Heterozygous inactivation of Gnas in adipose-derived mesenchymal progenitor cells enhances osteoblast differentiation and promotes heterotopic ossification

Abstract

Human genetic disorders sharing the common feature of subcutaneous heterotopic ossification (HO) are caused by heterozygous inactivating mutations in GNAS, a gene encoding multiple transcripts including two stimulatory G proteins, the α subunit of the stimulatory G protein (G(s)α) of adenylyl cyclase, and the extralong form of G(s)α, XLαs. In one such disorder, progressive osseous heteroplasia (POH), bone formation initiates within subcutaneous fat before progressing to deeper tissues, suggesting that osteogenesis may involve abnormal differentiation of mesenchymal precursors that are present in adipose tissues. We determined by immunohistochemical analysis that GNAS protein expression is limited to G(s)α in bone-lining cells and to G(s)α and XLαs in osteocytes. By contrast, the GNAS proteins G(s)α, XLαs, and NESP55 are detected in adipocytes and in adipose stroma. Although Gnas transcripts, as assessed by quantitative RT-PCR, show no significant changes on osteoblast differentiation of bone-derived precursor cells, the abundance of these transcripts is enhanced by osteoblast differentiation of adipose-derived mesenchymal progenitors. Using a mouse knockout model, we determined that heterozygous inactivation of Gnas (by disruption of the G(s)α-specific exon 1) abrogates upregulation of multiple Gnas transcripts that normally occurs with osteoblast differentiation in wild-type adipose stromal cells. These transcriptional changes in Gnas(+/-) mice are accompanied by accelerated osteoblast differentiation of adipose stromal cells in vitro. In vivo, altered osteoblast differentiation in Gnas(+/-) mice manifests as subcutaneous HO by an intramembranous process. Taken together, these data suggest that Gnas is a key regulator of fate decisions in adipose-derived mesenchymal progenitor cells, specifically those which are involved in bone formation.

Figure 1

Detection of Gnas protein products (Gsα, XLαs, Nesp) in bone and fat tissue from wild-type mice by immunohistochemical analysis. Arrowheads, bone-lining cells; arrows, osteocytes.

Figure 2

Expression of Gnas transcripts in (A) bone marrow stromal cells (BMSC) and in (B) adipocyte soft tissue stromal cells (STSC). Specific Gnas transcripts were quantified by qRT-PCR after 14 days in culture in the presence or absence of osteogenic differentiation factors. Relative expression of each transcript was normalized to day 1 levels. Ob, osteoblast.

Figure 2

Expression of Gnas transcripts in (A) bone marrow stromal cells (BMSC) and in (B) adipocyte soft tissue stromal cells (STSC). Specific Gnas transcripts were quantified by qRT-PCR after 14 days in culture in the presence or absence of osteogenic differentiation factors. Relative expression of each transcript was normalized to day 1 levels. Ob, osteoblast.

Figure 3

Accelerated osteoblast differentiation of adipocyte soft tissue stromal cells (STSC) from Gnas^+/− mice compared to wild-type (WT) animals. (A) Mineralization of wild-type and Gnas^+/− STSC cultures by Alizarin Red S staining on day 20. (B) Quantification of mineralization at days 9, 17, and 20 during osteoblast differentiation in WT and Gnas^+/− STSC cultures. *, statistically significant (p < 0.05)

Figure 4

Expression of osteogenic markers during differentiation of STSCs derived from wild-type and Gnas^+/− mice. Expression of (A) alkaline phosphatase (ALP), (B)osteopontin (OP), and (C) osteocalcin (OC) mRNAs were quantified by RT-PCR. Transcript levels were normalized to GAPDH mRNA. Relative expression of the indicated transcripts was compared between genotypes. *, statistically significant (p < 0.05)

Figure 5

Gnas^+/− mice develop subcutaneous heterotopic ossification. (A) X-ray showing subcutaneous ossification in a 12 month old Gnas^+/− male mouse. (B) Area of interest from (A) at higher magnification. White arrowheads indicate sites of heterotopic ossification. (C) Hematoxylin & Eosin staining of a histological section showing intramembranous ossification. Note the appearance of heterotopic bone in the context of adjacent adipocytes. (D) Onset of heterotopic ossification (HO) with age. Male Gnas^+/− mice were euthanized and imaged at the ages indicated (3–24 months). Numbers of unaffected animals are indicated by grey bars and mice with HO by black bars. By 12 months of age, all Gnas^+/− mice demonstrate HO. B, bone, F, fat.

Figure 6

Possible mechanisms of heterotopic bone formation regulated by GNAS/Gnas. In tissues where bone doses not ordinarily form, Gnas may have a role in preventing extra-skeletal ossification in wild-type animals. Reduction in Gsα (and cAMP signaling) could promote superficial bone formation as seen in Gnas^+/− mice. In POH, where progressive heterotopic ossification occurs, a further deficit in cAMP signaling may be required, perhaps by inactivation of XLαs.