Osteocalcin in Cell Proliferation and Differentiation

The osteocalcin gene is expressed in consonance with the inhibition of cell proliferation and the onset of cell differentiation and ECM mineralisation (Y.P. Li et al. 1995). VD3, as discussed above, induces osteocalcin expression, but on the other hand, it inhibits cell proliferation, apparently with the mediation of cdk inhibitors. M.J. Campbell et al. (1997) synthesised a number of VD3 analogues and demonstrated that these were capable of inhibiting proliferation of the prostate cancer cell lines LNCaP, PC-3, and DU145. The inhibition of cell proliferation was accompanied by an up-regulation of the expression of the cdk inhibitors p21waf1 and p27kip1. An up-regulation of p21waf1 expression has also been encountered in VD3-induced differentiation of human MG-63 osteosarcoma cells, and this has been shown to be independent of p53 function (Matsumoto et al. 1998). Therefore, these observations seem to define a direct and novel pathway of inhibition of cell cycle progression by VD3.

Other hormones, such as thyroid hormones, that regulate the differentiation of osteoblasts also seem to function through the mediation of osteocalcin. Triiodothy-ronine (T3) has been found to inhibit the proliferation of the osteoblast-type MC3T3-E1 cells and, in parallel, induce the expression of osteocalcin mRNA and protein and alkaline phosphatase activity (Varga et al. 1997; Luegmayr et al. 1998). Oestrogen has been reported to increase cell proliferation in the early stages of in vitro culture of osteoblasts derived from mouse bone marrow. The effects of oestrogen seem to involve the osteoblast-specific transcription factor osf2 (cbfa1) (Sasaki-Iwaoka et al. 1999). Oestrogen increases the expression of osteocalcin, alkaline phosphatase, osteopontin, and transforming growth factor (TGF)p-1 as well as collagen type 1. Furthermore, exposure to oestrogen also increased the formation of bone nodules. Anti-oestrogens (Qu et al. 1998) blocked all these effects. Post-menopausal breast cancer patients treated with the anti-oestrogen tamoxifen have reduced (22%) levels of osteocalcin in serum (Marttunen et al. 1998).

Fibronectin (FN) is a component of the ECM that has been implicated in several biological activities. Thus FN influences cell adhesion to substratum, spreading, and modulation of cell shape. It influences membrane ruffling and cell motility and also is associated with cell differentiation (Sherbet, 1987). The pattern of FN expression in osteoblast differentiation has attracted some attention. Although there is very little information about its role in vivo, there seems to be some correlation between the expression of FN and osteocalcin, in relation to the state of proliferation and differentiation in vitro. In the initial period of growth of osteoblasts, derived from foetal rat calvaria, on a collagen-coated substratum, a 50 to 70% reduction of FN occurs, but the expression of osteocalcin, osteonectin, and osteopontin is up-regulated sev-eralfold (Lynch et al. 1995). Similarly, in chicken osteoblast cell cultures at 6 to 18 days, FN is associated with the cell membrane, but subsequently it remains associated with the ECM in a fibrillary form. Overall, FN increases in the early periods of cell culture and its levels are then maintained. In contrast, the major bone matrix ECM markers show increased expression with the onset of differentiation (Winnard et al. 1995). There is no suggestion, however, that these events are necessarily related, beyond the recognition that collagen type I could be involved in the signal transduction pathway. Possibly, in the initial stages where it is bound to the membrane, FN could be mediating extracellular signals through the occupancy of its specific integrin receptors. The subsequent association with ECM in a fibrillary form is clearly a postsignalling event. This suggestion seems to be upheld by the experiments described by Moursi et al. (1996). These authors found that anti-FN antibodies inhibited the formation of bone-like nodules and the expression of osteocalcin and alkaline phosphatase genes. Generally compatible with this is the ability of VD3 to regulate FN expression at the transcriptional level. A VDRE has been identified in the FN gene (Polly et al. 1996). Interestingly, osteoblast differentiation requires more than the RGD domain of FN that is functional in cell adhesion. Certainly, the experiments of Moursi et al. (1996) indicate that FN regulates the differentiation of osteoblasts.

There is now general acceptance that integrin receptors form an important link in the transduction of signals generated by ECM components (Sherbet and Lakshmi, 1997a). Integrin receptors are actively involved in the recognition of and interaction with ECM ligands occurring in the process of osteoblast differentiation (Uemura et al. 1997). The integrin asp1 has been identified as the critical component in FN interaction with osteoblasts (Moursi et al. 1997). The murine MC 3T3 cell line responds to ascorbic acid treatment by synthesising a collagen matrix, and collagen type 1 ligand seems to be essential for the subsequent expression of osteoblast markers as well as the activation of the osteocalcin promoter element, OSE2. OSE2 is recognised by the osteoblast-specific transcription factor osf2 (also known as cbfa1, AML3, PEBP2, and alpha A). The latter is expressed only in osteoblastic cell lines, such as MG63, ROS 17/2.8, and MC3T3-E1, but not in cell lines of nonos-teoblastic origin (Sasaki-Iwaoka et al. 1999). When the collagen type 1 receptor a2-subunit is blocked, the binding of osf2 to OSE2 is also blocked, with consequent inhibition of transcription of the osteocalcin gene (Xiao et al. 1998). These experiments indicate the importance of the interaction between collagen type 1 and its integrin receptor for transduction of the signal that can elicit osteocalcin gene activation. Much effort needs to be directed toward a dissection of the pathway of signal transduction in order to provide the crucial evidence that can link these events in a coherent fashion.

Intracellular adhesion as well as cell-substratum adhesion is determined by the components of the ECM. Their temporal and spatial expression is invariably associated with the alterations in the adhesive interactions, as well as changes in cell motility or migration on a substratum or invasive behaviour of cancers. The faculty of migration could be an important feature in bone resorption. Osteoclast precursors, for example, need to target sites of bone resorption and they do possess the ability of diapedesis across capillary endothelia. The modulation of certain ECM components such as FN, in conjunction with osteoblast differentiation and osteocalcin expression, has inevitably led to investigations of a potential association of osteo-calcin with cell migration. Stringa et al. (1995) set up osteoblast cultures from 7-day-old rat tibia fragments. They found that when the cells were exposed to parathyroid hormone they synthesised and secreted large amounts of osteocalcin together with collagen type III. The conditioned medium from these cultures stimulated the migration of EA HY-926 endothelial cells in vitro. Osteoblast cells obtained from rat calvaria adhere and show migratory behaviour upon plating on three-dimensional matrices. These migrating cells expressed osteocalcin as indicated by immunohistochemical staining (Attawia et al. 1995). TGFp-1 mRNA expression in regenerating tissue in distraction osteogenesis is said to coincide with osteoblast migration and ECM mineralisation (Mehrara et al. 1999). However, there is also much evidence that TGFp-1 can inhibit osteonectin expression. Hence, it is conceivable that, in this experimental model, this promotion of migration by TGFp-1 could be an effect mediated by means other than involving osteocalcin. TGFp-1 is a highly versatile cytokine that can elicit a wide-ranging cellular response. Not least among these is that it can induce the expression of S100A4 protein (Okada et al. 1997), which has itself been implicated in the induction of cell motility.

The identity of the ECM component that might be instrumental in osteoblast migration is uncertain at present. The two major adhesion mediating glycoproteins FN and laminin both regulate adhesive interactions involving osteoblasts. As seen earlier, FN expression does vary with the state of cell proliferation and differentiation, but it has not been directly implicated in the motile behaviour of osteoblasts. Laminin, on the other hand, does seem to mediate the adhesion to substratum as well as migration of osteoclasts. There is also a suggestion that there might be some form of cooperative functioning of laminin and FN in osteoblast migration. It has been suggested, for instance, that FN might be secreted in response to laminin-2-mediated adhesion (Colucci et al. 1996). This postulate needs to be tested further. There are a number of possibilities that can be tested, e.g., whether there is de novo synthesis of FN, whether there is a deletion of FN into the medium, whether there is a modulation in the expression of FN receptors, etc. But Colucci et al. (1996) have shown that osteocalcin induces the migration of osteoclasts on laminin-2- but not collagen-coated substratum. One could concede easily that osteocalcin can promote migration involving ECM components and their particular integrin receptors. However, it is unclear at present how one can envisage a physical mechanism that transduces the osteocalcin signal to the cytoskeletal machinery to bring about cell locomotion. Also relevant in this context is the question of whether other biological macromolecules, such as the cadherins, might be involved too. Cadherin has been studied extensively for its ability to suppress invasion by cancer cells and, indeed, it has often been described as an invasion suppressor gene. Now there is evidence that VD3 analogues up-regulate the expression of E-cadherin in prostate cancer cell lines (H.D. Campbell et al.1997). In osteoblastic cell lines, VD3 analogues up-regulate osteocalcin expression. It would be of much interest to examine the status of cadherin expression in osteoblasts and determine what effects VD3 exerts on cadherin.

Besides its effects on cell motility, TGFp is also a powerful modulator of growth and differentiation. TGFp peptides are known to inhibit proliferation of a number of cell types. More than coincidental is that the mechanism by which TGFp brings about growth inhibition involves cdk inhibitors, e.g., p27kip1 (see Sherbet and Lak-shmi, 1997b for references). TGFp isoforms subserve many functions, including bone turnover. Banerjee et al. (1996) found that TGFp-1 down-regulated osteocalcin expression in ROS 17/2.8 osteosarcoma cells. Similar effects have also been described in foetal mouse long bone cultures (Staal et al. 1998). Thus, although it would appear that TGFP could be regulating bone metabolism by inhibiting osteocalcin expression, its effects on cell proliferation are achieved by a different route involving cyclin/cdks (Figure 9). This conclusion is supported by the suggestion made by J.H. Liu et al. (1999) that TGFP might be inhibiting G1-S arrest partly by inactivating cyclin B/cdc2 kinase. TGFP treatment results in the phosphorylation of the cdc2 component in the TGFP receptor Il-cyclin B2-cdc2 complex and down-regulates its kinase activity. Despite this, it should be recognised that there is much obvious coordination in the functioning of osteocalcin, VD3, and TGFP in the inhibition of cell proliferation and induction of cell differentiation.

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