Identification of functional insulin‐like growth factor‐II/mannose‐6‐phosphate receptors in isolated bone cells

Abstract

The role of the IGF-II/cation independent mannose-6-phosphate (IGF-II/M6P) receptor in the transduction of cellular effects evoked by IGF-II has been extensively debated in the literature. Many reports suggest that IGF-II transduces its effects through the IGF-I receptor, while others show that IGF-II utilizes the type II receptor to affect cellular activity. This study 1) verifies the presence of the IGF-II/M6P receptor in rat calvarial osteoblasts, and 2) evaluates the ability of the receptor to initiate intracellular signals. Using conventional receptor binding assays, it was found that osteoblasts bind IGF-II with high affinity. Scatchard analyses indicated that there are 5.08 x 10(4) IGF-II/M6P receptors per osteoblast with a Kd near 2.0 nM). The receptor protein was further identified by cross-linking with 125I-IGF-II. Northern analysis was used to identify an mRNA transcript for the IGF-II/M6P receptor protein. To examine if the IGF-II/M6P receptor can initiate second messenger signals, the ability of IGF-II to evoke Ca2+ transients was evaluated. Osteoblasts pretreated with IGF-I did not lose their ability to respond to IGF-II. Further, a polyclonal antibody against the rat IGF-II/M6P receptor (R-II-PAB1) 1) was able to evoke its own Ca2+ response, and 2) was able to block the generation of Ca2+ transients caused by IGF-II. The data in this report show that the osteoblastic Ca2+ response to IGF-II appears to be caused by an intracellular release of Ca2+ which is mediated by the IGF-II/M6P receptor making it possible to envision how the receptor may be an important modulator of osteoblast mediated osteogenesis.

Use of a Phage Display Technique to Identify Potential Osteoblast Binding Sites Within Osteoclast Lacunae

“…Isolated osteoblasts were prepared from neonatal rat calvaria as previously described. (18) Biopanning procedure Two micrograms of TRAP in 0.1 M of sodium bicarbonate (pH 8.6) was used to coat microtiter plates for 16 h at 4°C. The wells were blocked for 1 h at 37°C with phosphate-buffered saline (PBS) containing 1% [wt/vol] bovine serum albumin (BSA).…”

TRIP-1: A regulator of osteoblast function

“…We isolated rat calvarial primary osteoblast (ROB) cells as described. (14) ROB cells were cultured in low glucose Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Grand Island, NY, USA) with 5% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 50 mg/mL ascorbic acid. For osteoblast differentiation assays, a final concentration of 10 mM b-glycerophosphate (BGP) was also added to the media.…”

Insulin-Like Growth Factor-II/Cation-Independent Mannose 6-Phosphate Receptor in Neurodegenerative Diseases

“…Because the IGF-II/M6P receptor lacks intrinsic catalytic activity, most of the biological effects of IGF-II have been attributed either to the IGF-I receptor [13] or IR-A [8]. Nonetheless, a number of studies over the years have suggested that certain biological effects of IGF-II are being triggered directly via the IGF-II/M6P receptor, including calcium influx in mouse embryo fibroblasts [104] and rat calvarial osteoblasts [105], increased protein phosphorylation [106] and alkalinization in proximal renal tubule cells [107], stimulation of Na + /H + exchange and inositol trisphosphate production in canine kidney cells [108], increased amino acid uptake in muscle cells [109], increased glycogen synthesis in hepatoma cells [110], proteoglycan synthesis in human chondrosarcoma cells [111], calcium mobilization in rabbit articular chondrocytes [112], cell motility in rhabdomyosarcoma cells [113, 114], aromatase activity in placenta cytotrophoblasts [115], migration of human extravillous trophoblasts [116], insulin exocytosis by pancreatic β cells [117], and regulation of endogenous acetylcholine and GABA release from the adult rat brain [118, 119]. …”