Complex pattern of insulin‐like growth factor binding protein expression in primary rat osteoblast enriched cultures: Regulation by prostaglandin E₂, growth hormone, and the insulin‐like growth factors

Abstract

Primary osteoblast-enriched (Ob) cultures from fetal rat bone synthesize insulin-like growth factor (IGF) I and IGF-II, which each enhance Ob function. While a number of agents modulate IGF-I production, IGF-II is constitutively expressed in this culture model. Independent of their expression, however, the activity of the IGFs can be modified by a small group of proteins termed IGF binding proteins (IGFBPs), but little is known about the regulation of individual IGFBPs that are synthesized by Ob cells. Northern blot analysis revealed that serum-deprived primary rat Ob cells express transcripts encoding IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, but undetectable levels of IGFBP-1 transcripts. Western ligand blots of Ob culture medium probed with 125I-IGF-I or 125I-IGF-II showed predominant IGFBPs migrating at 30/32 kDa, with minor bands at 24 and 38-47 kDa. Western antibody analysis identified IGFBP-2 and IGFBP-5 within the 30/32 kDa complex, while gel mobility shift on SDS-PAGE following deglycosylation determined that IGFBP-3 comprised the 38-47 kDa complex. By Northern analysis, 6 h treatment with prostaglandin E2 (PGE2), growth hormone (hGH), IGF-I, or IGF-II revealed a complex pattern of regulatory effects on steady-state IGFBP transcript expression. PGE2 increased the transcript levels of IGFBP-3, IGFBP-4, and IGFBP-5, (approximately 22-, approximately 2- and approximately 4-fold respectively), but had no effect on IGFBP-2 or IGFBP-6 transcripts. hGH enhanced IGFBP-3 and IGFBP-5 transcripts (each approximately twofold). IGF-I and IGF-II had no effect on IGFBP-2 steady-state transcript levels but enhanced the level of IGFBP-5 transcripts (approximately fourfold). By Western ligand blot analysis, 24 h treatment with PGE2 elevated the 24 and 38-47 kDa IGFBPs and to a lesser extent the 30/32 kDa complex, hGH elevated the 38-47 kDa IGFBPs, and IGF-I and IGF-II each increased the 30/32 kDa IGFBP complex. Therefore, a comparison of results obtained from Northern, Western ligand, and Western antibody studies indicates that multiple IGFBPs are expressed by primary rat Ob cultures. While IGFBP-2 and IGFBP-6 synthesis in Ob cultures is relatively unaffected by short-term treatment with PGE2, hGH, or the IGFs, these agents modify IGFBP-3, IGFBP-4, and IGFBP-5 expression with individual patterns of effects. In addition, some changes in IGFBP polypeptide levels that are independent of alterations in transcript expression may result from the formation of complexes between IGFs and certain IGFBPs, which could serve to store IGFs for future utilization in the formation phase of bone remodeling.

Promoter-dependent and -independent Activation of Insulin-like Growth Factor Binding Protein-5 Gene Expression by Prostaglandin E2 in Primary Rat Osteoblasts

“…Unstimulated fetal rat osteoblasts synthesize IGFBP-5 mRNA and protein (4,40,41). Results from transient transfection experiments confirm our earlier studies that near-maximal basal promoter activity resides within the first 156 bp of 5Ј-flanking DNA and that over 20% of basal activity is con-trolled by the proximal 75 bp of the promoter (32).…”

“…These results contrast with the lack of an effect of cycloheximide on the rise in IGF-I transcript abundance following PGE 2 treatment, 3 and demonstrate that inducible protein(s) contribute to the transcriptional and post-transcriptional effects of PGE 2 on IGFBP-5 gene expression in osteoblast-enriched cultures. DISCUSSION IGFBP-5 expression is activated through cAMP-dependent pathways in osteoblasts and in other cell culture models (4,16,39). While IGFBP-5 transcripts accumulate in response to PGE 2 and other agents that elevate cAMP, little is known about the mechanisms of hormone-induced IGFBP-5 synthesis in osteoblasts.…”

“…In bone cell cultures, both IGFBP-5 and IGF-I synthesis are regulated by prostaglandin E 2 (PGE 2 ), parathyroid hormone (PTH), other agents that stimulate cAMP synthesis, or by cAMP itself (4,16). PGE 2 is produced by osteoblasts in response to PTH, to cytokines such as interleukin-1 and transforming growth factor-␤, and to mechanical strain, and PGE 2 has been shown to mediate various biological actions on osteoblasts initiated by these stimuli (17)(18)(19)(20)(21)(22).…”

Long-chain polyunsaturated fatty acids: Selected mechanisms of action on bone

“…In osteoblasts, hormones such as growth hormone, parathyroid hormone and oestrogen modulate IGF-I expression [78]. In addition to IGF-I, osteoblasts also synthesise extracellular high affinity IGFbinding proteins (IGFBPs), which modify the interaction of IGF-I with its receptors by prolonging IGF stability and by influencing ligand-receptor interaction [79,80]. IGF-1 is deemed necessary for maintaining the normal interaction between the osteoblast and osteoclast to support osteoclastogenesis through regulating RANKL and RANK expression [81].…”

“…PGE 2 also promotes the expression of various IGF-binding proteins [77,80,82] suggesting that PGE 2 could keep IGF available for stimulation of osteoblasts at a later phase of bone remodeling [77]. McCarthy et al [48] suggested that the ability of PGE 2 to enhance osteoblastic IGF-I synthesis could explain its anabolic potential, and furthermore suggests a role for PGE 2 in coupled bone remodeling.…”