Elsevier

Bone

Volume 110, May 2018, Pages 221-229
Bone

Full Length Article
Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway

https://doi.org/10.1016/j.bone.2018.02.018Get rights and content

Highlights

  • Clusterin protein is expressed and secreted by mBMSCs.

  • sCLU inhibits the differentiation of BMSCs into osteoblasts versus adipocytes.

  • sCLU regulates the differentiation of BMSCs by modulating ERK1/2 signaling pathway.

Abstract

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation.

Introduction

Bone marrow skeletal (also known as stromal or mesenchymal) stem cells (BMSCs) are a subpopulation of adult stem cells that reside in the bone marrow within a specific perivascular niche and are characterized by their ability for self-renewal and multipotent differentiation into mesodermal cells, including osteoblast, adipocytes, and chondrocytes [[1], [2], [3], [4]]. Several pre-clinical and clinical studies have suggested the possible use of BMSC-based therapy for enhancing bone regeneration in a number of conditions, such as non-union fracture, bone reconstruction and augmentation in cranial, oral, maxillo-facial and long bone defects [5]. Thus, understanding the regulatory mechanisms underlying the differentiation of BMSCs into bone-forming osteoblastic cell lineage is important to provide novel therapeutic targets that can be used to direct the differentiation of BMSCs into the osteoblastic lineage to enhance bone formation.

In this context, we and others have demonstrated that the regulation of BMSCs differentiation into osteoblasts is mediated by the secreted factors produced by BMSCs [3,6]. These osteogenic secreted factors include the secreted Frizzled-related protein 1 (sFRP-1) [7], Delta like-1/Fetal antigen 1 (Dlk1/FA1) [8,9], Leukemia inhibitor factor (LIF) [10], Vascular endothelial growth factor A (VEGF) [11], WNT1-induced Secreted Protein-1 (WISP1) [12], Semaphorin 3A (Sema3A) [13] and Nel-Related Protein 1, NELL-1 [14]. We have also previously employed global, hypothesis-generating methods of transcriptomics or proteomics to identify novel factors important for BMSCs commitment to osteoblastic cells and to bone formation [15,16]. By comparing the transcriptome and secretome of BMSC-derived osteoprogenitor cells versus adipoprogenitor cells [17], we identified Clusterin and found that its expression was significantly upregulated in BMSCs-derived adipocytes (Abdallah BM and Kassem M, unpublished data).

Clusterin (CLU, also known as Apolipoprotein J), is a heterodimeric protein, that found in two forms: nuclear form (nCLU) and soluble form (sCLU). sCLU is ubiquitously expressed in many tissues including brain, liver, testis, ovary, and heart and is present in the circulation and in all biological fluids as a component of high density lipoprotein (HDL) complex with Apolipoprotein A-1 (ApoA1) [[18], [19], [20]]. sCLU is a pro-cell survival factor, that is involved in the regulation of cell proliferation, apoptosis, tissue remodeling, complement inhibition, lipid transport, and carcinogenesis [18,21,22]. sCLU has been reported to be protective against oxidative stress-induced apoptotic cell death in a variety of cells including BMSCs [23], [24] [25]. Furthermore, increased expression of CLU was shown to be associated with oxidative stress and inflammation in many diseases including neurodegenerative diseases, cancers and inflammatory diseases [26]. The function of sCLU as an anti-apoptotic factor is mediated by the modulation of NF-κB, PI3K/AKT and ERK1/2 signaling pathways [[27], [28], [29]]. Regarding bone metabolism, sCLU was reported to inhibit osteoclast bone resorption by suppressing macrophage colony-stimulating factor, M-CSF-mediated ERK activation [30]. However, the role of sCLU in osteoblast differentiation from BMSCs and in bone formation has not been reported. In this study, we demonstrated that sClu is expressed by BMSCs and that its steady-state gene expression is increased during adipocyte differentiation and decreased during osteoblast differentiation. Functional analysis revealed that sCLU stimulates cell proliferation and the early commitment of BMSCs toward the adipocytic lineage at the expense of the osteoblastic lineage, an effect mediated via ERK1/2 phosphorylation.

Section snippets

Animals

C57BL/6 mice were originally purchased from Charles River. Mice were bred and housed under standard conditions (21 °C, 55% relative humidity) on a 12-h light/12-h dark cycle at the animal housing unit and the Physiology Laboratory, College of Science, King Faisal University, Saudi Arabia, in accordance with the protocol approved by the Standing Research Ethics Committee. Ad libitum food (Altromin® Spezialfutter GmbH & Co. KG, Lage, Germany) and water were provided. Sera were collected from young

Gene expression and protein secretion of sCLU during mBMSCs differentiation

We have recently employed a combination of microarray and secretome approaches to identify secreted factors regulating BMSC differentiation into osteoblasts and adipocytes. Among the factors identified and that have not been previously studied in the context of BMSC biology, we chose CLU to perform a detailed study. We examined the expression of Clu mRNA in flat and long bones, and we compared its expression levels to the one of other tissues in adult mice. As shown in Fig. 1A, Clu mRNA

Discussion

In this study, we have identified sCLU as a novel regulator of BMSC lineage commitment, which inhibits the differentiation of BMSCs into the osteoblastic cell lineage versus adipocytic cell lineage. Furthermore, we demonstrated that the inhibitory effect of sCLU on osteoblasts versus adipocyte differentiation, is mediated by the modulation of the ERK1/2 signaling pathway.

The ubiquitously expressed secreted glycoprotein sCLU plays an important role in cell proliferation and differentiation in

Conclusions

There is a need to identify the regulatory factors present within the BMSC niche and to determine their role in regulating BMSC lineage commitment and differentiation, as a pre-requisite to develop therapeutic strategies to enhance bone regeneration and bone formation [3]. Our study identified sCLU as a novel protein present within the BMSC niche, which controls the commitment of BMSCs into the adipogenic or osteogenic cell lineages. It is plausible that inhibiting sCLU within the BMSC niche

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

All materials are available by the corresponding author.

Competing interests

The authors declare that he has no competing interests.

Funding

This work was funded by the Deanship of Scientific Research at King Faisal University, Saudi Arabia, Grant # (170050).

Authors' contributions

BMA conceived the project, designed the study, performed experiments, analyzed data and wrote the manuscript. AZ performed experiments, analyzed data and edited the manuscript. MK conceived the project, analyzed the data and edited the manuscript.

Acknowledgments

The Authors acknowledge the Deanship of Scientific Research at King Faisal University, Saudi Arabia for the financial support (under Grant # 170050).

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