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Task Last Updated: 06/10/2008 
Division Name: Human Research 
Program/Discipline: NSBRI 
Element/Subdiscipline: Musculoskeletal Alterations Team 
Project Title: Recovery of IGF-1 Signaling in Bone by Skeletal Reloading 
Joint Agency Name:  
PI Name: Long, Roger   PI Phone: 415-750-2089  
PI Email: Roger.Long@ucsf.edu  Fax: 415-750-6929 
PI Organization Type: UNIVERSITY 
Organization Name: Northern California Institute for Research & Education 
PI Address 1: 4150 Clement St, 111N 
PI Address 2:  
PI Web Page:  
City: San Francisco State: CA Zip Code: 94121 Congressional District: 8
Comments:  
Project Type: GROUND  Solicitation: NSBRI-RFP-06-01 
Start Date: 02/01/2007  End Date: 01/31/2009 
Fiscal Year: 2008     
No. of Post Docs: 0  No. of PhD Degrees: 0 
No. of PhD Candidates: 0  No. of Master' Degrees: 0 
No. of Master's Candidates: 0  No. of Bachelor's Degrees: 0 
No. of Bachelor's Candidates: 0  Monitoring Center: NSBRI 
Contact Monitor:   Contact Phone:  
Contact Email:      
Flight Program:  
Flight Assignment:

 
Key Personnel Changes/Previous PI:  
COI Name: COI Institution:
Bikle, Daniel   University of California, San Francisco 
Grant/Contract No.: NCC 9-58-PF01102 
Performance Goal No.:  
Performance Goal Text:

 
Task Description:  POSTDOCTORAL FELLOWSHIP.

Prolonged spaceflight causes osteopenia due to decreased bone formation secondary to impaired osteoblast proliferation and increased osteoblast apoptosis. Hindlimb unloading by tail suspension, a model for skeletal unloading of spaceflight, causes osteoblast precursor and skeletal tissue resistance to the effects of insulin-like growth factor-1 (IGF-1). The nature of this resistance is characterized by decreased activation of the IGF-1 receptor and downstream signaling pathways. Osteoblast precursors from unloaded bones demonstrate decreased expression of integrins, and treatment of normal osteoblasts with echistatin, an integrin antagonist, recreates the phenomena of unloading-induced IGF-1 resistance. Additionally, mechanical stimulation of human osteoblasts activates the IGF-1 receptor and augments the receptor response to IGF-1. These effects are abrogated by echistatin treatment. These findings suggest that integrin receptors have a role in the regulation of IGF-1 receptor in osteoblasts.

Our hypothesis is that interaction of integrin and IGF-1 receptor signaling cascades is required for IGF-1 activation of its receptor and intact IGF-1 signaling in osteoblasts. Mechanical loading stimulates the formation of an integrin/IGF-1 receptor complex, thus, enhancing IGF-1 signaling and enabling mechanically induced osteoblast proliferation and bone formation. To test the hypothesis, we propose the following specific aims:

1. Determine the means by which skeletal reloading regulates skeletal response to IGF-1. Tail suspended rats treated with IGF-1 will be skeletally reloaded to preserve bone mass and osteoblast IGF-1 signaling. Integrity of IGF-1 signaling will be correlated with the interaction between integrins and the IGF-1 receptor.

2. Determine the mechanism for regulation of the osteoblast IGF-1 receptor by integrins in response to mechanical loading by pulsatile fluid flow. Formation of the integrin and IGF-1 receptor complex in human osteoblasts will be stimulated by fluid flow loading in order to determine the components within the complex and their function.

The feasibility of intermittent reloading and IGF-1 infusion to prevent unloading-induced bone loss will be established. Furthermore, establishing the interaction between integrins and the IGF-1 signaling cascade in osteoblasts will provide potential targets for countermeasure treatments to preserve the effects of mechanical loading and prevent the bone loss of spaceflight.

 
Research Impact/Earth Benefits: The research project outlined in my post-doctoral fellowship focuses on understanding the role of the hormone insulin-like growth factor 1 (IGF-1) in the skeletal response to skeletal unloading and loading. Previous work from our lab has demonstrated that skeletal unloading by tail suspension, a disuse osteoporosis model that not only mimics the weightlessness of spaceflight but the skeletal unloading associated with prolonged bedrest or neurological injury, causes a resistance to the anabolic effects of IGF-1 both in vivo and in vitro. A disruption of integrin signaling has been associated with the resistance of the IGF-1 receptor and the role of the interaction between these two signaling pathways will be explored. A membrane associated complex of the integrin and IGF-1 receptors would enable mechanically induced bone formation and osteoblast proliferation via enhanced IGF-1 signaling. Understanding the interaction between integrin receptors with their ability to sense mechanical load and the powerfully anabolic effects of IGF-1 is vital for efforts to maximize bone accrual and minimize bone loss associated with skeletal unloading. We propose to test the hypothesis that the skeletal response to mechanical load requires the formation of a complex including selected integrins and IGF-1R. If this hypothesis turns out to be valid, efforts to maintain the integrin signaling pathway will prove efficacious in preventing the skeletal loss of responsiveness to IGF-1 associated with skeletal unloading. We envision the use of intermittent skeletal loading, to maintain the integrity of the integrin and IGF-1 receptor interaction and thus the skeletal sensitivity to IGF-1, combined with IGF-1 therapy incorporated into hospital stays to prevent acute bone loss and rehabilitation programs to stimulate recovery of bone loss from acute injury or illness.

Task Progress: Aim 1: During the first year of the post-doctoral fellowship the strain-load relationship of the adult male rat tibiae was established. This relationship was found to be uniformly consistent and allowed rats to be mechanically loaded with an applied load of 30 N which is predicted to elicit an anabolic compressive strain of 2000 microstrain.

Six episodes of intermittent cyclic loading (40 compression events per episode) were administered concurrently with skeletal unloading by tail suspension over a two week period. Hindlimb unloading by tail suspension is the animal model used to simulate the weightlessness of spaceflight. Evaluation of the left tibiae, not subjected to cyclic loading, from the unloaded and normal ambulatory animals reveals the profound bone loss caused by the skeletal disuse model, a significant change in trabecular bone volume to total volume ratio (BV/TV) from 12% to 5% as assessed by in vitro microCT. Intermittent cyclic loading of the right tibiae while animals were subjected to tail suspension significantly blunted the unloading-induced bone loss. The trabecular BV/TV from these tibiae were not significantly different from the tibiae from normal ambulatory animals. This remarkable protective effect was achieved by a cumulative period of only 42 minutes of skeletal loading during a two week period of continuous skeletal unloading. The animals tolerated both interventions well as indicated by stability of body weights and gross visual inspections of the hindlimbs.

The mechanism by which brief intermittent cyclic loading preserves bone during skeletal unloading will be evaluated. Dynamic and standard histomorphometric analysis of bone sections will provide valuable information as to the nature of the skeletal protection by assessing bone formation and bone resorption. The role of the anabolic hormone IGF-1 in the preservation of bone in response to cyclic skeletal loading will also be determined. Animals will be subjected to administration of IGF-1 in vivo concurrent with the unloading and cyclic loading processes and the changes in the tibiae assessed. The integrity of the IGF-1 signaling cascade in bone marrow stromal cells, osteoblast precursors, isolated from these mechanically stimulated tibiae will be assessed to elucidate the process at the cellular level.

Aim 2: The osteoblast-like human osteosarcoma cells were studied to evaluate the role of integrins in the regulation of the IGF-1 receptor. Initial results demonstrate that stimulation of integrins with ligands that bind integrins beta1 or beta3 augments the phosphorylation of the IGF-1 receptor and activation of downstream signaling components in response to ligand and mechanical stimulation. Additionally, knockdown of integrins beta1 and beta3 by siRNA technology significantly impairs the activation of IGF-1 signaling suggesting that both these integrins play a significant regulatory role of IGF-1 signaling in osteoblasts.

 
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