NELL-1 is a novel osteogenic factor identified from its upregulation in cranial bones of patients with craniosynostosis [1
]. Previous studies have demonstrated that NELL-1 is a secreted protein [5
] which likely interact with certain cell surface membrane receptor(s) thereby mediating its downstream signaling pathways. The original purpose of our work was to identify the membrane receptor(s) that physically interacts with NELL-1 and mediates NELL-1’s function on human osteosarcoma cell lines Saos2 and U2OS proliferation and osteoblastic differentiation. We constructed a phage library encoding various protein fragments from the Saos2 cell line and screened binding proteins using recombinant human NELL-1 protein as bait by multiple rounds of biopanning. Among the over 100 identified candidates, APR3, previously reported as a transmembrane protein and located on the cell surface of MCF-7 cells [7
], was a promising candidate as a “receptor-like” NELL-1 binding protein. Unexpectedly, our results indicated that APR3 was located dispersedly in the cytoplasm, but predominantly concentrated around/on the nuclear envelope in Saos2 and U2OS cells as well as in other non-osteoblastic cell lines including 293T and Cos-7. To make certain that the relatively large size of fluorescent tag in our APR3 construct would not affect its subcellular localization, we also used APR3 with small FLAG tag for transfection and observed a similar localization pattern in those same cell lines (Supplemental Fig. 1A
). Interestingly, the cytoplasmic localization of APR3 was also the major distribution pattern in MFC-7 cells transfected with our APR3 constructs although the cell membrane localization was seen in some transfected cells (Supplemental Fig. 1B
). This finding suggests that APR3 cellular localization may vary in different cell types, allowing it to regulate different signaling pathways for cell-type specific functions. Nevertheless, the finding of a nuclear envelope/membrane localization of APR3 directed our current study towards exploring an intracellular protein, rather than a cell surface protein, that interacts with NELL-1 by using plasmid co-transfection strategy. The subsequent effects of this interaction on proliferation and osteoblastic differentiation of Saos2 and U2OS cells were also analyzed. It is noteworthy that human NELL-1 has been identified not only as an extracellular signaling molecule [4
], but also as an intracellular protein capable of directly interacting with a specific subtype of protein kinase C (PKC) [5
]. It would not be surprising if the existence of a cytoplasmic form of human NELL-1 protein, similar to human NELL-2 [15
], is verified experimentally since an alternative promoter utility has been identified on transcription of human NELL-1
). Therefore, the intracellular interaction of NELL-1 with APR3 is unexpected, but not entirely unlikely, and its functional significance worthy of further exploration. The physical interaction between NELL-1 and APR3 was further confirmed by co-immunoprecipitation with Cos-7 cells co-transfected with NELL-1 and APR3-FLAG expression plasmids. However, the critical experiment to demonstrate the interaction between endogenous NELL-1 and APR3 in human Saos2 or U2OS cells was severely hampered due to the lack of specificity of all three anti-APR3 antibodies currently available in the field. Furthermore, the multiple cell lines with detectable RNA levels of either NELL-1 (Raji cells [5
]) or APR3 (HL-60 and 293T [7
]) were used to maximize the chance of endogenous binding of these two proteins. Unfortunately, these experiments did not result in any definitive data either (Supplemental Fig. 2
). Using confocal microscope imaging on Saos2 cells co-transfected with red and green fluorescent-tagged APR3 and NELL-1, the distinct alteration in the subcellular distribution of NELL-1 in NELL-1/APR3 double positive cells in comparison to NELL-1 single positive cells strongly indicated the formation of an intracellular NELL-1/APR3 complex on the nuclear envelope. We speculate that this direct intracellular interaction between NELL-1 and APR3 may play a significant role in regulating human osteoblastic cell differentiation as both molecules have been identified previously as critical components in the regulation of cell proliferation and differentiation [2
]. Notably, a subset of preliminary data presented in supplemental Fig. 3
, the TSPN and EGF-like domains of NELL-1 did not appear to play crucial roles in binding of NELL-1 to APR3 as the binding capacity was not changed significantly when the fragment-deleted NELL-1 constructs were transfected. However, the binding of NELL-1 without TSPN domain showed a relative weak affinity, suggesting that TSPN domain may be more important than the other examined domains for further investigation. More defined deletion of NELL-1 domains of TSPN and EGF will be carefully constructed and investigated in future studies for identifying interactive components of NELL-1 and APR3.
In ATRA treated cells, APR3 was significantly upregulated resulting in G1/S phase cell arrest leading to cell differentiation [7
]. It also has been reported that NELL-1 can promote osteoblastic differentiation without promoting cellular proliferation in adenoviral NELL-1 transduced MC3T3 and BMSCs [3
]. However, the underlying mechanisms and signal transduction pathways remain unclear. In this study, we focused on the changes on Cyclin D1 at the mRNA and protein levels after NELL-1 and APR3 were introduced into Saos2 and U2OS human osteosarcoma cells since Cyclin D1 has been identified as a critical factor in APR3 mediated suppression of cell cycle in G1/S phase. Interestingly, the significant inhibition of cell proliferation occurred only when APR3
were co-transfected into target cells. Both APR3
single transfection could also reduce the expression of Cyclin D1 to a certain extent however, this modest reduction, in contrast to the greater reduction achieved by co-transfection with both APR3
, was incapable of affecting cell proliferation significantly although slight suppression of cell growth was observed in both single transfection at certain time points. MTS assay was done after knock down of endogenous APR3 by siRNA, which further verified APR3’s inhibitory function on osteoblastic proliferation (). These results clearly demonstrate that binding to APR3 is required for NELL-1’s inhibitory role on the proliferation of Saos2 and U2OS human osteosarcoma cells partially through further down-regulation of Cyclin D1 expression.
In addition to their critical roles on cell proliferation, the significance of APR3 binding on NELL-1’s osteogenic effects was further evaluated by in vitro
osteoblastic differentiation of Saos2 cells and confirmed with APR3 siRNA knock down strategy. NELL-1 was previously identified as one of the downstream mediators of Runt-related transcription factor 2 (Runx2), which is essential for osteogenesis [16
]. We found that the mRNA levels of important bone marker genes such as Bsp and Ocn were significantly upregulated by APR3 and NELL-1 respectively, while ALP activity was increased by single transfection of APR3 or NELL-1, and co-transfection of both. Notably, a synergistic increase in both Bsp and Ocn mRNA was observed only when NELL-1
were co-overexpressed in Saos2 cells. Moreover, the mineralization of Saos2 cells was induced by either NELL-1 or APR3 alone at moderate levels, but co-transfection induced the highest level of mineralization among all treatment groups. On the other hand, the application of APR3 siRNA significantly inhibited the upregulation of bone marker mRNA expression and mineralization induced by either APR3
alone or co-transfection of APR3
in Soas2 cells. Collectively, these data indicate for the first time that APR3, a molecule directly regulated by two critical transcriptional factors NFAT and NFκB [18
], is capable of inducing critical bone marker genes and terminal osteoblastic differentiation of Saos2 cells in osteoblastic differentiation medium. More importantly, the synergistic effects on promoting osteoblastic differentiation of Saos2 cells by co-transfection of APR3
further demonstrated the functional significance of NELL-1 binding to APR3 with respect to NELL-1’s osteogenic property.
One of the proposed mechanisms underlying NELL-1’s osteogenic effects involves increasing the phosphorylation of Runx2, which in turn actively regulates osteogenesis [4
]. It has been reported that the level of Runx2 can be downregulated by Cyclin D1 through promotion of its degradation [19
]. It remains to be seen whether the significant decrease in Cyclin D1 level caused by APR3 binding to NELL-1 results in further stabilization of Runx2 and enhancement of its bioactivity, ultimately resulting in its synergistic effects in promoting Saos2 cell differentiation.
Prospectively, it is also of interest to further investigate whether the nuclear membrane localization of the NELL-1 and APR3 complex could function as the signaling bridge to the nucleus for cytoplasmic retinoic acid (RA), which is the core molecule that acts through binding nuclear receptors, in ATRA signaling pathway [20
]. In addition to its pro-osteoblastic property, the critical roles of the direct interaction between APR3 and NELL-1 in inducing tumor cell differentiation and/or apoptosis through ATRA and/or other pathways would not be surprising as both NELL-1 and APR3 are suggested to have such capacities in previous reports [7
- We constructed a cDNA library of Saos2 cells to identify NELL-1 interacting proteins.
- APR3 was identified as a direct binding protein of NELL-1 through phage display.
- The inhibitory effect of APR3 on osteoblast proliferation is enhanced by NELL-1.
- NELL-1 and APR3 binding can promote osteoblast differentiation and mineralization.