The placental trophoblastic epithelium functions to transport nutrients required by the developing fetus, including calcium, which is needed in the greatest amounts during the last third of pregnancy when the majority of fetal skeletal mineralization occurs. While the exact mechanism of trophoblast calcium transport is incompletely understood, there is evidence suggesting that a developmentally expressed, trophoblast-specific, high-Mr 57 kDa cytosolic Ca-binding protein (CaBP) plays an important role in regulating and/or shuttling cytosolic calcium. The objectives of this thesis research are to: (1) determine the sequence and structure of the mouse placental CaBP cDNA; (2) examine the mechanisms regulating the expression of CaBP; (3) analyze the function/role of CaBP in calcium transport. We report here the cloning of a full length cDNA of the mouse CaBP. Structural analysis shows that CaBP has significant homology with calreticulin, an endoplasmic reticulum. associated calcium binding protein. To analyze the regulation of CaBP expression, placenta organ cultures and Rcho-1 cells were treated with a series of candidate regulatory agents. In both placenta organ cultures and in undifferentiated as well as differentiated Rcho-1 cells, treatment with parathyroid hormone related protein (PTHrP 67-84) substantially stimulated CaBP mRNA and protein levels. PTHrP 67-84 treated Rcho-1 cells also exhibited higher calcium uptake activity than untreated control cells. To examine the functional role of CaBP in trophoblast calcium transport and differentiation, we (1) recombinantly overexpressed CaBP in an inducible manner and (2) downregulated CaBP expression using antisense technology, in the Rcho-1 cell line. The results show that overexpression of COP stimulates both calcium uptake and calcium transport in Rcho-1 cells. Those cells stably expressing CaBP also exhibit higher levels of steady state intracellular calcium and enhanced calcium buffering ability. In addition, prolonged overexpression of CaBP in Rcho-1 cultures promotes trophoblast differentiation. Conversely, downregulation of CaBP expression had a negative effect on calcium uptake, calcium transport, and trophoblast differentiation in Rcho-1 cells. These data indicate that CaBP plays a direct role in trophoblast calcium transport, functioning both as an intracellular calcium buffer and a shuttle as well as possible involvement of this CaBP in the process of trophoblast differentiation.
