Title

The role of insulin-like growth factor-I (IGF-I) in regulating bone mass and its application in the cell-based gene therapy of bone

Date of Completion

January 2003

Keywords

Biology, Molecular|Biology, Animal Physiology|Health Sciences, Medicine and Surgery

Degree

Ph.D.

Abstract

Insulin-like growth factor-I (IGF-I) is an important growth factor involved in embryonic and postnatal growth, skeletal development and bone remodeling. The goals of this work were to understand the role of IGF-I in growth and bone mass regulation during various stages of postnatal life, and to evaluate the use of IGF-I in cell-based gene therapy of bone. First, we analyzed the growth and skeletal phenotype in cohorts of wild type (igf1 +/+, WT) and igf1 heterozygous null (igf1 +/−, HET) mice in the outbred CD-1 background at 1, 2, 4, 8, 12, 15, and 18 months of age. HET mice were healthy and bred normally. They had decreased serum IGF-I levels (20–30% lower than WT) accompanied by reduced body weight, femur length, areal bone mineral density (BMD) and cortical bone area in most age groups with little change in trabecular bone volume. Serum IGF-I levels showed strong positive correlation with body weight, femur length and BMD. Primary osteoblasts from neonatal HET mice had significantly reduced IGF-I production, proliferation and differentiation capacity compared to WT cells. These results suggest that both circulating and skeletal IGF-I signaling are important in the acquisition and the maintenance of bone mass. ^ Next, to explore the therapeutic potential of IGF-I as an anabolic agent to stimulate bone formation, we developed a bicistronic retroviral system to deliver functional IGF-I in a bone-targeted manner for cell-based gene therapy. ROSA2.3-IGF-I/eGFP retrovirus was constructed to express both IGF-I and a visual marker—enhanced green fluorescent protein (eGFP) under the control of a 2.3 kb fragment of the rat Col1a1 promoter. Primary murine osteoblasts transduced with ROSA2.3-IGF-I/eGFP showed enhanced osteoblast differentiation. The effects of retroviral-delivered IGF-I on bone formation in vivo were assessed by an ectopic bone formation assay in which bone marrow stromal cells transduced with ROSA2.3-IGF-I/eGFP or control ROSA2.3-GFP retroviruses were implanted subcutaneously into nude CD-1 recipients. The expression of viral-delivered eGFP and IGF-I was detected in implant sections by fluorescent microscopy and immunohistochemistry six weeks after the surgery. Implants with ROSA2.3-IGF-I/eGFP-transduced cells showed higher bone volume than controls. In conclusion, characterization of HET mice revealed that both circulating and skeletal IGF-I play a critical role in the regulation of postnatal bone mass; bone-targeted overexpression of IGF-I by a retroviral gene delivery system may be applicable to cell-based somatic gene therapy to increase bone mass. ^