CONSTITUTIVE EXPRESSION OF CALRETICULIN IN OSTEOBLASTS INHIBITS MINERALIZATION

Recent studies have shown that the multifunctional protein calreticuli n can localize to the cell nucleus and regulate gene transcription via its ability to bind a protein motif in the DNA-binding domain of nucl ear hormone receptors. A number of known modulators of bone cell funct ion, including vitamin D, act through this receptor family, suggesting that calreticulin may regulate their action in bone cells. We have us ed a gain-of-function strategy to examine this putative role of calret iculin in MC3T3-E1 osteoblastic cells. Purified calreticulin inhibited the binding of the vitamin D receptor to characterized vitamin D resp onse elements in gel retardation assays. This inhibition was due to di rect protein-protein interactions between the vitamin D receptor and c alreticulin. Expression of calreticulin transcripts declined during MC 3T3-E1 osteoblastic differentiation. MC3T3-E1 cells were transfected w ith calreticulin expression vectors; stably transfected cell Lines ove rexpressing recombinant calreticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Cons titutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expressi on was unaffected. This pattern mimicked the gene expression pattern o bserved in parental cells before down-regulation of endogenous calreti culin expression. In longterm cultures of parental or vector-transfect ed cells, 1 alpha,25-dihydroxyvitamin D-3 (1,25[OH]D-2(3)) induced a t wo- to threefold stimulation of Ca-45 accumulation into the matrix lay er. Constitutive expression of calreticulin inhibited the 1,25(OH)(2)D -3-induced Ca-45 accumulation. This result correlated with the complet e absence of mineralization nodules in long-term cultures of calreticu lin-transfected cells. These data suggest that calreticulin can regula te bone cell function by interacting with specific nuclear hormone rec eptor-mediated pathways.