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

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.

Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site.

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D3 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D3) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys71-->Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at -171/-163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5'-TGTCGGTCA-3', is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.