Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh - PubMed (original) (raw)
Chondrocytic Atf4 regulates osteoblast differentiation and function via Ihh
Weiguang Wang et al. Development. 2012 Feb.
Abstract
Atf4 is a leucine zipper-containing transcription factor that activates osteocalcin (Ocn) in osteoblasts and indian hedgehog (Ihh) in chondrocytes. The relative contribution of Atf4 in chondrocytes and osteoblasts to the regulation of skeletal development and bone formation is poorly understood. Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages. Unexpectedly, this genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect. Together, these data indicate that Atf4 in chondrocytes is required for normal Ihh expression and for its paracrine effect on osteoblast differentiation. Therefore, the cell-autonomous role of Atf4 in chondrocytes dominates the role of Atf4 in osteoblasts during development for the control of early osteogenesis and skeletal growth.
Figures
Fig. 1.
Generation of Col2a1-Atf4 transgenic (Tg) mice. (A) Schematic of the Col2a1-Atf4 transgenic construct. Restriction sites, primers used for genotyping, and cDNA probe covering the coding region of the Atf4 gene are indicated. E, exon; I, intron. (B) Southern hybridization of tail genomic DNA identified five founders (2, 3, 5, 9 and 11). (C) qRT-PCR of tail RNA revealed two transgenic lines (Tg#2 and Tg#9) overexpressing Atf4. Data were normalized to endogenous Atf4 level in WT mice and 18S rRNA (_n_=3). Error bars represent s.e.m. *P<0.05 by paired Student’s _t_-test. (D) qRT-PCR of cartilage and bone RNA demonstrated that Atf4 is specifically overexpressed in cartilage but not in bone in Col2a1-Atf4 (Tg) and _Atf4_–/–;Col2a1-Atf4 (–/–;Tg, #9) mice. (E) In situ hybridization of E15 sections of humeri showing Atf4 expression pattern. Note that Atf4 expression is ubiquitous in WT (a) but absent in _Atf4_–/– (b) limb. The Col2a1-Atf4 expression in Col2a1-Atf4 (Tg, c) and _Atf4_–/–;Col2a1-Atf4 (–/–;Tg, d) limbs is restricted to chondrocytes and at a higher level than that in WT limbs. (e) Higher magnification of the ossification center region of E15 _Atf4_–/–;Col2a1-Atf4 humeral sections showing the absence of Col11-Atf4 expression in osteoblasts. _n_=3. Scale bars: 0.2 mm.
Fig. 2.
Overexpression of Atf4 in chondrocytes rescues shortened statures in _Atf4_–/– mutant mice. (A) Alizarin Red and Alcian Blue staining of skeletons of embryos and newborn pups at the indicated developmental stages. Scale bars: 0.5 mm. _n_=3. (B) Quantification of humeral length in embryos and pups of indicated genotypes at indicated developmental stages. Error bars represent s.e.m. _n_=3. *P<0.05 by paired Student’s _t_-test. Tg, Col2a1-Atf4; –/–;Tg, _Atf4_–/–;Col2a1-Atf4.
Fig. 3.
Overexpression of Atf4 in chondrocytes restores growth plate chondrocyte defects in _Atf4_–/– mice. (A) H&E staining of humeral sections of indicated genotypes with proliferative chondrocyte zones (PZ) and hypertrophic chondrocyte zones (HZ) of growth plate chondrocytes are indicated. Scale bars: 0.1 mm. (B,C) Quantification of the length of the PZ and HZ in growth plates of indicated genotypes. Error bars represent s.e.m. _n_=3. *P<0.05 by paired Student’s _t_-test. (D) BrdU immunohistochemistry of humeral sections showing BrdU-positive (brown) proliferating growth plate chondrocytes at indicated developmental stages. Scale bars: 0.05 mm. (E,F) Quantification of the BrdU immunohistochemistry results shown in D. Error bars represent s.e.m. _n_=3. *P<0.05 by paired Student’s _t_-test. Tg, Col2a1-Atf4; –/–;Tg, _Atf4_–/–;Col2a1-Atf4.
Fig. 4.
Overexpression of Atf4 in chondrocytes corrects the reduced expression of Ihh and its target genes in _Atf4_–/– mice. (Aa-x) In situ hybridization of E15 humeral sections. Note that the decrease in Ihh, Ptch1 and Gli1 expression in _Atf4_–/– humeri was completely rescued in _Atf4_–/–;Col2a1-Atf4 humeri, whereas the expression levels of Col2a1, Col10a1 and PPR were not affected. _n_=3. Scale bars: 0.5 mm. (B-E) qRT-PCR analysis showing expression levels of Ihh, Ptch1 Gli1 and PPR in P0 cartilages (black bars) and bones (gray bars). Error bars represent s.e.m. _N_=3. *P<0.05 by paired Student’s _t_-test. Tg, Col2a1-Atf4; –/–;Tg, _Atf4_–/–;Col2a1-Atf4.
Fig. 5.
Overexpression of Atf4 in chondrocytes restores osteoblast differentiation and bone formation in _Atf4_–/– mice. (Aa-l) In situ hybridization of sections through E15 humeri showing that the decreased level of Ocn and Bsp in _Atf4_–/– bones was restored to normal level in _Atf4_–/–;Col2a1-Atf4 bones. von Kossa staining of sections through E15 humeri showing rescued mineralization in _Atf4_–/–;Col2a1-Atf4 primary ossification centers. Scale bars: 0.5 mm. (B,C) qRT-PCR analysis of P0 bones quantifying Ocn (B) and Bsp (C) expression. Error bars represent s.e.m. _n_=3. *P<0.05 by paired Student’s _t_-test. (D) Microtomographic image showing rescued bone formation in 1- (1 mon) and 3-month (3 mons) _Atf4_–/–;Col2a1-Atf4 femur heads. _n_=6. Scale bars: 1 mm. (E,F) Quantification of 1- and 3-month femur bone volume/tissue volume (BV/TV) measured by μCT. Error bars represent s.e.m. _N_=6. *P<0.05 by paired Student’s _t_-test. Tg, Col2a1-Atf4; –/–;Tg, _Atf4_–/–;Col2a1-Atf4.
Fig. 6.
Cartilage conditioned media contain osteogenic activities. (A,B) Quantification of CFU-ap (A) and CFU-ob (B) formed in BMSC cultures under osteoblast differentiation condition. Note that _Atf4_–/– BMSC failed to properly differentiate into mature osteoblasts, which can be partially rescued by Atf4_–/– cartilage CM and completely rescued by CM of WT and Atf4_–/–;Col2a1-Atf4 cartilages. Error bars represent s.e.m. _n_=3. *P<0.05 by paired Student’s _t_-test. (C,D) Quantification of CFU-ap (C) and CFU-ob (D) formed in BMSC cultures in osteoblast differentiation assays. Note that purmorphamine (Pur.) increases CFU-ap and CFU-ob formed by _Atf4_–/– BMSCs supplemented with _Atf4_–/– cartilage CM and cyclopamine (Cyc.) and 5E1, but not anti-PTHrP, decrease the CFU-ap and CFU-ob formed by _Atf4_–/– BMSCs supplemented with _Atf4_–/–;Col2a1-Atf4 cartilage CM. Error bars represent s.e.m. _N_=3. *P<0.05 by paired Student’s t_-test. Tg, Col2a1-Atf4; –/–;Tg, Atf4_–/–;Col2a1-Atf4.
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