Osteoblast-restricted Disruption of the Growth Hormone Receptor in Mice Results in Sexually Dimorphic Skeletal Phenotypes

Abstract

Growth hormone (GH) exerts profound anabolic actions during postnatal skeletal development, in part, through stimulating the production of insulin-like growth factor-1 (IGF-1) in liver and skeletal tissues. To examine the requirement for the GH receptor (GHR) in osteoblast function in bone, we used Cre-LoxP methods to disrupt the GHR from osteoblasts, both in vitro and in vivo. Disruption of GHR from primary calvarial osteoblasts in vitro abolished GH-induced signaling, as assessed by JAK2/STAT5 phosphorylation, and abrogated GH-induced proliferative and anti-apoptotic actions. Osteoblasts lacking GHR exhibited reduced IGF-1-induced Erk and Akt phosphorylation and attenuated IGF-1-induced proliferation and anti-apoptotic action. In addition, differentiation was modestly impaired in osteoblasts lacking GHR, as demonstrated by reduced alkaline phosphatase staining and calcium deposition. In order to determine the requirement for the GHR in bone in vivo, we generated mice lacking the GHR specifically in osteoblasts (ΔGHR), which were born at the expected Mendelian frequency, had a normal life span and were of normal size. Three week-old, female ∆GHR mice had significantly reduced osteoblast numbers, consistent with the in vitro data. By six weeks of age however, female ΔGHR mice demonstrated a marked increase in osteoblasts, although mineralization was impaired; a phenotype similar to that observed previously in mice lacking IGF-1R specifically in osteoblasts. The most striking phenotype occurred in male mice however, where disruption of the GHR from osteoblasts resulted in a "feminization" of bone geometry in 16 week-old mice, as observed by μCT. These results demonstrate that the GHR is required for normal postnatal bone development in both sexes. GH appears to serve a primary function in modulating local IGF-1 action. However, the changes in bone geometry observed in male ΔGHR mice suggest that, in addition to facilitating IGF-1 action, GH may function to a greater extent than previously appreciated in establishing the sexual dimorphism of the skeleton. Keywords: growth hormone; osteoblasts; knockout mice; bone; sexual dimorphism coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. During postnatal development, the activity and lifespan of osteoblasts is regulated by many local growth factors, cytokines, and systemic hormones (1,2). Among these, growth hormone (GH) and insulin-like growth factor-1 (IGF-1) exert anabolic activity, particularly during the rapid phases of bone acquisition that occur during the pubertal growth [22][23][24].In many tissues, GH actions are mediated by its ability to stimulate the production of IGF-1, a widely expressed polypeptide that bears homology to pro-insulin. IGF-1 signals primarily via the heterotetrameric type-1 IGF-1 receptor (IGF-1R) to trigger ERK and PI3K/Akt activation via SHC and insulin receptor substrate-1 and -2 (IRS-1/2) (25,26). IGF-1 is an important growth and survival factor for many cell types, including osteoblasts (27,28). IGF-1 ...

Estrogens and Androgens in Skeletal Physiology and Pathophysiology

“…Hence, the effects of sex steroids on cortical bone expansion in male pubertal mice rely largely on GH/IGF-I signaling (81,82,530). Similarly, osteoblast-specific deletion of GH-receptor results not only in reduced osteoblastic sensitivity to IGF-I in vitro but also in a striking feminization of cortical bone geometry in male mice in vivo (467). Nevertheless, castration and replacement studies reveal that androgens still enhance cortical bone development in GH-deficient rats and mice (263,530,583) without clear IGF-I changes (82,530).…”

Skeletal effects of growth hormone and insulin-like growth factor-I therapy

“…Besides the effects of increased IGF-I expression, GH can directly stimulate the differentiation of prechondrocytes (Lindahl et al, 1987, 1986; Ohlsson et al, 1992), leading to longitudinal growth at the epiphyseal growth plate, data which have been recently confirmed by growth plate-specific postnatal knockout of the IGF-IR in mice (Wu et al, 2015). Additionally, targeted disruption of the GHR in osteoblasts suggests that, besides its stimulation of local IGF-I action, GH may play a role in directly regulating the development of skeletal sexual dimorphism in mice (Singhal et al, 2013). The intracellular molecular mechanisms that contribute to IGF-independent, growth-promoting effects of GH are yet to be identified.…”

Bone and Skeletal Muscle: Neighbors With Close Ties

“…Some differences are apparent, however, in the activity of these growth factors between muscle and bone. GH appears to uniquely interact with sex steroids in determining the sexually dimorphic patterns of the skeleton …”

“…GH appears to uniquely interact with sex steroids in determining the sexually dimorphic patterns of the skeleton. (23) Wnt signaling is also critical for the development of both muscle and bone and exhibits significant overlap in function between the two tissues. In muscle, Wnts control myogenic regulatory factor (MRF) expression during early embryogenesis to initiate the myogenic program, as well as regulating satellite cell differentiation, self renewal, and muscle fiber growth in response to loading (the latter two through noncanonical signaling) in adult life.…”