DIFFERENTIAL EXPRESSION OF PLASMA-MEMBRANE CALCIUM-PUMP MESSENGER-RNAISOFORMS IN RAT OSTEOBLAST-LIKE CELLS

This study was conducted to identify the plasma membrane Ca2+ transpor t ATPase (PMCA) mRNA isoforms expressed in rat osteoblast-like cells a s PMCAs are likely to participate in calcium deposition in bone. We de signed oligonucleotide primers for each PMCA isoform based on rat cDNA sequences in order to develop reverse transcription polymerase chain reaction (RT-PCR) assays, Rat kidney total RNA was used to validate th e assays as we have shown that each isoform is present in kidney. When used in RT-PCR assays each primer pair gave rise to a single major pr oduct of the appropriate size. Southern blot analysis of the PCR produ cts with oligonucleotide probes specific for each isoform revealed tha t each probe hybridized only to the expected product. Reamplification of purified PCR products with probe and antisense primers gave rise to products of appropriate size, further confirming the identity of the products. Using these primers we have identified the presence of trans cripts for PMCA1, 2 and 4 in RNA from UMR-106 osteoblasts and PMCA1 in RNA from ROS 17/2.8 osteoblasts. We conclude that the two major rat c ell lines used as models to study osteoblast function differentially e xpress PMCA mRNA isoforms.