Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Abstract

Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45 Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating the phosphatases were, in part, providing a source of inorganic phosphate.To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study

“…Therefore, the DPP phosphorylation pattern could be a code for the configurations of the peptides and hence their affinity for potential ligands. In previous studies, we showed the important role of phosphorylation in mineralized tissues and its role in the biomineralization process (17,18); this study showed the specific behavior of the DPP peptides when they are phosphorylated. The precise role of each of the peptide structures remains unclear.…”

14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1

“…An indirect effect on the signal transduction pathways was ruled out, as IL-1-mediated MMP-1 expression was not hindered by dephosphorylation of intact cells. Reports show that ECM proteins (osteopontin, sialoprotein, dentin matrix protein-1, phosphophoryn) as well as many secretory proteins (casein, enkephalin precursor, progastrin, chromogranin) become phosphorylated during their movement through the secretory pathway (Boskey et al, 2008;Turner et al, 1993). In addition, the extracellular phosphorylation of the ectodomains of integral proteins is not unprecedented.…”

MOVAS-1 cell line: A new in vitro model of vascular calcification

“…Levamisole reversibly but non-competitively inhibits ALP (39), and is a potent inhibitor of the calcification of osteoblasts (bone forming cells) (44,45) and VSMCs (19,46). The bisphosphonate etidronate, an inhibitor of hydroxyapatite formation, has also been reported to inhibit calcification of VSMcs and osteoblasts.…”