Vanadium and Bone - American Chemical Society

Chapter 15

Vanadium and Bone: Relevance of Vanadium Compounds in Bone Cells 1,2

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Downloaded by UNIV OF MINNESOTA on August 3, 2013 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch015

Susana B. Etcheverry and Daniel A, Barrio

Cátedra de Bioquimica Patológica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina Centro de Química Inorgánica (CEQUINOR), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina 1

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Bone is one of the main tissues for vanadium storage in vertebrates. Vanadium compounds exert interesting biological effects as well as potential pharmacological actions. Herein we review the bioactivity of vanadium with emphasis on results obtained in bone-related cells in culture. Vanadium compounds behaved as antitumoral agents, as growth factor mimetic compounds and as osteogenic drugs. We report for the first time, the effects of a complex of vanadyl(IV) cation with Trehalose on long-term cultures of non transformed osteoblasts. The complex induces cell proliferation, glucose consumption, osteoblast differentiation and the mineralization of the extracellular matrix.

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© 2007 American Chemical Society

In Vanadium: The Versatile Metal; Kustin, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


205 Bone is a dynamic tissue with a great capacity to remodel and repair throughout life (1). The integrity of the skeleton depends on a series of events that regulate the coordinated activity of osteoblasts (bone-forming cells) and osteoclasts (cells involved in bone resorption). Other cells that play an important role in bone tissue are macrophages (2). A l l these cells are placed in a mineralized matrix whose main moieties are the mineral phase hydroxyapatite and the organic part with collagen as one of the principal components. Numerous growth factors and cytokines regulate osteogenesis and bone repair (3). Misbalance in skeletal cell activity may cause bone diseases. Vanadium compounds are an interesting group of drugs with potential effects on bone tissue (4.5). Different studies demonstrate the accumulation of vanadium in bone (6-8). Vanadium accumulation in bone tissue may be due to its analogy with phosphate (9). Vanadium stimulates D N A and collagen synthesis in fibroblasts and promotes bone formation and repair in vivo (10 - 12). Moreover, vanadium derivatives display interesting potential pharmacological actions as insulin- and growth factor-mimetic compounds (12,13), as antitumoral agents (14 -16) and as osteogenic drugs (17,18). We have previously demonstrated that vanadium(V) and vanadium(IV) species as well as different vanadium complexes regulate the proliferation and differentiation of osteoblast-like cells in culture (17-19). Several mechanisms have been proposed to explain the bioactivity of vanadium (20,21). As a consequence, different cellular cascades can be triggered and translated into biological actions, including the regulation of osteoblast-specific gene expression, proliferation, matrix mineralization, and cell adhesion to the matrix (20-22). Herein, we present new and interesting results of the complex of vanadyl(IV) cation with the disaccharide trehalose (TreVO) on non-transformed osteoblasts that in long-term cultures develop the stages and characteristics of normal bone tissue.

Potential Pharmacological Effects of Vanadium Compounds As part of a project devoted to the synthesis, characterization and the study of the bioactivity of vanadium derivatives in bone-related cells, we have used Swiss 3T3 fibroblasts, two osteoblast-like cell lines (MC3T3-E1 that are nontransformed osteoblasts derived from mouse calvaria and UMR106 osteoblastlike cells derived from a rat osteosarcoma) (19, 23, 24) and the murine macrophage cell line R A W 264.7 (2).


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Insulin Mimetic Effects of Vanadium Compounds


Effects of Vanadium on Osteoblast Glucose Consumption

The most interesting pharmacological action of vanadium is its behavior as insulin mimics. Different models of diabetic rats as well as different vanadium compounds were used (26 - 28). On the other hand, the first studies in human beings were developed later (20-31). The activation of phosphorylation cascades has been proposed as one of the most important mechanisms involved in the insulin mimetic actions of vanadium compounds. In vivo, insulin signaling transduction pathway is mediated through a complex network of phosphorylation and dephosphorylation reactions. (32,33). In vivo and in vitro studies demonstrated that vanadium influences different parts of the insulin-signaling pathway. In vitro studies carried out in our laboratory using a set of inhibitors of different cellular pathways, have allowed us to demonstrate that insulin and a complex of vanadyl(IV) with trehalose stimulate glucose consumption in the osteoblasts in culture through a mechanism independent of PI3-K in contrast with previous results in other cell types (17). Besides, we have recently reported for the first time the effect of TreVO on the phosphorylated and nonphosphorylated forms of GSK-3 (kinase 3 of the glycogen synthase) in osteoblast-like cells. This compound stimulated glucose consumption and the phosphorylation of this protein and a cytosolic protein kinase would be involved in this effect (17,34).

Vanadium Compounds as Growth Factor Mimetic Compounds

Effects of Vanadium on Osteoblast

Proliferation

Several research groups have reported that vanadium derivatives behave as growth factor mimetic compounds, resembling EGF, F G F and insulin actions, promoting bone formation and repair (11,19). As an overview, at low concentrations most vanadium compounds behave as weak mitogens in comparison to insulin, while at high doses they exert cytotoxicity (17). Bioactivity of vanadium compounds involves a series of complex events that depends on the cellular type, the vanadium concentration, the oxidation state of vanadium and the nature of the ligands. The model of two osteoblastic lines in culture has allowed us to demonstrate that, in general, vanadium(V) compounds are more cytotoxic than vanadium(IV) derivatives and MC3T3E1 cells are more sensitive to the cytotoxicity than



207 UMR106 cells. At present, the signaling mechanisms by which vanadium compounds produced their biological effects are under exhaustive research. One of the pathways that seems to be involved in vanadium bioactivity is the regulation of phosphotyrosine protein levels (35). The insulin mimetic effects of vanadium may be attributed to its analogy with phosphate and to the stimulation of protein tyrosine phosphorylation through the inhibition of protein tyrosine phosphatases (PTPases) (20,36). The activation of the extracellular regulated kinases (ERK) by vanadyl sulphate has been shown to be dependent on PI3-K activation (21). It is assumed that the stimulation of the PI3-K/ras/ERK pathway plays a key role in mediating the insulin-mimetic and growth factor like effects of inorganic vanadium salts. Moreover, it has been shown that several vanadium salts activate ERKs and two ribosomal protein kinases, including p90 , in a maner independent of the kinase of the insulin receptor (IR) (37,38). On the other hand, different authors have pointed to the formation of reactive oxygen species (ROS) as the molecular mechanism by which vanadium exerts its biological effects (39 - 43). Among the vanadium(V) complexes synthesized and characterized in our group, the complex with trehalose (TreVO) has shown very interesting effects and it may be considered as a promising pharmacological compound. At low doses it induced cell proliferation in MC3T3E1 non-transformed osteoblasts while it caused inhibitory effects on the tumoral cell line. TreVO also stimulates E R K phosphorylation in the MC3T3E1 osteoblast cell line (17). This effect was totally blunted by an inhibitor of M E K (PD98059) and by wortmannin (an inhibitor of PI3K) but not by a mixture of vitamins E and C. Low doses of the complex, which are mitogenic for MC3T3E1 cells, could act though the PI3KM E K - E R K pathway and by a mechanism independent of free radicals. High concentrations of TreVO strongly increased E R K phosphorylation, an effect that was partially blocked by wortmannin, PD98059 or a mixture of vitamins E and C (17). These results suggest that E R K pathway may be involved in the proliferative effects of TreVO. The activation of the E R K cascade by low doses of the complex seems not to be mediated by ROS. On the contrary, the activation of this pathway by high doses of TreVO would be mediated by ROS. rsk

Effects of Vanadium on Osteoblast

Differentiation

Studies in fibroblasts have shown that vanadium stimulates D N A and collagen synthesis, suggesting that it promotes osteoblastic differentiation (22,23). On the other hand, alkaline phosphatase (ALP) is involved in the mineralization process of bone. Vanadium compounds inhibit A L P activity in varying degrees^/ 7,19,44). Mineralization takes place in the E C M where type I collagen is one of the more important components. Vanadium compounds display opposing actions on



208 these two markers of osteoblastic phenotype: they inhibit A L P activity and some of them enhance the synthesis of collagen. As a consequence between these two aspects, the probability of a vanadium compound acting as a stimulating agent for bone formation would depend on the relationship between the induction of osteoblast proliferation and collagen synthesis with a slight inhibition on A L P activity. The complex of vanadyl(IV) cation with trehalose behaves as a weak inhibitor of A L P (17) but has an stimulatory effect on collagen synthesis in UMR106 cells (45), as well as in long-term cultures of MC3T3E1 (see below). These results indicate that this compound can be considered as a good osteogenic agent. As it is well known, diabetic patients with a bad control of the hyperglycemia present important alterations in bone tissue (46). We have also demonstrated that A L P activity is decreased upon a great level of glycosylation (47). The osteogenic action of TreVO together with its effect as a very good promoter of glucose uptake by the osteoblasts, allow us to strongly suggest that this complex is a worthy candidate for clinical trials.

Long-term Studies on the Effects of Vanadium in MC3T3E1 Osteoblasts The process of maturation from preosteoblasts into osteoblasts requires the expression of several specific markers along bone development (48). This cell line is a good in vitro model of preosteoblasts that in long-term cultures differentiate into mature osteoblasts, going through developmental stages similar to the growth of bone in vivo. Ascorbic acid (AA) and P-glycerol phosphate (PGP) are two compounds required for differentiation of MC3T3R1 cell line. The differentiation begins approximately after two weeks of culture and the cells express some proteins such as A L P and collagen. From previous results obtained in short-term cultures in this cell line and in the UMR106 tumoral osteoblasts, TreVO has shown interesting potential pharmacological actions as insulin mimetics and as an osteogenic compound. It stimulated collagen production and caused a weak inhibition of A L P , a key enzyme for the mineralization process (45). TreVO stimulated MC3T3E1 cell proliferation in the range of 5 - 25 p M in 24 h culture. For the long-term studies (up to 25 days), we used the minor effective dose in the induction of cell proliferation (5 pM). We have also included a control with A A and P-GP as inducers of differentiation. The cells were cultured in 24 well/plates in D M E M medium supplemented with 10 % fetal bovine serum (FBS). When the monolayers reached the confluence, the medium was replaced every two days for a fresh one. The assay conditions were: D M E M plus 10 % FBS (Basal), D M E M plus 10 % FBS plus 5 p M of the complex (TreVO), and D M E M plus 10% F B S plus 140 p M A A and 5 m M p-GP (AA+P-GP). At different culture stages, we



209 determined the cell number, collagen and glucose consumption. In another assay we investigated the effect of 5 p M of TreVO on the process of mineralization induced by A A and P-GP. Figure 1 shows the effect of the treatment on cell proliferation (Crystal violet assay) (49). As can be seen, the cells grow and proliferate until approximately 10 days of culture. Then the cell number remains constant through 25 days. After 10 days no significant differences in the cell number were observed for each culture condition. The effects on collagen production (Sirius Red staining) (50) and on the specific A L P activity (19) are shown in Figures 2 and 3, respectively. From figure 2 it can be seen that the mixture of A A and PG P induced collagen production in this cell line. TreVO also produced an increase in collagen in these cells at 1 6 - 2 5 days of culture. Besides, it can be observed that the basal collagen production significantly increased during 25 days. When the results on the A L P activity were analyzed, we can see that the mixture of AA+P-GP stimulated cell differentiation of MC3T3E1 since it induced the collagen production and the activity of A L P . TreVO enhanced collagen synthesis but it partially inhibited the A L P activity. Figure 4 shows the effects on the glucose consumption from MC3T3E1 osteoblasts at different culture times (Glucose oxidase assay) (17). The basal value was very low after 10 days of culture (2 pg/8h/well), but it increased markedly at 16 days (32 pg/8h/well) and then remained constant. The addition of AA+p-GP caused a high increment in glucose consumption (48 pg/8h/well) after 16 days. This effect is maintained during the mineralization stage at 25 days. On the other hand, TreVO also significantly increased the glucose consumption by the osteoblasts at 16 day culture (42 pg/8h/well) and a great increase was detected after 25 days of culture (45 pg/8h/well). As we can mention above, A L P specific activity and collagen production are markers for the differentiation of the preosteoblastic stage into the mature osteoblast. During the first days of culture, F A L activity was undetectable in this cell line. Nevertheless, after 10 days a little A L P activity could be observed. Between 10 and 16 days, the activity of F A L significantly increased for the three experimental conditions mentioned above. The mixture of AA+ P-GP induced a 400% of increment over basal activity. TreVO (5pM) caused a 50 % of inhibition of F A L activity. Nevertheless, when the cells were incubated with the mixture of A A and p-GP plus TreVO (5pM), the inhibition of A L P was only 30 %, remaining at 70 % of the enzyme activity under this condition. Between 16 25 days (mineralization step), F A L activity increased only 20 % for AA+ p-GP mixture while the treatment with TreVO did not produce any change in this period. These results suggest that TreVO could affect the mineralization of MC3T3E1 cells. To evaluate this hypothesis, we incubated the cells for 30 days with A A + p-GP (control) and with A A + p-GP + 5 p M TreVO and analized the mineralization nodule formation by an hystochemical method. The effect of 5



Figure 1. Cell proliferation assay.

Figure 2. Collagen content. *p

Vanadium and Bone - American Chemical Society

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