Abstract
Polycystin-1 (PC1) may play an important role in skeletogenesis through regulation of the bone-specific transcription factor Runx2-II. In the current study we found that PC1 co-localizes with the calcium channel polycystin-2 (PC2) in primary cilia of MC3T3-E1 osteoblasts. To establish the role of Runx2-II in mediating PC1 effects on bone, we crossed heterozygous Pkd1(m1Bei) and Runx2-II mice to create double heterozygous mice (Pkd1(+/m1Bei)/Runx2-II(+/-)) deficient in both PC1 and Runx2-II. Pkd1(+/m1Bei)/Runx2-II(+/-) mice exhibited additive reductions in Runx2-II expression that was associated with impaired endochondral bone development, defective osteoblast-mediated bone formation, and osteopenia. In addition, we found that basal intracellular calcium levels were reduced in homozygous Pkd1(m1Bei) osteoblasts. In contrast, overexpression of a PC1 C-tail construct increased intracellular calcium and selectively stimulated Runx2-II P1 promoter activity in osteoblasts through a calcium-dependent mechanism. Site-directed mutagenesis of critical amino acids in the coiled-coil domain of PC1 required for coupling to PC2 abolished PC1-mediated Runx2-II P1 promoter activity. Additional promoter analysis mapped the PC1-responsive region to the "osteoblast-specific" enhancer element between -420 and -350 bp that contains NFI and AP-1 binding sites. Chromatin immunoprecipitation assays confirmed the calcium-dependent binding of NFI to this region. These findings indicate that PC1 regulates osteoblast function through intracellular calcium-dependent control of Runx2-II expression. The overall function of the primary cilium-polycystin complex may be to sense and transduce environmental clues into signals regulating osteoblast differentiation and bone development.
Highlights
Ulates the differentiation of mesenchymal precursors into osteoblasts and hypertrophic chondrocytes (4 –10)
Consistent with Pkd1 regulation of Runx2-II expression, we observed further reductions of Runx2-II expression in combined Pkd1ϩ/m1Bei and Runx2IIϩ/Ϫ mice to levels that were 50% that observed in wild-type mice (Fig. 1)
These changes were selective for P1-Runx2-II, since we observed no differences in P2-Runx2-type I (Runx2-I) isoform expression in Pkd1-deficient mice (Fig. 1)
Summary
Mice—Pkd1m1Bei heterozygous mice, which have an inactivated point mutation in the Pkd gene leading to the substitution of an arginine for methionine in the first transmembrane domain [28], were obtained from the Mutant Mouse Regional Resource Center (University of North Carolina, Chapel Hill, NC). The selective Runx2-II-deficient mice were generated in our laboratory as previously described [11]. We used a PCR strategy to generate previously described amino acid substitutions at sites 148, 149, and 155 of PC1-AT to create a mutant construct (PC1-ATmutant) that was unable to couple to PC2 [31]. Real-time RT-PCR—For quantitative real-time RT-PCR, 2.0 g of total RNA isolated from newborn mice and the long bone of 6-week-old mice were reverse-transcribed as previously described [15]. Quantitative Chromatin Immunoprecipitation (ChIP) Analyses—The ChIP analyses were performed using the ChIP-IT kit (Active Motif, Carlsbad CA) and modifications of previously described methods [34]. Primers amplifying sequences in exon 4 of Runx were used to normalize for DNA content and to calculate the relative ratio of the P1 promoter sequences over control sequences. All computations were performed using the Statgraphic statistical graphics system (STSC Inc.)
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