CREB activation in hypertrophic chondrocytes is involved in the skeletal overgrowth in epiphyseal chondrodysplasia Miura type caused by activating mutations of natriuretic peptide receptor B.

Abstract

Natriuretic peptide receptor B (NPRB) produces cyclic guanosine monophosphate (cGMP) when bound by C-type natriuretic peptide (CNP). Activating mutations in NPRB cause a skeletal overgrowth disorder, which has been named epiphyseal chondrodysplasia, Miura type (ECDM; OMIM #615923). Here we explored the cellular and molecular mechanisms for the skeletal overgrowth in ECDM using a mouse model in which an activating mutant NPRB is specifically expressed in chondrocytes. The mutant mice (NPRB[p.V883M]-Tg) exhibited postnatal skeletal overgrowth and increased cGMP in cartilage. Both endogenous and transgene-derived NPRB proteins were localized at the plasma membrane of hypertrophic chondrocytes. The hypertrophic zone of growth plate was thickened in NPRB[p.V883M]-Tg. An in vivo BrdU-labeling assay suggested that some of the hypertrophic chondrocytes in NPRB[p.V883M]-Tg mice continued to proliferate, although wild-type (WT) chondrocytes stopped proliferating after they became hypertrophic. In vitro cell studies revealed that NPRB activation increased the phosphorylation of cyclic AMP-responsive element binding protein (CREB) and expression of cyclin D1 in matured chondrocytes. Treatment with cell-permeable cGMP also enhanced the CREB phosphorylation. Inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A pathway had no effects on the CREB phosphorylation induced by NPRB activation. In immunostaining of the growth plates for the proliferation marker Ki67, phosphorylated CREB and cyclin D1, most signals were similarly observed in the proliferating zone in both genotypes, but some cells in the hypertrophic zone of NPRB[p.V883M]-Tg were also positively stained. These results suggest that NPRB activation evokes its signal in hypertrophic chondrocytes to induce CREB phosphorylation and make them continue to proliferate, leading to the skeletal overgrowth in ECDM.