PTH effects on osteoblasts in vitro

2.2.1 On osteoblast proliferation

In vitro, PTH induces various contradicting responses. In osteoblast-like cells such as UMR 106 and ROS 17/2.8 osteosarcoma cells, incubated with PTH at 10^-8 M for 24 hours, DNA synthesis was decreased (126, 127). On the contrary, at a dose of 10^-11 M, PTH was able to induce cell proliferation of the same cell line, i.e. UMR 106 cells (128). Moreover, in mouse non cancerous osteoblasts such as MC3T3 and in primary osteoblasts, PTH induced cell proliferation (129). Thus, PTH-induced cell proliferation in vitro depends on the dose and the cell type.

In vivo, intermittent injection of PTH in rats increase the number of osteoprogenitor cells from bone marrow (130, 131). However, the in vivo assessment of [³ H]-thymidine incorporation into osteoblastic cells in rats showed no difference in the percentage of labeled osteoblasts between vehicle- and PTH-treated animals, whereas incorporation of [³H]-proline in the distal epiphysis was increased (120).

These observations suggest that PTH does not stimulate osteoblastic proliferation but rather stimulates differentiation of osteoprogenitor cells in vivo. It is commonly accepted that differentiation requires exit from the cell cycle, and numerous in vitro studies have shown that proliferation decreases as osteoblast differentiation proceeds. Consistent with the possibility that PTH increases osteoblast differentiation by suppressing proliferation, in vivo and in vitro studies have shown that short-term exposure to PTH causes exit of osteoblast progenitors from the cell cycle. Thus, PTH reduced the expression of histone H4, a marker of the cell cycle (132), as well as expression of the cell cycle inhibitors p27KIP1 and p21Cip1 (133), in metaphyseal bone of young rats, a site rich in replicating osteoblast progenitors (134).

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2.2.2 On osteoblast differentiation

In vitro, expression levels of specific genes associated with the various stages of osteoblastic differentiation, such as alkaline phosphatase (ALP), type I collagen (COLL I) and osteocalcin (BGP), can also be either stimulated or inhibited by PTH depending on the maturation stage of cell culture and on the duration of treatment (135-137). Indeed, PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts and rather inhibit it in mature osteoblasts. Moreover, the longer osteoblasts are exposed to PTH, the less they may differentiate. However, under well-defined experimental conditions, intermittent PTH treatment increases mineralized nodule formation in cultures of mouse (MC3T3) osteoblastic cells as well as in primary osteoblastic cell cultures from mice, rats and humans (135, 137). This mineralized nodule formation is associated with high levels of expression of ALP, COLL I and BGP.

It has been shown in vitro, that some of the anabolic effects of PTH may also be mediated by an autocrine production of Insulin-like growth factor 1 (IGF-1) (138).

Thus in osteoblastic cells from newborn rat calvariae and exposed to PTH, osteoblast differentiation was inhibited by anti-IGF-1 neutralizing antibodies (137). In another model, using cultured marrow cells treated with PTH, osteoblast differentiation was also inhibited by anti-IGF-1 antibodies (139). In vivo, IGF-1 knocked mice showed an impaired bone formation induced by PTH (140, 141).

Among the genes implicated in osteoblast differentiation, the transcription factor Cbfa1 (core-binding protein a-1, also known as Runx2) is essential (9-11). Cbfa1-deficient mice die at birth and exhibit a complete lack of bone formation (10), and intermittent PTH administration in vivo stimulates Cbfa1 protein in the rat proximal tibiae metaphysis (142). However, in vitro, PTH regulates expression of Cbfa1 in a time- and dose-dependent manner in UMR-106. Regulation occurred both at the Cbfa1 mRNA level, and at the posttranslational level to increase the activity of Cbfa1 protein. The dose-dependent activity on mRNA and protein expression suggests an anabolic function for PTH at lower doses. However, higher doses of PTH decreased Cbfa1 mRNA and protein level. Moreover, it was found that PTH also activates a 38

PKA-dependent increase in Runx2 activity (142). Increased Runx2 levels may be secondary to cAMP-dependent down-regulation of cyclin D1, which besides controlling the cell cycle, also targets Runx2 for proteasomal degradation (143).

2.2.3 On osteoblast apoptosis

In vitro and in vivo, PTH is anti-apoptotic and increases life-span of mature osteoblasts, without affecting the generation of new osteoblasts. This antiapoptotic effect was sufficient to account for the increase in bone mass measured by BMD on distals femurs of mice. The anti-apoptotic effect of PTH was confirmed (in the same study) in vitro in cultures of calvarial osteoblasts, MC3T3 murine osteoblastic cells and MG-63 human osteoblastic cells. In fact, osteoblasts incubated with dexamethasone which induces a pro-apoptotic effect, was inhibited with the addition of PTH (122). Another in vitro study showed that PTH effects on apoptosis were dramatically dependent on cell status. In preconfluent MC3T3 murine osteoblasts and multipotential mesenchymal cells (C3H10), PTH protected against dexamethasone-induced reduction cell viability, whereas in postconfluent cells PTH resulted in reduced cell viability (144).

2.2.4 On osteoblast-mediated activation of osteoclasts

The discovery of the osteoprotegerin (OPG)/RANK ligand (RANKL)/RANK system and of its actions on osteoclastogenesis represents an important step in the understanding of bone physiology. Whereas RANKL promotes osteoclast formation by binding to RANK on the osteoclast surface, OPG inhibits RANKL activity by binding it, thereby inhibiting osteoclat differentiation and activation (33, 34, 37).

In vitro and in vivo studies have clearly demonstrated that PTH increases the number and activity of osteoclasts by upregulation of RANK/RANKL system (145, 146). PTH has been found to stimulate RANKL mRNA levels, to decrease OPG production, and 39

to increase the RANKL/OPG ratio in osteoblasts/stromal cells (147-149). These findings have been confirmed also by in vivo demonstration that PTH inhibits the expression of OPG in bone with the kinetics of an immediate early gene (150).

Furthermore, OPG has been shown to inhibit bone resorption induced by administration of PTH (151). In summary, PTH dose- and time-dependently increases RANKL and decreases OPG expression in vivo and in vitro by osteoblastic cells, while low-dose intermittent PTH increases OPG and reduces RANKL/OPG ratio (139, 147).

In summary, all these findings clearly show that the actions of PTH depend on multiple mechanisms and various factors such as the dose, method of administration, and the osteoblastic cell model used. Such conflicting results have plagued the scientific community and hinder our understanding of PTH action in bone.

Nevertheless all these studies tend to indicate that transient activation of PTH1R activates multiple interconnected pathways leading to decreased proliferation of osteoblastic progenitors, increased osteoblastic survival, increased osteoblastic differentiation and the production and/or activation of osteogenic growth factors.

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