Mode of Growth Hormone Action in Osteoblasts

Douglas J Digirolamo,Aditi Mukherjee,Keertik Fulzele,Yujun Gan,Xuemei Cao,Stuart J Frank,Thomas L Clemens

doi:10.1074/jbc.m705219200

Abstract

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R(flox/flox)) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.

Highlights

The process of osteogenesis and remodeling of the skeleton is orchestrated by a constellation of local growth factors, cytokines, and systemic hormones (1, 2), of which growth hormone (GH)2 and insulin-like growth factor type 1 (IGF-1) are key
Disruption of IGF-1 receptor (IGF-1R) in Osteoblasts—To establish a system for Cre-mediated gene excision of the IGF-1R in vitro, primary osteoblasts from calvaria of ROSA26 reporter mice were infected with adenoviral Cre (AdCre) or GFP control (AdGFP) vectors and stained by X-gal staining
GHR Signaling in the Absence of IGF-1R—To examine the effect of IGF-1R on GH signaling, calvarial osteoblasts from mice carrying the floxed IGF-1 receptor (Igf1r) allele were infected with adenovirus-encoding green fluorescent protein (AdGFP) or adenovirus-encoding Cre recombinase (AdCre) and treated with GH or IGF-1

Summary

Mode of Growth Hormone Action in Osteoblasts*

To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in treated mice lacking the IGF-1R in osteoblasts were not significantly affected These results indicate that direct IGF1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo. We used a Cre-loxP method to selectively disrupt the IGF-1R in osteoblasts Results from this model suggest that IGF-1R-independent actions of GH to inhibit osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for normal GH action in osteoblasts in vivo, presumably through its ability to stimulate the production of IGF-1

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS