Paternally inherited gsα mutation impairs adipogenesis and potentiates a lean phenotype in vivo

Abstract

Paternally inherited inactivating mutations of the GNAS gene have been associated with a rare and disabling genetic disorder, progressive osseous heteroplasia, in which heterotopic ossification occurs within extraskeletal soft tissues, such as skin, subcutaneous fat, and skeletal muscle. This ectopic bone formation is hypothesized to be caused by dysregulated mesenchymal progenitor cell differentiation that affects a bipotential osteogenic-adipogenic lineage cell fate switch. Interestingly, patients with paternally inherited inactivating mutations of GNAS are uniformly lean. Using a mouse model of Gsα-specific exon 1 disruption, we examined whether heterozygous inactivation of Gnas affects adipogenic differentiation of mesenchymal precursor cells from subcutaneous adipose tissues (fat pad). We found that paternally inherited Gsα inactivation (Gsα(+/p-) ) impairs adipogenic differentiation of adipose-derived stromal cells (ASCs). The Gsα(+/p-) mutation in ASCs also decreased expression of the adipogenic factors CCAAT-enhancer-binding protein (C/EBP)β, C/EBPα, peroxisome proliferator-activated receptor gamma, and adipocyte protein 2. Impaired adipocyte differentiation was rescued by an adenylyl cyclase activator, forskolin, and provided evidence that Gsα-cAMP signals are necessary in early stages of this process. Supporting a role for Gnas in adipogenesis in vivo, fat tissue weight and expression of adipogenic genes from multiple types of adipose tissues from Gsα(+/p-) mice were significantly decreased. Interestingly, the inhibition of adipogenesis by paternally inherited Gsα mutation also enhances expression of the osteogenic factors, msh homeobox 2, runt-related transcription factor 2, and osteocalcin. These data support the hypothesis that Gsα plays a critical role in regulating the balance between fat and bone determination in soft tissues, a finding that has important implications for a wide variety of disorders of osteogenesis and adipogenesis.

Figure 1

Paternally inherited Gsα mutation impairs adipogenesis in vitro. (A, B): Adipose stromal cells (ASCs) from subcutaneous fat pads from Gsα^+/p− or WT mice were cultured under adipogenic conditions for 7 days then stained with oil red O and quantified at OD₅₀₀. **, p < .01. (C): Quantitative reverse-transcriptase polymerase chain reaction analysis of adipogenic markers in ASCs from fad pads on days 1, 3, and 7 after adipogenic induction. *, p < .05. Four independent experiments used cells from three WT and three mutant mice (analyzed individually, in triplicate) for each experiment. Abbreviations: aP2, adipocyte protein 2; C/EBP, CCAAT-enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; WT, wild type.

Figure 2

Expression of Gnas transcripts during adipocyte differentiation. Quantitative reverse-transcriptase polymerase chain reaction analysis of Gnas transcripts Gsα (A), XLαs (B), and Nesp (C) in fat pad-derived adipose stromal cells from Gsα^+/p− and WT mice on days 1, 3, and 7 after induction of adipogenesis. **, p < .01 and *, p < .05. Three independent experiments used cells from three WT and three mutant mice (analyzed individually, in triplicate) for each experiment. Abbreviations: Gsα, guanine nucleotide-binding protein G(s) subunit alpha; Nesp, neuroendocrine secretory protein of mol. wt. 55,000; WT, wild-type; XLαs, guanine nucleotide-binding protein G(s) subunit alpha extralarge isoform.

Figure 3

Adenylyl cyclase activation rescues the adipogenic impairment of Gsα^+/p− adipose stromal cells (ASCs) during an early stage of adipogenesis. (A): Schematic of treatment with forskolin, an activator of adenylyl cyclase. (B): On day 10 of adipogenic treatment, ASCs were fixed, stained with oil red O, and quantified at OD₅₀₀. Di-methyl sulfoxide was added to cells without forskolin treatment as a solvent control. *, p < .05. Two independent experiments used cells from three WT and three mutant mice (analyzed individually in triplicate) for each experiment. Abbreviation: WT, wild type.

Figure 4

Inactivating Gsα mutation reduces adipogenic tissues in vivo. (A): Adipose tissues from subcutaneous fat pads, abdominal white fat, and interscapular brown fat from Gsα^+/p− (n = 3) and WT (n = 3) mice (left panels) were examined histologically with hematoxylin and eosin staining (right panels). (Supporting Information Fig. 2). (B): Expression of adipose markers was quantified using reverse-transcriptase polymerase chain reaction. *, p < .05; **, p < .01. Three independent experiments used cells from three WT and three mutant mice (analyzed individually in triplicate) for each experiment. Abbreviations: aP2, adipocyte protein 2; PPAR, peroxisome proliferator-activated receptor; UCP1, uncoupling protein 1; WT, wild type.

Figure 5

Paternally inherited Gsα mutation potentiates osteogenesis during adipogenic induction. Expression of the osteogenic markers, Msx2 (A), Runx2 (B), and Osteocalcin (C) were detected by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in adipose stromal cells from fat pads of Gsα^+/p− and WT mice grown under adipogenic differentiation conditions. Samples were analyzed by qRT-PCR on days 0, 1, 3, and 7. *, p < .05. Three independent experiments used cells from three WT and three mutant mice (analyzed individually in triplicate) for each experiment. Abbreviations: Msx2, msh homeobox 2; Runx2, runt-related transcription factor 2; WT, wild type.