Expression of mouse osteocalcin transcripts, OG1 and OG2, is differently regulated in bone tissues and osteoblast cultures

Abstract.

Osteocalcin is a noncollagenous protein that is abundant in mineralized bone matrix. Mice have a gene cluster of osteocalcin that consists of OG1, OG2, and ORG. We established a new method to directly analyze the expression levels of OG1, OG2, and ORG mRNAs relative to total osteocalcin mRNA. They were amplified as 371-bp fragments by reverse transcription-polymerase chain reaction (RT-PCR) at the same time using common primers, digested with ApaLI, and separated in a polyacrylamide gel. ApaLI digestion did not affect the mobility of the OG1-derived 371-bp fragment, whereas both 371-bp fragments, derived from OG2 and ORG, were digested into 350 bp. Total RNA prepared from mouse bone was then subjected to RT-PCR followed by ApaLI digestion. OG1 and OG2 mRNAs were found to be expressed at ratios of 80%–86% and 14%–20%, respectively, to the total osteocalcin mRNA in mouse bone. The ratios were almost constant in various bones in vivo, independent of the animal's genetic background, age, or gender, or different parts of bone. RT-PCR using specific primers revealed that mouse bone tissues strongly expressed osteocalcin mRNA derived from OG1 and OG2, but not ORG. In contrast, cells cultured in vitro showed different expression ratios of osteocalcin mRNA: 53%–65% for OG1 and 35%–47% for OG2 to the total osteocalcin mRNA in the osteoblast cell line and primary osteoblasts in culture even though they formed many mineralized bone nodules. Similar results were obtained in both KS483 osteoblasts and C2C12 myoblasts, when they were cultured with bone morphogenetic protein-2 (BMP-2) to induce osteocalcin mRNA. Taken together, these findings indicate that OG1 is the predominant transcript among the three osteocalcin genes in mouse bone in vivo. It is also suggested that the expression of OG1 and OG2 is regulated differently in bone tissues and osteoblast cultures.