Stimulatory G protein directly regulates hypertrophic differentiation of growth plate cartilage in vivo

Abstract

Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The alpha subunit of Gs (Gsalpha) is encoded by GNAS (Gnas in mice), and heterozygous Gsalpha inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gsalpha in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 (Gnas(E2-/E2-)) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice (Gnas(+/E2-)). We generated chimeric mice containing wild-type cells and either Gnas(E2-/E2-) or Gnas(+/E2-) cells. Gnas(E2-/E2-) chondrocytes phenocopied PTH/PTHrP receptor (PPR)(-/-) cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gsalpha, one of several gene products that include Gnas exon 2, into Gnas(E2-/E2-) cells prevented premature hypertrophy. Gsalpha mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal Gnas(+/E2-) growth plate chondrocytes, indicating biallelic expression of Gsalpha in these cells. Hypertrophy of Gnas(+/E2-) chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and Gnas(+/E2-) cells. These data suggest that Gsalpha is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gsalpha signaling in Gnas(+/E2-) chondrocytes.

Fig. 1.

Isolation of Gnas-mutant ES cells and generation of chimeras. (A) Schematic diagram of the Gnas locus. S, translation start site; X, termination codon. 1, 2, and 3–12 denote the number of the exons. Exons of the antisense and the 1A transcripts are not shown. (B Left) Southern blot analysis of isolated ES cells for the mutation in Gnas exon 2. Wt, wild-type; Het, heterozygous mutant; Hom, homozygous mutant. (B Right) The restriction map of the Gnas locus. XmnI denotes the restriction site by XmnI endonuclease.

Fig. 2.

Gnas^E2–/E2– cells phenocopy PPR^–/– cells in cartilage. (A) In situ hybridization for type X collagen mRNA of tibial sections from E17.5 wild-type, PPR^–/–/wild-type chimera, and Gnas^E2–/E2–/wild-type chimera embryos. pp, Periarticular proliferating chondrocytes; cp, columnar proliferating chondrocytes; ph/h, prehypertrophic and hypertrophic chondrocytes. (Scale bars, 100 μm.) (B) Staining for β-galactosidase activity plus H&E staining of sections from E17.5 Gnas^E2–/E2–/wild-type chimeric embryo. Wild-type cells were stained blue because of the presence of the β-galactosidase transgene. Arrowheads indicate ectopic hypertrophic chondrocytes. (Scale bar, 100 μm.) (C) In situ hybridization for Indian hedgehog (Ihh), Patched 1 (Ptc1), osteopontin (OP), PTHrP, and PPR mRNAs of tibial sections from E17.5 wild-type and Gnas^E2–/E2–/wild-type chimera embryos. (Scale bar, 100 μm.)

Fig. 3.

Abnormal hypertrophy of Gnas^+/E2– chondrocytes. (A) H&E staining of the radii from E17.5 wild-type/wild-type (Left), Gnas^matE2–/+/wild-type (Center), and Gnas^+/patE2–/wild-type (Right) chimeric embryos. Cells derived from host blastocysts were stained blue with 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal). The earliest hypertrophic chondrocytes (detected by their characteristic morphology) derived from host blastocysts and ES cells were marked with blue and white arrowheads, respectively. (Scale bar, 100 μm.) (B) In situ hybridization for Gsα mRNA and XLαs mRNA of tibial sections from E17.5 wild-type embryos. Upper panels, bright fields; lower panels, dark fields. pp, Periarticular proliferating chondrocytes; cp, columnar proliferating chondrocytes; ph/h, prehypertrophic and hypertrophic chondrocytes. (Scale bar, 100 μm.) (C) Real-time RT-PCR analysis of Gsα and XLαs mRNAs expressed in limb chondrocytes isolated from wild-type (Wt), Gnas^+/patE2– (P–), or Gnas^matE2–/+ (M–) newborn mice.

Fig. 4.

Rescue of Gs signaling in Gnas^E2–/E2– cells by rat Gsα. (A) Integration of pCMV-rGsα transgene was detected by PCR of genomic DNA for rat Gsα (Top), and expression of the transgene was detected by Western blot analysis for HA tag (Middle), in ES cell lines differentiated into fibroblast-like cells. (B) cAMP response of various differentiated ES cell lines to PTH. Absolute basal and stimulated cAMP levels are shown on the left, and fold-stimulation is shown on the right. Clones 21, 6, 12, and 44 were Gnas^E2–/E2– ES cell lines stably transfected with pCMV-rGsα. (C) Comparison of expression levels of Gsα and endogenous mouse Gsα proteins detected by Western blot for Gsα. Arrowhead indicates the longer variant.

Fig. 5.

Rescue of Gnas^E2–/E2– chondrocytes by rat Gsα. Shown is staining for β-galactosidase activity plus H&E staining of a section of the radii from an E17.5 Gnas^E2–/E2–; pCMV-rGsα/wild-type chimeric embryo. Cells derived from host blastocysts were stained blue with 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal). The earliest hypertrophic chondrocytes (detected by their characteristic morphology) derived from host blastocysts and ES cells were marked with blue and white arrowheads, respectively. The lower portions of the panels for clones 21 and 44 show in situ hybridization for type X collagen mRNA in serial sections. (Scale bar, 100 μm.)