Differential expression of plasma membrane calcium pump mRNA isoforms in rat osteoblast-like cells.
This study was conducted to identify the plasma membrane Ca2+ transport ATPase (PMCA) mRNA isoforms expressed in rat osteoblast-like cells as PMCAs are likely to participate in calcium deposition in bone. We designed 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 the 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 product of the appropriate size. Southern blot analysis of the PCR products with oligonucleotide probes specific for each isoform revealed that 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 transcripts 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 cell lines used as models to study osteoblast function differentially express PMCA mRNA isoforms.
Abramowitz
J
J
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Gonzalez
J M
JM
Rouse
D
D
Suki
W N
WN
D000818:Animals; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D015342:DNA Probes; D007536:Isomerism; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D015335:Molecular Probes; D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases