NELL‐1 promotes cell adhesion and differentiation via integrinβ1

NELL‐1 (Nel‐like molecule‐1) is a secreted osteogenic growth factor first identified in human craniosynostosis (CS) patients. NELL‐1 protein has been observed to promote bone and cartilage differentiation and to suppress adipogenesis in both in vitro and in vivo models. Despite these findings, the cell surface receptors of NELL‐1 have remained unknown. In this study, we observed for the first time that NELL‐1 promotes cell adherence in multiple cell lines, including ST2, C3H10T1/2, M2‐10B4, ATDC5, and MC3T3 cells. Additionally, we found that NELL‐1 binds to extracellular Integrinβ1 and induces cell focal adhesion. By utilizing siRNA methods, we determined that NELL‐1 cell surface binding and enhanced cell attachment were dependent on Integrinβ1 expression. Finally, we observed that pre‐coating of culture dishes or PLGA (polylactic‐co‐glycolic acid) scaffold with NELL‐1 resulted in a significant increase in both cell attachment and osteogenic differentiation. Our results identify for the first time a cell surface target of NELL‐1, Integrinβ1, and elucidate new functions of NELL‐1 in promoting cell adherence and osteogenic differentiation. J. Cell. Biochem. 113: 3620–3628, 2012. © 2012 Wiley Periodicals, Inc.     

Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells

NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways. We have previously produced NELL1 using an insect’s cell expression system; however, the protein was relatively unstable and was degraded by proteases released from dead cells. In the present study, NELL1 protein was expressed in human embryonic kidney 293-F cells. Stable cell lines expressing NELL1 fused to a C-terminal hexahistidine-tag were obtained by G418 selection of transfected cells. Cells grown in serum-free medium showed high levels of NELL1 protein production (approximately 4 mg/l cell culture) for up to 6 months. NELL1 protein was purified from culture medium using a one-step nickel-chelate affinity chromatography protocol. Purified NELL1 protein immobilized onto culture dishes induced the expression of both early and late osteogenic markers on mouse mesenchymal C3H10T1/2 cells. When NELL1-expressing 293-F cells were grown on gelatin-coated glass cover slips, recombinant NELL1 was deposited in the extracellular matrix after detachment of cells. These results suggest that NELL1 acts as an extracellular matrix component. Recombinant NELL1 formed multimers and was glycosylated. An abundant source of functionally active NELL1 protein will be useful for more advanced studies, such as the development of novel techniques for bone regeneration.     

The C-terminal region of NELL1 mediates osteoblastic cell adhesion through integrin α3β1

NELL1 is a secretory osteogenic protein containing several structural motifs that suggest that it functions as an extracellular matrix component. To determine the mechanisms underlying NELL1-induced osteoblast differentiation, we examined the cell-adhesive activity of NELL1 using a series of recombinant NELL1 proteins. We demonstrated that NELL1 promoted osteoblastic cell adhesion through at least three cell-binding domains located in the C-terminal region of NELL1. Adhesion of cells to NELL1 was strongly inhibited by function-blocking antibodies against integrin α3 and β1 subunits, suggesting that osteoblastic cells adhered to NELL1 through integrin α3β1. Further, focal adhesion kinase activation is involved in NELL1 signaling.     

The roles of circRFWD2 and circINO80 during NELL‐1‐induced osteogenesis

Bone defects caused heavy social and economic burdens worldwide. Nel‐like molecule, type 1 (NELL‐1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL‐1‐induced osteogenesis of human adipose‐derived stem cells (hASCs). In this study, we performed RNA‐sequencing to investigate the expression profiles of circRNAs in recombinant NELL‐1‐induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up‐regulated during recombinant NELL‐1‐induced osteogenesis, and knockdown of them affected the positive effect of NELL‐1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa‐miR‐6817‐5p, which could inhibit the osteogenesis. Silencing hsa‐miR‐6817‐5p could partially reverse the negative effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL‐1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.     

The cytosolic splicing variant of NELL2 inhibits PKCβ1 in glial cells

NELL2 is an abundant glycoprotein containing EGF-like domain in the neural tissues where it has multiple physiological functions by interacting with protein kinase C (PKC). There are two different splicing variant forms of NELL2 identified so far. One is secreted NELL2 (sNELL2) which is a neuron-specific variant and the other is cytosolic NELL2 (cNELL2) which is non-secreted splicing variant of NELL2. Although cNELL2 structure was well characterized, the expression pattern or the cellular function of cNELL2 is not fully determined. In this study, we found that cNELL2 specifically interacts with PKCβ isotypes and inhibits PKCβ1 through direct binding to the N-terminal pseudosubstrate domain of PKCβ1. Here, we also demonstrate that cNELL2 is predominantly expressed and has inhibitory effects on the PKC downstream signaling pathways in astrocytes thereby establishing cNELL2 as an endogenous inhibitor of PKCβ1 in glia.     

The molecular mechanism of NELL2 movement and secretion in hippocampal progenitor HiB5 cells

Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.     

BMP-2 promotes osteogenic differentiation of mesenchymal stem cells by enhancing mitochondrial activity

Objectives:Mesenchymal stem cells (MSCs) have become seed cells and basic elements for bone regeneration and bone tissue engineering. The aim of the present study was to investigate the roles and mechanisms of bone morphogenetic protein 2 (BMP-2) on osteogenic differentiation of MSCs.Methods:Primary MSCs were isolated from the femur and tibia bone of rats and then transfected with BMP-2 and PGC-1α adenovirus vectors. Alkaline phosphatase (ALP) activity and alizarin red staining were used to measure osteogenic differentiation of MSCs. Real-time PCR and western blot assays were performed to assess osteogenic differentiation-related proteins levels. The activities of mitochondrial respiratory chain complexes I and II and mitochondrial fluorescence intensity were used to explore mitochondria status during osteogenic differentiation of MSCs.Results:We found that the ability of BMP-2 overexpressed (OE) group osteogenic differentiation was significantly improved, compared with the negative control (NC) group. The results also indicated that BMP-2 can promote the activity of mitochondria. We further used the gain- and loss-of-function approaches to demonstrate that BMP-2 promotes mitochondrial activity by up-regulating PGC-1α to promote osteogenic differentiation of MSCs.Conclusions:These results explored the important role of BMP-2 in the osteoblast differentiation of MSCs from a new perspective, providing a theoretical and experimental basis for bone defect and repair.     

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.     

Identification and characterization of a truncated isoform of NELL2

NELL2 is a neuron-specific secreted glycoprotein containing an N-terminal thrombospondin I-like domain (TSP-N). In this study, we describe NELL2-Tsp, a novel alternative splice variant of rat NELL2. NELL2-Tsp uses an alternate stop codon resulting in a C-terminal truncated form of NELL2, containing a signal peptide and a TSP-N domain. NELL2-Tsp is a glycosylated protein specifically expressed in brain tissue. NELL2-Tsp and NELL2 are secreted, likely due to the putative signal peptide. However, due to the truncation, the secreted portion of NELL2-Tsp is smaller than that of NELL2. Immunoprecipitation analysis confirmed that NELL2-Tsp was able to associate with NELL2 and with itself. In addition, expression of NELL2-Tsp notably reduced secretion of NELL2 and inhibited NELL2-mediated neurite outgrowth. These results suggest that NELL2-Tsp may act as a negative regulator of wild-type NELL2.     

microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation

Studies have indicated that Nel-like molecule-1 (NELL-1) was an osteoblast-specific cytokine and some specific microRNAs (miRNAs) could serve as competing endogenous RNA (ceRNA) to partake in osteogenic differentiation of human adipose-derived stem cells (hASCs). The aim of this study was to explore the potential functional mechanisms of recombinant human NELL-1 protein (rhNELL-1) during hASCs osteogenic differentiation. rhNELL-1 was added to osteogenic medium to activate osteogenic differentiation of hASCs. High-throughput RNA sequencing (RNA-Seq) was performed and validated by real-time quantitative polymerase chain reaction. Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed to detect the functions of differentially expressed miRNAs and genes. Coding-noncoding gene co-expression network and ceRNA networks were constructed to predict the potential regulatory role of miRNAs. A total of 1010 differentially expressed miRNAs and 1762 differentially expressed messenger RNAs (mRNAs) were detected. miRNA-370-3p, bone morphogenetic protein 2 (BMP2), and parathyroid hormone like hormone (PTHLH) were differentially expressed during NELL-1-induced osteogenesis. Bioinformatic analyses demonstrated that these differentially expressed miRNAs and mRNAs enriched in Rap1 signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, Glucagon signaling pathway, and hypoxia-inducible factor-1 signaling pathway, which were important pathways related to osteogenic differentiation. In addition, miRNA-370-3p and has-miR-485-5p were predicted to interact with circ0001543, circ0002405, and ENST00000570267 in ceRNA networks. Based on the gain or loss of functional experiments by transfection, the results showed that miR-370-3p was a key regulator in osteogenic differentiation by targeting BMP2 and disturbing the expression of PTHLH, and participated in NELL-1-stimulated osteogenesis. The present study provided the primary data and evidence for further exploration on the roles of miRNAs and ceRNAs during NELL-1-induced ossification of hASCs.     

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.     

An Akt-dependent increase in canonical Wnt signaling and a decrease in sclerostin protein levels are involved in strontium ranelate-induced osteogenic effects in human osteoblasts

Sclerostin is an important regulator of bone homeostasis and canonical Wnt signaling is a key regulator of osteogenesis. Strontium ranelate is a treatment for osteoporosis that has been shown to reduce fracture risk, in part, by increasing bone formation. Here we show that exposure of human osteoblasts in primary culture to strontium increased mineralization and decreased the expression of sclerostin, an osteocyte-specific secreted protein that acts as a negative regulator of bone formation by inhibiting canonical Wnt signaling. Strontium also activated, in an apparently separate process, an Akt-dependent signaling cascade via the calcium-sensing receptor that promoted the nuclear translocation of β-catenin. We propose that two discrete pathways linked to canonical Wnt signaling contribute to strontium-induced osteogenic effects in osteoblasts.     

Cellular retinoic acid binding protein 2 inhibits osteogenic differentiation by modulating LIMK1 in C2C12 cells

Cellular retinoic acid binding protein 2 (CRABP2) is essential for myoblast differentiation, however, little is known about its role in osteogenic differentiation. This study mainly aims to explore the biological functions and the underlying molecular mechanisms of CRABP2 in osteogenesis. Using quantitative polymerase chain reaction and western blot assays, we found that the expression of CRABP2 at both mRNA and protein levels were downregulated during osteogenesis. Furthermore, CRABP2 knockdown displayed significant changes in the cell phenotype and the actin filaments (F‐actin) polymerization in C2C12 cells treated with BMP2. Moreover, the western blotting of osteogenic differentiation biomarkers, alkaline phosphatase (ALP) staining and Alizarin red staining showed that CRABP2 dramatically inhibited osteogenic differentiation. The following investigation of molecular mechanisms implicated that CARBP2 specifically interacted with LIMK1, a key factor in acin cytoskeletal rearrangements in osteogenesis, to interrupt its activity and stability in an ubiquitin‐proteasome pathway to prevent C2C12 cells from osteogenic differentiation in response to BMP2. Above all, our data suggest a novel function of CRABP2 in regulating actin remodeling and osteogenic differentiation via LIMK1, thus presenting a possible molecular target for promoting the osteogenic differentiation in bone degenerative diseases.