Matrix IGF-1 regulates bone mass by activation of mTOR in mesenchymal stem cells

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, regulates bone mass in adulthood. We report that IGF-1 released from bone matrix stimulates osteoblastic differentiation of mesenchymal stem cell (MSCs) by activation of mTOR during bone remodeling. Mice knockout of IGF-1 receptor (Igf1r) in the preosteoblastic cells exhibited low bone mass and reduced mineral deposition rates. The MSCs recruited to the bone surface were unable to differentiate into osteoblasts. In age-related osteoporosis in humans, we found that marrow IGF-1 levels were 40% lower than controls. Similarly, the levels of IGF-1 in the bone matrix and marrow of aged rats were also decreased and directly correlated with the age-related decrease in bone mass. Notably, injection of IGF-1 with IGF binding protein 3 (IGFBP3), not IGF-1 alone, increased the level of IGF-1 in the bone matrix and stimulated new bone formation in old rats. Thus, IGF-1 released during bone resorption from bone matrix activates mTOR to induce osteoblast differentiation of MSCs in maintaining bone micro-architecture and mass.


Adenoviral infection.
For the adenoviral (Ad) infections, we plated MSCs at a density of 5 × 10 5 cells in 100 mm plates. We added Ad-Cre-GFP viruses to the MSCs at a multiplicity of infection (MOI) of 100, suspended in PBS at 37 °C in 5% CO 2 atmosphere. Plates were swirled every 15 minutes and fresh α-MEM with 10% FBS was added after 1 hour of incubation. We exposed the cells to fresh α-MEM containing adenovirus for 48 hours. The control group Ad-GFP was treated with the same method. We obtained the adenovirus preparations from Vector Biolabs (Philadelphia, PA).

RNA interference.
We transfected MSCs with siRNA targeting Irs1 or scrambled control siRNA that has been tested without leading to the specific degradation of any known cellular mRNA (Santa Cruz) using Lipofectamine RNAiMAX in Opti-MEM (Invitrogen) according to the protocol recommended by the manufacturer (Invitrogen).
Experiments were performed 48 hours following transfection. Scrambled siRNA and siRNA targeting Irs1 had no significant effect on cellular morphology. We validated the successful knockdown of IRS1 using western blot analysis.

CFU-F and CFU-Ob assays.
At the time of euthanasia, we collected bone marrow from femoral, tibial, and humeral medullary cavities, and determined cell numbers with Zapoglobin (Coulter Corp.) after removal of red blood cells. The numbers of CFU-Fs and CFU-Obs in murine bone marrow isolates and cocultures with irradiated guinea pig marrow cells were as reported 39 . We obtained marrow cells from the femurs and tibiae of 2 months old female Hartley guinea pigs by flushing with a 22 gauge needle and then resuspended. We γ-irradiated cells with a Co57 source for 50 minutes at 1.2 Grays min -1 as reported 40 . After rinsing by centrifugation, cells were resuspended in α-MEM medium with 20% FBS, counted and cultured at 2.5 × 10 6 per well of a six-well plate. For assay of CFU-F and CFU-Ob number, we plated 0.1, 0.5, or 1×10 5 murine marrow cells into six-well plates in 3 ml α-MEM supplemented with glutamine (2 mM), penicillin (100 U ml -1 ), streptomycin sulfate (100 µg ml -1 ), and 20% lot-selected FBS. We also established duplicate cultures. After 2 to 3 hours of adhesion, we removed unattached cells, and added 2.5 × 10 6 irradiated guinea pig feeder cells (provided by Dr. Brendan J. Canning) to cultivation medium of adherent cultures just after washing. On day 14, we fixed and stained the cultures with 0.5% Crystal Violet. We counted colonies that contained 50 or more cells. For CFU-Ob assay, we cultured the cells with osteogenic medium as described above for 21 days and analyzed with Alizarin Red staining. We determined the colony-forming efficiency by quantifying number of colonies per 10 5 marrow cells plated.
Bone marrow cavity transplantation. We used 2 months old male Rag2 -/mice with an immune-deficient background as recipients. We injected GFP-labeled cells in 10 µl of α-MEM media into the bone marrow cavity of the left femora, as previously described. The mice were euthanized 2 or 4 weeks after transplantation and processed the distal femora for staining. In some cases, we collected cells from bone Subrenal capsule transplant. We utilized 2 months old male Rag2 -/mice with an immune-deficient background as recipients. 5×10 3 GFP-labeled cells in the matrigel (with 100 ng ml -1 IGF-1) were inoculated underneath the renal capsule of 2 months old Rag2 -/mice injected subcutaneously with rapamycin (3 mg kg -1 per day) or vehicle for 4 weeks. We euthanized the mice 4 weeks after transplantation and processed the kidneys for frozen histology staining.
In vitro osteoblastic differentiation. We plated MSCs cells at a density of 1,000 cells per well in the six-well plates and expanded in growth medium for 3 days. We induced osteoblastic differentiation in low serum conditions (2% FBS) in differentiation medium (αMEM supplemented with 50 μg ml -1 of ascorbic acid and 10 mM β-glycerol phosphate). We added mouse recombinant IGF-1(Abcam) to the differentiation medium at 100 ng ml -1 with or without PI3K inhibitor LY294002 (10 μM) or mTOR inhibitor rampamycin (20 nM). We changed the medium every 3 days and assayed cellular differentiation 20 days after induction through Alizarin Red staining. and 100 ng ml -1 of RANKL. Under these conditions, osteoclasts began to form and resorb bone at 6 to 7 days. The cultured cells were stained for TRAP activity at 8 to 10 days using a commercial kit (Sigma-Aldrich). We determined the resorption of the bovine cortical bone slices by scanning electronic microscopy. We harvested the conditioned media from the osteoclast-mediated resorption at 8 to 10 days with centrifugation. We added neutralizing antibodies for IGF-1, IGF-II, TGF-β1, PDGF (BD Biosciences) to the conditioned media or depleted the conditioned media (BRCM) of active IGF-1 by three cycles of immunoprecipitation using a monoclonal antibody specific for IGF-1 tagged to protein G-Sepharose.
Bone marrow cavity injection of IGF-1, IGFBP3 and IGF-1 with IGFBP3. We randomly divided forty rats into four groups with ten animals per group. We purchased recombinant IGF-1 and IGFBP3 from Abcam and R&D Systems. We delivered 8 μg of IGF-1, 42 μg IGFBP3, IGF-1 with IGFBP3 (8 μg of IGF-1 and 42 μg of IGFBP3, molar ratio 1:1) or vehicle (PBS) into bone marrow cavity once a week injected from the medial side of patellar tendon using 0.5 ml syringes with 27 gauge needles. We collected whole blood samples by cardiac puncture for biochemical assessments immediately after euthanasia by CO 2 asphyxiation after 4 weeks. We collected blood samples by centrifugation to obtain the sera, which were then stored at -80 °C until assayed.
Bone matrix extraction and bone marrow serum collection. We exposed bone marrow from freshly sacrificed animals at the growth plate and placed samples for centrifugation with trabecular part of the bone at top. We removed bone marrow by centrifugation of the samples for 15 minutes at 5,000 rpm at 4 °C. We immersed the resected femurs in liquid nitrogen and mechanically crushed specimens into small fragments of several millimeters in diameter. The fragments were washed repeatedly in cold distilled water until they were free of blood and defatted in cold isopropylether.

Measurement of IGF-1 and IGFBP3 levels.
We determined the concentration of IGF-1 by a mouse or rat IGF-1 Immunoassay kit (ELISA) (R & D Systems) according to the instructions provided. This assay was designed to eliminate interference by binding proteins. We diluted peripheral blood serum 1000 folds; bone marrow serum 500 folds and bone matrix protein 10 folds with the Calibrator Diluent RD5-38 before detection. The assay sensitivity was 3.5 pg ml -1 ; intra-and interassay coefficients of variation were 5.6% and 4.3%. We assessed Human IGF-1 levels by ELISA after an acid-ethanol extraction. The inter-assay variation was 5.3% and the intra-assay variation was 2.3%. We determined the concentration of IGFBP3 by a mouse or rat IGFBP3 Immunoassay kit (ELISA) (BioVendor, Candler) according to the instructions provided. The assay sensitivity was 18 pg ml -1 ; intra-and interassay coefficients of variation were 4.6% and 8.4%.
Collection of human bone marrow samples. We collected bone marrow samples from individual subjects with osteoporosis, as defined by hip fractures and low bone mass (n=12), and individual subjects with osteoarthritis and normal bone mineral density of similar ages (n=12). The bone marrow collection was performed during total hip replacement with informed consent and approval by the Institutional Review