The p38α MAPK Function in Osteoprecursors Is Required for Bone Formation and Bone Homeostasis in Adult Mice

<div><p>Background</p><p>p38 MAPK activity plays an important role in several steps of the osteoblast lineage progression through activation of osteoblast-specific transcription factors and it is also essential for the acquisition of the osteoblast phenotype in early development. Although reports indicate p38 signalling plays a role in early skeletal development, its specific contributions to adult bone remodelling are still to be clarified.</p><p>Methodology/Principal Findings</p><p>We evaluated osteoblast-specific deletion of p38α to determine its significance in early skeletogenesis, as well as for bone homeostasis in adult skeleton. Early p38α deletion resulted in defective intramembranous and endochondral ossification in both calvaria and long bones. Mutant mice showed reduction of trabecular bone volume in distal femurs, associated with low trabecular thickness. In addition, knockout mice also displayed decreased femoral cortical bone volume and thickness. Deletion of p38α did not affect osteoclast function. Yet it impaired osteoblastogenesis and osteoblast maturation and activity through decreased expression of osteoblast-specific transcription factors and their targets. Furthermore, the inducible Cre system allowed us to control the onset of p38α disruption after birth by removal of doxycycline. Deletion of p38α at three or eight weeks postnatally led to significantly lower trabecular and cortical bone volume after 6 or 12 months.</p><p>Conclusions</p><p>Our data demonstrates that, in addition to early skeletogenesis, p38α is essential for osteoblasts to maintain their function in mineralized adult bone, as bone anabolism should be sustained throughout life. Moreover, our data also emphasizes that clinical development of p38 inhibitors should take into account their potential bone effects.</p></div

p38α is necessary for adult bone homeostasis.

<p>(A) Scheme of doxycyline (doxi) administration and sacrifice of the different mice groups analyzed. (B) The distal femoral epiphysis of control and knockout mice after different time of doxycycline (doxi) treatments (3 or 8 weeks) were analyzed by μCT at 30 or 60 weeks of age. Trabecular bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp.) were determined (n = 5 (KO) and 8 (FF)). (C) Cortical bone volume (BV), bone area (B.Ar), bone perimeter (B.Pm) and thickness (Ct.Th) analysis was performed in femur midshafts from these animals (n = 5–8). Data are presented as means ± SEM. (*p<0.05). Differences between values from KO and FF mice (expressed as %) are also included. (D) qRT-PCR showing gene expression of calvariae from 30-week old FF and KO mice treated with doxycycline until 3 weeks of age (Doxi 3–30). Graph shows levels of <i>p38a</i> (<i>Mapk14</i>), <i>Runx2</i>, <i>bone sialoproteïna</i> (<i>Ibsp</i>), <i>osterix</i> (<i>Osx</i>) and <i>osteocalcin</i> (<i>Oc</i>) (FF n = 4, KO n = 5) (*p<0.05; **p<0.01).</p

p38α deletion in osteoblasts affects the expression of osteogenic markers.

<p>(A) Graphs show fibroblast-colony forming units (CFU-F) (upper panel) and osteoblast-colony forming units (Ob-CFU) of mesenchymal stem cells from bone marrow of FF and KO long bones (n = 9 per genotype). Representative images are also shown. (B&C) qRT-PCR of mRNA extracted from calvariae (B) and long bones (bone-marrow flushed out tibiae and femurs) (C) of 12-week-old mice (n = 4 (KO) and 8 (FF) independent animals). (D) Protein levels of OSX and p38α were decreased in calvaria from knockout mice. Left panel: western blot of calvarial bones showing Osterix (OSX), RUNX2, p38α and alpha-TUBULIN as control. Right panel: graph depicting densitometric analysis of western blots (n = 3(KO) and 6(FF)) (*p<0.05; **p<0.01; ***p<0.001).</p

Histological analysis of bones from control and p38α knockout mice.

<p>(A) Representative images of calvarial sections stained with H&E. Right panel shows quantification of calvarial thickness (n = 5 independent animals). (B) Representative images of long bone sections stained with H&E. Quantification of trabecular area and trabecular number from tibiae sections are shown in the right panel (n = 3 (KO) and 4 (FF) independent animals). (C) Thickness and osteocyte count from cortical areas of long bone stained with toluidine blue (n = 3–6) (*p<0.05; **p<0.01; ***p<0.001).</p

p38α specific-deletion in osteoblast progenitors.

<p>(A) PCR of calvarial bones of control (FF) and knockout (KO) mice using specific primers against <i>Mapk14</i> (<i>p38a</i>) demonstrate gene deletion in KO mice after Cre-mediated recombination. (B) qRT-PCR of mRNA from calvaria or tibia displays <i>p38α</i> (MAPK14) reduced expression in knockout versus control littermates (n = 7 for KO and 13 for FF). (C) Expression of p38 isoforms in primary osteoblasts (p38α/MAPK14; p38β/MAPK11; p38γ/MAPK12; p38δ/MAPK13) (n = 6 independent animals). (D) Calvarial protein levels of p38, phospho-p38, Creb, and phospho-Creb show impaired MAPK signalling in knockout animals (n = 3 independent animals) (*p<0.05; **p<0.01; ***p<0.001).</p

p38α blockage does not affect bone resorption parameters.

<p>(A) <i>Rankl</i>, <i>Opg</i> and <i>Trap</i> mRNA levels in long bones from knockout and control mice (n = 5 (KO) and 8 (FF) independent animals). (B) The <i>Rankl/Opg</i> expression ratio was not modified. (C) N-terminal telopeptides of collagen from serum of 12-weeks-old mice do not display significant differences between control and knockout mice (n = 4 (KO) and 10 (FF) independent animals). (*p<0.05; **p<0.01). (D) TRAP staining of proximal tibiae from FF and KO mice (n = 3 per genotype) showed no differences in osteoclast parameters (N.Oc./B.S., numbers of osteoclast/Bone perimeter; Oc. S./B.S, osteoclast surface/Bone perimeter). Data is presented as means ± SEM. Image shows representative images counterstained with fast green (20× and 40× magnification).</p

p38α deletion is associated with bone developmental defects.

<p>(A) Alcian blue/alizarin red staining of P7 mice pups. Alcian blue and alizarin red stain cartilage and calcified structures respectively (FB: frontal bone; PB: parietal bone; PF: posterior fontanel) (B) Radiographic lateral projections of 8-week old mice show maxilla and mandible hypoplasia (# marks maxilla and * marks mandible).</p

<i>In vitro</i> analysis of primary cultures reproduces the gene expression disturbances seen <i>in vivo</i>.

<p>(A) Images show bone chips and isolated osteoblasts from KO animals after collagenase treatment. From left to right: 1, 2, 3 and 5 days post-digestion. Endogenous GFP expression from the Osx1-GFP::Cre transgene is lost as culture advances. (B) BrdU incorporation on primary osteoblasts cultures. (C) qRT-PCR reveals the comparative mRNA expression levels of <i>Col1a1</i>, <i>Bglap</i> (OC), <i>Osx</i> and <i>Runx2</i> in primary cultures of osteoblasts from control and knockout pups (n = 6 independent animals per genotype). (*p<0.05; **p<0.01).</p