Osteoblasts are derived from multipotential progenitor cells within the bone marrow stroma that can also differentiate into cells of other mesenchymal lineages. 1,25-(OH)2D3 plays a role in the regulation of these early stages of human osteoblast differentiation by promoting an osteogenic differentiation (in human bone marrow stromal cell cultures;  and in clonal cell lines derived from human trabecular bone) although the induction of both osteogenic and adipocytic pathways have been described as well (29,30). In bone marrow stromal cultures from species other than human (rat and mouse), 1,25-(OH)2D3 was clearly found to favor the differentiation into the osteoblastic instead of the adipocytic lineage (31-35). However, it has been reported that in primary rat calvaria cells, 1,25-(OH)2D3 stimulated differentiation into adipocytes (36). 1,25-(OH)2D3 has been shown to regulate the expression of genes and proteins involved in the developmental control and regulation of differentiation, cbfa1 (37,38), I-mfa (inhibitor of the MyoD family; ref. 39), and Notch (40).
In vitro studies using osteoblast-like cells of various origin demonstrated clear effects of 1,25-(OH)2D3 on mRNA and protein expression and on enzyme activities. However, some contradictory effects have been described, for example, stimulation as well as inhibition of osteocalcin (41-43) and cbfa1 expression (37,38). This can be attributed to various causes among other species differences, which will be discussed here with primary focus on data obtained with human osteoblasts. Initially, the effects on alkaline phosphatase, mineralization, apoptosis, and proliferation and next the effects of 1,25-(OH)2D3 on the regulation of collagen type I and various noncollagenous proteins, plasmino-gen, prostaglandins, and growth factors will be discussed.
Alkaline phosphatase activity, an important biochemical marker of bone formation (44) with a possible role in mineralization because of the hypophosphatasia observed in alkaline phosphate knockout mice, is shown in many in vitro osteoblast studies to be regulated by 1,25-(OH)2D3.
In human osteoblasts, 1,25-(OH)2D3 has been reported to stimulate the alkaline phosphatase activity (45), alkaline phosphatase mRNA, and protein expression (30) during both proliferation and differentiation stages. But the stimulatory effect of 1,25-(OH)2D3 on alkaline phosphatase protein expression was not always found in late-stage postconfluental cultures (46). Other studies, particularly on mature human osteoblasts, described synergistic effects of 1,25-(OH)2D3 and transforming growth factor (TGF)-p on alkaline phosphatase activity (47-50).
1,25-(OH)2D3 treatment also was found to stimulate alkaline phosphatase activity in rat osteoblasts (51,52). When different differentiation stages of rat osteoblasts were compared, acute 1,25-(OH)2D3 treatment inhibited alkaline phosphatase mRNA expression during the highest basal expression levels (early phase) but stimulated expression at the lowest basal expression levels (during the mineralization period of the cells; ref. 53).
In contrast, in mouse osteoblastic cells, alkaline phosphatase activity was stimulated by 1,25-(OH)2D3 in the early phase of differentiation, and no effect was found during the late phase when mineralization occurs (54) or when alkaline phosphatase activity was reduced by 1,25-(OH)2D3 during confluency (55).
Alkaline phosphatase activity is important for mineralization of bone and even of other tissues (56,57). Speculations have been made that a stimulatory effect of 1,25-(OH)2D3 on the alkaline phosphatase activity of osteoblasts indicates a direct involvement of 1,25-(OH)2D3 in bone mineralization (51). In rat bone, the cells with the highest alkaline phosphatase activity and the highest intracellular calcium content (regulated by 1,25-(OH)2D3) were the cells closest to the forming bone (58). Electron microscopy showed that the origin of alkaline phosphatase-positive bone matrix vesicles was polarized to the mineral-facing side of osteoblasts (59). In contrast, it has also been reported that when alkaline phosphatase activity is repressed, mouse osteoblasts still differentiate to a mineral-secreting phenotype (60).
In human cultured osteoblasts, 1,25-(OH)2D3 has been reported to stimulate mineralization of the extracellular matrix, which was promoted by vitamin K (61), and increased with advancing donor age (62).The relation with multipotent stem cell differentiation is demonstrated by the vitamin D enhancement of extracellular mineralization by cells derived from adipose tissue (63). Low doses of 1,25-(OH)2D3
stimulated the activity of ecto-NTP pyrophosphatase, that is involved in the regulation of mineralization in bone, whereas higher doses had no effect (64) or have even been reported to inhibit the mineralization of human osteoblasts (65).
In rats, it has been found that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating phosphatidylserine synthesis, which is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-phosphatidyl-serin-phosphate complexes (66). Furthermore 1,25-(OH)2D3 is proposed to have a role in calcium transport from osteoblasts towards sites of active bone mineralization via the stimulation of calbindin-D9K synthesis (67). However, it has been found that a 1,25-(OH)2D3-induced upregulation of alkaline phosphatase and osteocalcin genes in rat osteoblasts cultured on a collagen matrix was accompanied by an inhibited mineralization (68).
In mouse osteoblast-like MC3T3 cells, treatment with 1,25-(OH)2D3 stimulated calcium accumulation during the mineralization stage of the cell culture (54), and this could be blocked by constitutive calreticulin expression, which negatively interacts with the VDR (69). However, it also has been shown that 1,25-(OH)2D3 downregulates mineralization in primary mouse osteoblast cultures, together with downregulation of Phex mRNA and protein, which are shown to be involved in cell differentiation and p-glycerophosphate-induced mineralization (70). Also in mice with impaired function of the 25-hydroxyvitamin D-24-hydroxylase enzyme and therefore elevated levels of 1,25-(OH)2D3, deficient mineralization of intramembranous bone was detected (71). These differences could possibly be explained by the necessity of an interplay with 24,25-(OH)2D3 or 1,24,25-(OH)2D3 for optimal mineralization of bone (72).
In addition to bone, vitamin D may also be involved in the calcification of other tissues as has been recently shown for the aorta and the femoral, mesenteric, hepatic, renal, and carotid arteries (73). Interestingly, these authors demonstrated that the osteoclast inhibitor osteoprotegerin completely blocked calcification in each of these arteries and reduced the levels of calcium and phosphate in the abdominal aorta to control levels.
The process of mineralization has been shown to be associated with apoptosis of osteoblast/osteo-cyte-like cells from fetal rat calvaria (74). Apoptosis was found to be related to the differentiative response of rat osteoblasts in fracture healing (75) and has been associated with differentiation (collagen I expression and mineralization) in mouse craniosynostosis (76), although others have reported differently (77).
In human osteoblastic cells 1,25-(OH)2D3 and several of its analogs has no clear effect on apoptosis; however, induction of apoptosis by camptothecin and staurosporin is strongly reduced by 1,25-(OH)2D3 (78). In contrast, 1,25-(OH)2D3 has been reported to induce tumor necrosis factor-a-mediated apoptosis in parallel to an increased cell differentiation, shown by a stimulation of osteocalcin and alkaline phosphatase (79). Also in canine osteosarcoma cells, 1,25-(OH)2D3, was involved in the induction of apoptosis and cell differentiation (80,81).
In relation to the effects of 1,25-(OH)2D3 on the induction of apoptosis are the studies that show an inhibition of osteoblast proliferation after treatment with 1,25-(OH)2D3. Proliferation of human osteoblast-like cells (MG63) was found to be inhibited by 1,25-(OH)2D3 (41). Effects on human osteoblast proliferation, however, can be dependent on the concentration of 1,25-(OH)2D3: high doses (5 x 10"9 to 5 x 10_6 M) showed a decreased proliferation, and low doses (5 x 10"12 M) showed an increased proliferation (82).
In rat osteoblasts, differences have been reported in the regulation of proliferation either inhibition (83,84) or stimulation (41) by 1,25-(OH)2D3. The stage of differentiation of the osteoblasts might be important because it has been reported that 1,25-(OH)2D3 treatment increased the amount of p57(Kip2), a member of the cyclin-dependent kinase inhibitors in rat calvarial primary osteoblasts in the transit from proliferation toward differentiation (85).
In mouse osteoblasts, inhibitory actions of 1,25-(OH)2D3 on the proliferation of osteoblasts have been described (83,86,87). However, studies with mouse osteoblasts have shown that cell proliferation rate can determine the cellular responses to 1,25-(OH)2D3 via a change in receptor levels (88).
The effect of 1,25-(OH)2D3 on collagen I, the major component of the extracellular matrix formed by osteoblasts, is investigated by numerous studies. Collagen I consists of a triple-helix formation, containing two identical Ia1 chains and a structurally similar, but genetically different, Ia2 chain. Collagens are synthesized as procollagen molecules with an extracellular removal of their N- and C-propeptides. These propeptides can be detected as byproducts of collagen synthesis and are clinically used as markers of bone formation.
1,25-(OH)2D3 has been described to stimulate the synthesis of type I collagen in human osteoblasts (MG-63 osteosarcoma cells), both the Ia1 and Ia2 components (89). This positive effect of 1,25-(OH)2D3 on type I collagen has also been shown in other human osteoblast culture systems, both at the mRNA (90) and protein level (30) after long-term 1,25-(OH)2D3 treatment. The effect of 1,25-(OH)2D33 could be enhanced by coincubation with TGF-p (48) or sodium fluoride (91). However, some studies did not find any effect of 1,25-(OH)2D3 on collagen synthesis in human osteoblasts (30, 46,49) mainly after short-term treatment. Osteoblasts do not only synthesize collagen type I but also collagenases that allow the initiation of bone resorption by generating collagen fragments that activate osteoclasts (92). With 1,25-(OH)2D3, an upregulation of collagenase was found in human osteo-blasts (93). This may reflect the dual role of osteoblasts in bone metabolism, on the one hand bone formation, and on the other hand bone resorption via control of osteoclast formation and activity.
In rat osteoblastic cells, 1,25-(OH)2D3 has been shown to reduce type I collagen synthesis and procollagen mRNA, and this reduction is even stronger in the presence of dexamethasone (94,95). These 1,25-(OH)2D3 effects in rat osteoblasts on collagen I synthesis have been shown to be dependent on differentiation and duration of 1,25-(OH)2D3 treatment: an inhibitory effect during proliferation (high basal levels), and stimulatory effect during the mineralization period (low basal levels) after acute hormone treatment (53).
In mouse osteoblasts, 1,25-(OH)2D3 inhibited the collagen Ia1 promoter activity (96). 1,25-(OH)2D3 also inhibited the collagen I synthesis in mouse calvarial osteoblasts grown on collagen I coatings; this was accompanied by an increased collagenase and gelatinase secretion and a reduction in free tissue inhibitor of metalloproteinases (ref. 97; although this effect was not observed when serum-free medium was used without plasminogen; ref. 98). In contrast, it has also been reported that 1,25-(OH)2D3 could inhibit collagenolysis in mouse osteoblasts, with a reduction in collagenase activity and increase in free tissue Inhibitor of metalloproteinases (99). These different effects of 1,25-(OH)2D3 might again be to the result of different differentiation stages of the mouse osteoblasts, because in early phase MC3T3-E1 cells, 1,25-(OH)2D3 has been found to stimulate collagen synthesis, whereas in late phase osteoblasts, no effects were found (54).
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