Investigation of the expression of insulin-like growth factor 1 splice variants in bone and primary rat osteoblasts
Insulin-like growth factor 1 (IGF-1) acts as a mediator for several hormones in bone. In rat the IGF-1 gene is processed into distinct mRNA molecules which differ in their 5' and 3' ends. Two major classes of mRNA transcript are produced which contain either exon 1 (Class 1) or exon 2 (Class 2). The main aims of this study were to identify the IGF-1 splice variants produced in bone and investigate possible effects of GH on transcript expression. IGF-1 transcripts were analysed in bone using reverse transcription polymerase chain reaction (RT-PCR) using forward specific primers for exon 1 and exon 2 and reverse primers for exon 5 and 6. Analysis of RNA extracted from either whole tibia, growth plate, primary osteoblasts, and clonal cell lines UMR 106 and ROS 17.6 showed distinct patterns of expression. All IGF-1 splice variants were expressed in bone tissue and primary osteoblasts however none of the variants were detected in UMR 106 cell line and only Class 1 Ea was detected in ROS 17.6 cell line. No obvious change in this pattern of expression was observed on GH stimulation of these primary cells and osteosarcoma cell lines. However, GH was found to stimulate osteoblast growth by enhancing proliferation of a subpopulation of cells but GH did not effect alkaline phosphatase production. GH did not increase total IGF-1 mRNA levels in osteoblasts as detected using RNase Protection assay but there was a change in the splicing profile such that Class 2 transcripts were increased by 150% (p=0.05) and Class 1 transcripts decreased by 35% (p=0.02). Furthermore antisense ODNs directed against the common exon 4 ofIGF-1 caused a dramatic increase in osteoblast apoptosis whereas sense and scrambled control ODNs had no effect. Our data show that IGF-1 is an important constitutively expressed factor in osteoblasts and GH may exert its actions by increasing Class 2 transcripts in bone which could have paracrine effects on neighbouring cells.