To examine the functional role

To examine the functional role of IGFBP-2 and -3 we conducted the experiments described in Fig. 4 where the effects of IGFBP-2 and -3 were examined either uncomplexed or complexed with IGF1. The data indicated that IGF1 stimulated ALP activity in DPCs in a dose dependant manner and although there were no statistically significant effects of IGFBPs on their own, at equimolar concentrations of IGF: IGFBP, IGF1 activity is enhanced by IGFBP-2 and inhibited by IGFBP-3. Such effects are consistent with a functional role for both IGFBPs and are consistent with the changes in IGFBP mRNA and protein concentrations seen during the differentiation of DPCs. At higher concentrations of IGF1 maximum stimulation of ALP occurs and at this higher IGF: IGFBP molar ratio (10) the inhibitory effect of IGFBP-3 is overcome. Interestingly DPCs appeared less sensitive to stimulation by IGF2 (Supplementary Fig. 4).
Discussion We have demonstrated reciprocal changes in IGFBP-2 and IGFBP-3 mRNA and protein expression in dental pulp stromal aniracetam during differentiation to a mineralising phenotype and show that these changes are co-ordinated with the functions of both IGFBPs to enhance (IGFBP-2) or inhibit (IGFBP-3) the mineralising activity of IGF1 in these cells. Although IGF1 has been reported previously to promote the differentiation of human dental pulp stem cells via mTor (Feng et al., 2014) and MAPK/Stat-3 (Vandomme et al., 2014) signalling pathways there is only a limited literature describing IGFBP-2 and/or IGFBP-3 expression and activity during osteogenic differentiation. A direct effect of IGFBP-2 on the osteoblastic differentiation of rat calvarial cells via a receptor tyrosine phosphatase β (RPTPβ) based mechanism was recently reported (Xi et al., 2014). Whether such a mechanism is associated with the potentiation of IGF1 activity by IGBP-2 reported in the current study is unknown. A potentiating effect of IGFBP-2 on IGF-2 stimulation of alkaline phophatase activity in differentiating rat tibial osteoblast cultures (Palermo et al., 2004) was reported although we found that DPCs were less sensitive to IGF2 stimulation compared to IGF1 in terms of stimulation of ALP activity (Supplementary Fig. 4). This suggests that the pro-mineralising action of IGFs is principally signalled through the IGF1R although confirmation of this should be sought through the use of specific IGF1R inhibitors. There are two reports which demonstrate upregulation of IGFBP-5 expression during mineralisation of dental pulp cells. Microarray analysis indicated an 8-fold increase in IGFBP-5 expression in DPCs derived from non-carious wisdom teeth following 10days treatment with mineralisation medium although this group did not confirm IGFBP-5 protein expression (Mori et al., 2011; Mori et al., 2010). Although we found that IGFBP-5 was expressed under both basal and mineralising conditions in DPCs we did not find any changes in gene expression under our experimental conditions in any of our donors. Although it is difficult to reconcile these results factors such as differences in precise tissue culture conditions or donor profiles may be involved. The IGF axis may also play an indirect role(s) in the differentiation of dental tissues. For example IGF-1 increased extra-cellular matrix secretion by dental-pulp derived fibroblasts (Nakashima, 1992) via the induction of bone morphogenetic protein (BMP)-2 expression (Li et al., 1998). Although our qRT-PCR data indicated a low abundance of IGF-1 mRNA expression in DPCs, in vivo dental pulp tissue is well vascularised and would therefore have access to systemic IGF1 and our data would suggest that growth factor activity could be modulated by locally expressed IGFBP-2 and -3. Conversley IGF-2 and IGF-1R were expressed at moderate to high levels in our experiments confirming previous reports (Caviedes-Bucheli et al., 2004; Shi et al., 2001). Both IGF-1 and IGF-2 (acting via the IGF-1R) can induce ALP activity in canine dental pulp cells (Onishi et al., 1999) and IGF-2 secretion was reported during matrix mineralisation of human dental pulp derived fibroblasts (Reichenmiller et al., 2004). IGF axis components are present in other dental structures with IGF-1 and -2 along with all six IGFBPs present in the ECM of the periodontal ligament and IGF-1R present on the surface of periodontal ligament derived fibroblasts (Gotz et al., 2006). A very recent study using stem cell populations isolated from apical papillae (SCAP) reported the stimulation of cell proliferation, ALP expression and mineralisation activity by IGF-1. Simultaneously, expression of odontogenic markers (dentin sialoprotein and dentin sialophosphoprotein) was downregulated arguing for a bias in IGF-1 action toward bone formation and away from odontogenic differentiation in this tissue niche (Wang et al., n.d.). However it should be noted that microscopic confirmation of appropriate bone tissue structure was not rigorously established in these studies and this area requires further investigation. In a detailed study of IGF axis expression in differentiating ameloblasts, position specific expression of IGF-1, IGF-2, IGF-1R and IGF-2R toward the outer enamel layer and away from pulp facing ameloblasts was reported arguing for the importance of the IGF axis in development of this tissue (Caviedes-Bucheli et al., 2009; Yamamoto et al., 2006) and an elegant study demonstrating the developmental stage-dependent expression of IGF-1 in the continually erupting rat incisor model (Joseph et al., 1996) also argues strongly for a role of the IGF axis in the development of dental tissues.