Fibroblast Growth Factor Receptor 3 Gene Transcription Is Suppressed by Cyclic Adenosine 3′,5′-Monophosphate

Abstract

Signaling through fibroblast growth factor receptors (FGFRs) is critical for the development and patterning of the vertebrate skeleton. Gain-of-function alleles of fgfr2 and fgfr3 have been linked to several dominant skeletal disorders in humans, while null mutations in fgfr3 result in the overgrowth of long bones in a mouse model system. Interestingly, the expression pattern of fgfr3 in growth plate chondrocytes overlaps that of the parathyroid hormone (PTH)-related peptide (PTHrP) receptor, a signaling molecule that also regulates endochondral ossification. The coincident expression of these two receptors suggests that their signaling pathways may also interact. To gain insight into the regulatory mechanism(s) that govern the expression of the fgfr3 gene in chondrocytes, we have identified a cellspecific transcriptional regulatory element (CSRh) by measuring the activity of various promoter fragments in FGFR3-expressing (CFK2) and nonexpressing (RCJ) chondrocyte-like cell lines. Furthermore, we demonstrate that activation of PTH/PTHrP receptors, either by stimulation with PTH or through the introduction of activating mutations, represses CSRh-mediated transcriptional activity. Finally, the transcriptional repression of the CSRh element was mimicked by treatment with forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine or by overexpression of the catalytic subunit of protein kinase A. Together, these data suggest that protein kinase A activity is a critical factor that regulates fgfr3 gene expression in the proliferative or prehypertrophic compartment of the epiphyseal growth plate. Furthermore, these results provide a possible link between PTHrP signaling and fgfr3 gene expression during the process of endochondral ossification.Elaboration of the vertebrate skeleton occurs via two overlapping, yet distinct developmental pathways. Intramembranous ossification, the primary pathway for flat bone development, relies upon the direct differentiation of condensed mesenchyme into osteoblasts. These osteoblasts then secrete various matrix components until they are encapsulated by calcified bone, thus linking the rate of intramembranous bone growth to the rate of osteoblast differentiation. Alternatively, endochondral ossification, the primary pathway for formation of the axial and appendicular skeleton, differs from intramembranous ossification in that condensed mesenchymal cells (chondrocytes) elaborate a complex cartilaginous template as they progress through a series of developmental stages at the epiphyseal growth plate. The epiphyseal growth plate is organized into distinct cellular compartments: the resting zone, which serves as a renewable source of chondrocytes; the proliferative zone, where rapid cell division results in stacked columns of chondrocytes; and the hypertrophic zone, where the cells terminally differentiate, hypertrophy, and secrete a specialized matrix. After the encapsulated hypertrophic chondrocytes mature, the associated extracellular matrix is rapidly invaded by blood vessels and bone-for...

FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth

“…In vitro analysis of the Fgfr3 promoter identified a regulatory sequence that results in decreased promoter activity in response to cAMP (McEwen et al, 1999). These in vitro data suggested that the observed decrease in Pthlh expression could contribute to increased Fgfr3 expression.…”

“…Analysis of Fgfr3 promoter function in vitro shows that Fgfr3 expression could be directly regulated (suppressed) by PTHLH activation of protein kinase A (PKA) (McEwen et al, 1999). To test whether parathyroid hormone (PTH) signaling could suppress Fgfr3 expression in vivo in Osx-Cre;DCKO mice that highly overexpress Fgfr3, Osx-Cre;DCKO mice were injected intermittently (daily) with PTH [PTH(1-34) peptide] from P15 to P21, a treatment regimen known to stimulate the anabolic effects of PTH signaling on bone (Esen et al, 2015;Xie et al, 2012).…”

The Wnt-inducible Transcription Factor Twist1 Inhibits Chondrogenesis

“…Transfection efficiencies were normalized to ␤-galactosidase activity derived from a co-transfected, constitutively expressed ␤-galactosidase plasmid. Lysates were assayed as described (21).…”

Fibroblast growth factor signaling in skeletal development and disease

“…In cultured cells, PTH treatment inhibited phosphorylation of FGFR3, suggesting that PTH may function in part through regulation of FGFR3 signaling . Additionally, analysis of the Fgfr3 promoter identified a transcriptional regulatory element, CSRh, which was repressed by PTH through a cAMP and protein kinase A (PKA)-dependent mechanism (McEwen et al 1999). A third mechanism that could regulate FGFR3 is hypoxia, a characteristic feature of growth plate chondrocytes.…”