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Constitutive Expression of Calreticulin in Osteoblasts Inhibits Mineralization

René St-Arnaud, Josée Prud'homme, Chungyee Leung-Hagesteijn and Shoukat Dedhar
The Journal of Cell Biology
Vol. 131, No. 5 (Dec., 1995), pp. 1351-1359
Stable URL: http://www.jstor.org/stable/1617227
Page Count: 9
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Constitutive Expression of Calreticulin in Osteoblasts Inhibits Mineralization
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Abstract

Recent studies have shown that the multifunctional protein calreticulin can localize to the cell nucleus and regulate gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors. A number of known modulators of bone cell function, including vitamin D, act through this receptor family, suggesting that calreticulin may regulate their action in bone cells. We have used a gain-of-function strategy to examine this putative role of calreticulin in MC3T3-E1 osteoblastic cells. Purified calreticulin inhibited the binding of the vitamin D receptor to characterized vitamin D response elements in gel retardation assays. This inhibition was due to direct protein-protein interactions between the vitamin D receptor and calreticulin. Expression of calreticulin transcripts declined during MC3T3-E1 osteoblastic differentiation. MC3T3-E1 cells were transfected with calreticulin expression vectors; stably transfected cell lines overexpressing recombinant calreticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Constitutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expression was unaffected. This pattern mimicked the gene expression pattern observed in parental cells before down-regulation of endogenous calreticulin expression. In long-term cultures of parental or vector-transfected cells, 1α,25-dihydroxyvitamin D3 (1,25[ OH]2 D3) induced a two- to threefold stimulation of 45 Ca accumulation into the matrix layer. Constitutive expression of calreticulin inhibited the 1,25( OH)2 D3-induced 45 Ca accumulation. This result correlated with the complete absence of mineralization nodules in long-term cultures of calreticulin-transfected cells. These data suggest that calreticulin can regulate bone cell function by interacting with specific nuclear hormone receptor-mediated pathways.

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