Altered Bone Development and an Increase in FGF-23 Expression in <em>Enpp1-</em>/- Mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PP(i)), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1(-/-)) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1(-/-) mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1(-/-) mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1(-/-) mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1(-/-) tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1(-/-) null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1(-/-) mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1(-/-) mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1(-/-) mice compared to controls. These results indicate that Enpp1(-/-) mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization

Conditional mesenchymal disruption of pkd1 results in osteopenia and polycystic kidney disease.

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1(flox/flox) and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1(flox/flox) and Pkd1(flox/+), heterozygous Col1a1(3.6)-Cre;Pkd1(flox/+) and Pkd1(flox/null), and homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) and Col1a1(3.6)-Cre;Pkd1(flox/null) mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1(flox/null) mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1(flox/+) and Col1a1(3.6)-Cre;Pkd1(flox/flox) mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1(flox/flox) mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1(flox/flox) mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1(flox/flox) mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation

Osteocyte-specific deletion of Fgfr1 suppresses FGF23.

Increases in fibroblastic growth factor 23 (FGF23 or Fgf23) production by osteocytes result in hypophosphatemia and rickets in the Hyp mouse homologue of X-linked hypophosphatemia (XLH). Fibroblastic growth factor (FGF) signaling has been implicated in the pathogenesis of Hyp. Here, we conditionally deleted FGF receptor 1 (FGFR1 or Fgfr1) in osteocytes of Hyp mice to investigate the role of autocrine/paracrine FGFR signaling in regulating FGF23 production by osteocytes. Crossing dentin matrix protein 1 (Dmp1)-Cre;Fgfr1null/+ mice with female Hyp;Fgfr1flox/flox mice created Hyp and Fgfr1 (Fgfr1Dmp1-cKO)-null mice (Hyp;Fgfr1Dmp1-cKO) with a 70% decrease in bone Fgfr1 transcripts. Fgfr1Dmp1-cKO-null mice exhibited a 50% reduction in FGF23 expression in bone and 3-fold reduction in serum FGF23 concentrations, as well as reductions in sclerostin (Sost), phosphate regulating endopeptidase on X chromosome (PHEX or Phex), matrix extracellular phosphoglycoprotein (Mepe), and Dmp1 transcripts, but had no demonstrable alterations in phosphate or vitamin D homeostasis or skeletal morphology. Hyp mice had hypophosphatemia, reductions in 1,25(OH)2D levels, rickets/osteomalacia and elevated FGF2 expression in bone. Compared to Hyp mice, compound Hyp;Fgfr1Dmp1-cKO-null mice had significant improvement in rickets and osteomalacia in association with a decrease in serum FGF23 (3607 to 1099 pg/ml), an increase in serum phosphate (6.0 mg/dl to 9.3 mg/dl) and 1,25(OH)2D (121±23 to 192±34 pg/ml) levels, but only a 30% reduction in bone FGF23 mRNA expression. FGF23 promoter activity in osteoblasts was stimulated by FGFR1 activation and inhibited by overexpression of a dominant negative FGFR1(TK-), PLCγ and MAPK inhibitors. FGF2 also stimulated the translation of an FGF23 cDNA transfected into osteoblasts via a FGFR1 and PI3K/Akt-dependent mechanism. Thus, activation of autocrine/paracrine FGF pathways is involved in the pathogenesis of Hyp through FGFR1-dependent regulation of FGF23 by both transcriptional and post-transcriptional mechanisms. This may serve to link local bone metabolism with systemic phosphate and vitamin D homeostasis

Genetic interactions between polycystin-1 and Wwtr1 in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice

Abstract Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and Wwtr1 have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1 flox/+;Wwtr1 flox/+, Pkd1 Oc-cKO , Wwtr1 Oc-cKO , and Pkd1/Wwtr1 Oc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and Wwtr1 in bone in vivo, Pkd1/Wwtr1 Oc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either Wwtr1 Oc-cKO or Pkd1 Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in Pkd1/Wwtr1 Oc-cKO mice compared to either Pkd1 Oc-cKO or Wwtr1 Oc-cKO mice. Pkd1/Wwtr1 Oc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to Pkd1 Oc-cKO or Wwtr1 Oc-cKO mice. Moreover, we found that Pkd1/Wwtr1 Oc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic, MS2 that activates the polycystin complex resulted in marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, Pkd1/Wwtr1 Oc-cKO mice were resistant to the anabolic effects of MS2. These findings suggest that PC1 and Wwtr1 form an anabolic mechanotransduction signaling complex that mediates mechanical loading responses and serves as a potential novel therapeutic target for treating osteoporosis

Effects of <i>Col1a1(3.6)</i>-Cre-mediated conditional deletion of <i>Pkd1</i> on osteoblastic proliferation and maturation, as well as gene expression profiles <i>ex vivo</i>.

<p>(A) Total <i>Pkd1</i> transcripts by real-time RT-PCR. All <i>Pkd1</i> transcripts were dose-dependently reduced in primary osteoblast cultures from heterozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/+ and homozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice. (B) BrdU incorporation. A gene dose-dependent increase of BrdU incorporation was observed during 6 h of primary osteoblast culture from heterozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/+ and homozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice. (C) ALP activity. Osteoblasts from control, heterozygous, and homozygous mice displayed time-dependent increments in ALP activity during 21 days of culture, but ALP activity was gene dose- and time-dependently decreased in heterozygous and homozygous osteoblasts compared to control osteoblasts. (D) Quantification of mineralization. Alizarin Red-S was extracted with 10% cetylpyridinium chloride and quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046038#s4" target="_blank">Materials and Methods</a>. A time-dependent increment of Alizarin Red-S accumulation was observed in control, heterozygous, and homozygous osteoblasts during 21 days of culture, but the accumulation was gene dose-dependently decreased in heterozygous and homozygous osteoblasts cultures compared to control osteoblasts at day 21 of culture. (<i>E–I</i>) Gene expression profiles by real-time RT-PCR. Osteoblastic markers such as <i>Runx2</i>, <i>Akp2</i>, and <i>FGF23</i> were gene dose-dependently reduced during 18 days of osteogenic culture from heterozygous and homozygous osteoblasts. In contrast, a marked increase of adipocyte markers such as <i>PPARγ2</i> and <i>aP2</i> was observed from heterozygous and homozygous osteoblasts under the same osteogenic media when compared with control osteoblasts. Data are expressed as the mean ± SD from three independent experiments. *Significant difference from control (<i>Pkd1</i>^flox/flox); ^#significant difference from heterozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/+ mice at <i>P<</i>0.05, respectively.</p

<i>Col1a1(3.6)</i>-Cre-mediated conditional deletion of <i>Pkd1</i> results in enhanced adipogenesis in bone marrow and in bone stromal cell cultures.

<p>(A) Histology of adipocytes in decalcified tibias. Oil Red O staining (upper panel) showed that the numbers of adipocytes and fat droplets in tibia bone marrow were greater in 6-week-old <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice compared with age-matched control <i>Pkd1</i>^flox/flox mice. Osmium tetroxide (OsO4) staining by μCT analyses (lower panel) showed that adipocyte volume/marrow volume (Ad.V/Ma.V, %) and adipocyte number (Ad.N, mm⁻³) were much higher in the proximal tibia from 6-week-old <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice compared with age-matched control <i>Pkd1</i>^flox/flox mice. (B) Adipocytic differentiation in BMSC cultures. An increase of adipogenesis potential was observed in 6-week-old <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox BMSC cultures, evidenced by a significant increase of Oil Red O staining in adipogenic cultures. (C and D) Expression of adipogenic markers by real-time RT-PCR. Significantly increased levels of <i>PPARγ</i> and <i>aP2</i> mRNAs were observed in 6-week-old <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox BMSC cultures compared with control (<i>Pkd1</i>^flox/flox) cultures. Data are expressed as the mean ± SD from three independent experiments. ^# Significant difference from control (<i>Pkd1</i>^flox/flox) at <i>P<</i>0.05.</p

Effect of <i>Col1a1(3.6)</i>-Cre-mediated conditional deletion of <i>Pkd1</i> in kidney on expression of epithelia-mesenchymal-transition (EMT) and fibrosis markers.

<p>(A–C) Expression of EMT markers in polycystic kidneys of 6-week-old mice by real-time RT-PCR. A panel of EMT markers including transforming growth factor β (TGF-β), <i>snail1</i>, and <i>vimentin</i> were significantly up-regulated in polycystic kidney from homozygous <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/flox mice compare with age-matched control <i>Pkd1</i>^flox/flox mice. (D–F) Expression of fibrosis markers in polycystic kidneys of 6-week-old mice by real-time RT-PCR. A panel of fibrosis markers such as α-smooth muscle actin (α-SMA) and precollagen type I (<i>Col1a1</i>) was markedly increased in the kidney from homozygous <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/flox mice compare with age-matched control <i>Pkd1</i>^flox/flox mice. 18 S was served as an internal control for gene expressions. There were no differences in EMT and fibrosis markers between heterozygous and control <i>Pkd1</i>^flox/flox mice. Data represent the mean ± SD from five to six individual mice. *Significant difference from control (<i>Pkd1</i>^flox/flox); ^#significant difference from <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/+ mice at <i>P<</i>0.05, respectively.</p

<i>Col1a1(3.6)</i>-Cre-mediated conditional deletion of <i>Pkd1</i> from the floxed <i>Pkd1</i> allele (<i>Pkd1</i>^flox) in different tissues.

<p>(A) Kaplan–Meier survival curve. Heterozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/+ had normal survival identical to control (<i>Pkd1</i>^flox/flox ) mice, homozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice began to die 1 week after birth and only half of these mice survived beyond 6 weeks. (B) Body weight of <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/+ and control (<i>Pkd1</i>^flox/flox) mice were not different, but the body weight of both male and female <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/flox mice was reduced by ∼30% and ∼36% compared with the other two genotypes. (C) Genotyping PCR analysis of different tissues harvested from 6-week-old homozygous <i>Col1a1(3.6)</i>-Cre; <i>Pkd1</i>^flox/flox mice showed that both <i>Pkd1</i>^flox and <i>Pkd1</i>^Δflox alleles existed in all tested tissues including bone and nonskeletal tissues, indicating that <i>Col1a1(3.6)</i>-Cre promoter is not specific for bone. (D–E) Real-time RT-PCR analysis of total <i>Pkd1</i> transcripts in calvaria from both <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/flox and <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/null models, and in kidney and liver from <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/flox model at 6 weeks of age. Total <i>Pkd1</i> transcripts were expressed as the fold changes relative to the housekeeping gene <i>β-actin</i> subsequently normalized to control <i>Pkd1</i>^flox/flox or <i>Pkd1</i>^flox/+ mice. Data represent the mean ± SD from five or six individual mice. Values sharing the same superscript are not significantly different at <i>P</i><0.05. *Significant difference from control (<i>Pkd1</i>^flox/flox); ^#significant difference from heterozygous <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/+ mice at <i>P<</i>0.05, respectively.</p

Biochemistry analysis of serum in 6-week-old mice.

<p>Data are mean ± S.D. from 6–8 individual mice. * and # indicates significant difference from control <i>Pkd1</i>^flox/flox and <i>Col1a1(3.6)</i>-Cre;<i>Pkd1</i>^flox/+ mice at <i>p<</i>0.05, respectively. Osteocalcin is produced by osteoblasts, and TRAP is produced by osteoclasts.</p