAK1 NF-κB Monocytes and macrophages Inflammatory response (À) [ 34] Mouse USP18 Mesenchymal stem cell Osteogenic differentiation (À) [ 13] Mouse USP21 Mesenchymal stem cell Osteogenic differentiation (+) [ 13] Human USP25 TRAF6 NF-κB Osteoclast Differentiation (+) [ 14] Mouse USP26 β-catenin WNT/β-catenin Mesenchymal stem cell Osteogenic differentiation (+) [ 13] Mouse USP26 IκBα NF-κB Bone myelomonocytes Osteoclastogenesis (À) [ 13] Mouse USP34 IκBα NF-κB Osteoclast Differentiation (À) [ 12] Human USP34 Smad1 and RUNX2 BMP Mesenchymal stem cell Osteogenic differentiation (+)…”