Analysis of cell‐mediated mineralization in culture of bone‐derived embryonic cells with neurofibromatosis

Abstract

von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone-derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell-mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + beta-glycerophosphate (beta GP) (Dex medium) coordinately with a decrease in cell proliferation. In In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding beta GP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the beta GP-induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis-hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone-derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex-treated cells, at days 0-11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11-specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone-derived cells from 12 weeks gestation, cultured in the absence of beta GP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require beta GP for mineralization and positively respond to dexamethasone. Therefore, bone-derived NF1 cells may be useful for studying the regulation of the mineralization process.

Ras-MAPK Signaling in Osteogenic Differentiation: Friend or Foe?

“…Cultured bone marrow osteoprogenitors (30) and calvarial/long bone osteoblasts (31) from NF1‐deficient mice possess reduced os teogenic potential compared with wildtype mouse cells. A similar deficiency was also noted in cultured bone‐derived cells isolated from NF1 human fetuses (32) . These data suggest that increased Ras‐MAPK activity in NF1 may have a mild but negative affect on osteoblast differentiation.…”

Bone mineral metabolism in patients with neurofibromatosis type 1 (von Recklingausen disease)

“…We also showed a significant increase in renal excretion of calcium (290 ± 70.9 vs. 146.8 ± 82.8 mg/24 h; P = 0.05), and reduction in both total (9.0 ± 0.4 vs. 9.8 ± 0.4 mg/dl) and ionized serum calcium levels (1.18 ± 0.05 vs. 1.26 ± 0.04 mmol/l) and increased circulating levels of PTH (55 ± 18.3 vs. 32.3 ± 15.3 pg/ml), leading to typical Bone alterations in NF1 patients are probably due to involvement of bone remodeling, by osteoclasts and osteoblasts, in which neurofibromin plays an important role in the differentiation and functional activity, with higher bone resorption and lower bone formation, induced by altered production of mineralized matrix and incomplete maturation of osteoblast precursors [19,21,23,33,37] and increased resorption, migration and survival of osteoclasts [14,29,31,32,45,46]. Other cellular elements can be involved into altered bone growth, such as chondrocytes, fibroblasts and endothelial cells, all expressing physiologically neurofibromin [12,30,40].…”

Decreased bone mineralization in children with Noonan syndrome: Another consequence of dysregulated RAS MAPKinase pathway?

“…In so doing, mutations can affect proliferation and differentiation in a cell type specific manner. Several research groups have shown that osteoprogenitors and osteoblasts from Nf1 +/− mice, and NF1 haploinsufficient human embryonic bone cells showed reduced expression of bone markers and/or produced less mineralized matrix when grown under osteogenic conditions [26-29]. When cultured ex vivo, myeloid precursors from Nf1 +/− mice generated more osteoclasts at lower concentrations of MCSF/RANKL than precursors from wild type mice.…”