Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines

Abstract

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.