Systems Genetic Analysis of Osteoblast-Lineage Cells

Abstract

The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types.

Bicc1 is a genetic determinant of osteoblastogenesis and bone mineral density

“…Of the other 20 modules, Bicc1 was also highly correlated with the module 9 (M9) eigengene (r = 0.45; P = 2.9 × 10 -6 ) ( Figure 3D). This was not unexpected, because like the M6, we have demonstrated that the M9 is specific to cells of the osteoblast lineage (35). Based on its membership in the M6 and its association with the M9, we measured the expression of the 21 genes in the M6 and the 13 genes in the M9 with "osteoblast differentiation" GO annotations in the 15 HMDP strains that displayed the lowest and highest Bicc1 expression.…”

“…A full description of the network used to characterize Bicc1 in this study is provided in ref. 35. To generate the coexpression network, we first calculated Pearson's correlation coefficients for all gene-gene comparisons across all microarray samples.…”

“…The details of isolation and culture of primary calvarial osteoblast, siRNA transfection, qPCR, and osteoblast differentiation assays are described in ref. 35. Briefly, primary calvarial osteoblasts were isolated from 3-to 9-day-old neonates using sequential collagenase P digestions.…”

Multifarious effects of 17-β-estradiol on apolipoprotein E receptors gene expression during osteoblast differentiation <i>in vitro </i>

“…Many independent studies of gene expression patterns during osteoblast differentiation have been described (10,33,44,45), in which apoE was the only apolipoprotein strongly induced during this process (10,33). ApoE regulated bone metabolism in mice is possible due to uptake of lipid and lipid soluble vitamins such as vitamin K into osteoblasts (10).…”

17-β-estradiol up-regulates apolipoprotein genes expression during osteoblast differentiation <i>in vitro </i>

“…This process of differentiation has been subdivided into three developmental stages: proliferation, extracellular matrix synthesis and maturation, and mineralization, each of which as characteristic changes in gene expression (8). Many independent studies of patterns of gene expression during osteoblast differentiation have been reported (9)(10)(11), and those studies found that some apolipoproteins were induced during this process (9).…”