Clock genes influence gene expression in growth plate and endochondral ossification in mice

Abstract

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.

FIGURE 1.

Rhythmic expression of clock genes in growth plates. A, mouse costal chondrocytes were cultured for 7 or 21 days followed by RT-PCR using primers specific for each clock gene. B, mice were bred under 12-h light and dark cycle conditions for a week followed by extraction of total RNA from rib growth plates at different times after the last light cycle initiation for real time-based RT-PCR. **, p < 0.01, significantly higher than the lowest control value for each gene.

FIGURE 2.

Suppression of chondrogenic differentiation in stable PER1 transfectants. A and B, ATDC5 cells were stably transfected with Per1 expression vectors or EV followed by determination of expression levels of Per1 by real time-based RT-PCR (A) and PER1 protein by Western blotting analysis (B). C, ATDC5 cells were stably transfected with Per1 expression vectors or EV followed by transient transfection with Per1 promoter, which contains three tandem copies of E-box element, linked to a luciferase construct in either the presence or the absence of Bmal1/Clock expression vector and subsequent incubation for 48 h. D, ATDC5-PER1 or ATDC5-EV cells were cultured for 7–21 days followed by fixation with 10% formalin and subsequent Alcian blue staining for acidic polysaccharide. E, stable transfectants were cultured for 14–21 days followed by determination of ALP activity. F, total RNA was extracted from stable transfectants cultured for 14 days followed by quantitative determination of mRNA expression for differentiation markers such as Col II and Col X with Northern blotting. G, total RNA was extracted from each stable transfectant cultured for 7–21 days followed by determination of Sox9, Runx2, and Ihh by real time-based RT-PCR analysis. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in ATDC5-EV cells. ##, p < 0.01, significantly different from the value obtained in ATDC5-EV cells with Bmal1/Clock. N. D, not detectable. H, HEK293T cells were transfected with V5-tagged Per1 expression vector in either the presence or the absence of two types of LV vectors for Per1 shRNA followed by determination of expression levels of V5-PER1 by Western blotting. I and J, chondrocytes cultured for 6 days were infected with LV-Per1 shRNA for 1 day followed by determination of Per1 (I) and Col II, Col X, Sox9, Runx2, and Ihh (J) by real time-based RT-PCR analysis 7 days after infection. **, p < 0.01, significantly different from each control value obtained in chondrocytes infected with LV-empty.

FIGURE 3.

Transactivation by clock proteins of Ihh. A, ATDC5 cells were transiently transfected with expression vectors of Bmal1 and Clock in either the presence or the absence of an expression vector of Per1 followed by determination of Ihh levels 10 days after transfection with real time-based RT-PCR. B, HEK293 cells were transiently transfected with a reporter plasmid linked to intact and mutated Ihh promoters with different lengths including −2380 to +120, −1792 to +120, and −1129 to +120 bp in either the presence or the absence of expression vectors of Per1, Per2, Cry1, and Cry2 followed by further culture for an additional 48 h and subsequent determination of luciferase activity. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in cells transfected with EV. #, p < 0.05, ##, p < 0.01, significantly different from the value obtained in cells transfected with Bmal1/Clock. C, DNA-protein complex was prepared from ATDC5 cells and treated with the anti-CLOCK antibody for ChIP assay. Typical pictures are shown in the figure, whereas similar results were invariably obtained in at least three independent determinations. D, costal chondrocytes cultured for 6 days were infected with both LV-Bmal1 and LV-Clock for 1 day followed by determination of Ihh by real time-based RT-PCR analysis 7 days after infection. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in chondrocytes infected with LV-empty.

FIGURE 4.

Phenotypes in Bmal1^−/− mice. A, tibiae were isolated from Bmal1^+/+ or Bmal1^−/− mice at 1 day of age followed by immunohistochemical analysis using an antibody against BMAL1 or CLOCK. Typical micrographic pictures are shown in the figure, whereas similar results were invariably obtained in at least three independent determinations. Scale bars, 100 μm. B, general appearance of 4-week-old male Bmal1^+/+ and Bmal1^−/− mice. C, body weight curves of male Bmal1^+/+ and Bmal1^−/− mice. D, body and bone lengths in 4-week-old male Bmal1^+/+ and Bmal1^−/− mice. E, rib growth plates were isolated from Bmal1^+/+ and Bmal1^−/− mice followed by determination of expression levels of Col II, Col X, Sox9, Runx2, and Ihh by real time-based RT-PCR. F and G, tibiae were isolated from Bmal1^+/+ and Bmal1^−/− mice at 1 day after birth followed by sectioning for Alcian blue and ALP staining (F) or in situ hybridization using cRNA probes (G) for Col II, Col X, Ihh, and Alp. H, quantitative data are shown with mRNA expression in the panel. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in Bmal1^+/+ mice. Scale bars, 100 μm.

FIGURE 5.

Suppression of chondrogenic differentiation in Bmal1^−/− mice. A, costal chondrocytes were prepared from the ribs of Bmal1^+/+, Bmal1^+/−, and Bmal1^−/− mice followed by culture for several days and subsequent determination of BMAL1 expression by Western blotting. B, costal chondrocytes were prepared from homozygous Bmal1^−/− mice followed by culture for 14 days and subsequent determination of expression levels of Per1, Per2, Cry1, and Cry2. C–F, costal chondrocytes were also cultured for 3–21 days for subsequent determination of MTT reduction (C), Alcian blue staining (D), ALP activity (E), and expression levels (F) of Col II, Col X, Ihh, Sox9, Runx2, and Pthrp. **, p < 0.05, **, p < 0.01, significantly different from each control value obtained in chondrocytes isolated from Bmal1^+/+ mice. N. D, not detectable. G–I, costal chondrocytes were prepared from heterozygous Bmal1^+/− mice followed by culture for 3–21 days and subsequent determination of Alcian blue staining (G), ALP activity (H), and expression levels (I) of Col II, Col X, Ihh, Sox9, and Runx2.

FIGURE 6.

Rhythmic expression of Ihh in rib growth plate. A and B, mice were bred under 12-h light and dark cycle conditions for a week followed by extraction of total RNA from the rib growth plates different times after the last light cycle initiation and subsequent determination of Ihh (A) and Ptch1 (B) levels with real time-based RT-PCR. **, p < 0.01, significantly higher than the lowest control value. C, rib growth plates were isolated from Bmal1^+/+ and Bmal1^−/− mice at ZT4 and ZT12 followed by determination of Per1 and Ihh levels by real time-based RT-PCR. D, costal chondrocytes were prepared from the ribs of neonatal Bmal1^+/+ mice followed by culture for 14 days and subsequent exposure to 100 μm Dex for 2 h. Cells were then subjected to determination of Ihh levels every 4 h for 48 h after Dex real time-based RT-PCR. E, prechondrogenic ATDC5 cells were exposed to 100 μm Dex for 2 h followed by determination of Ihh expression every 4 h. *, p < 0.05, **, p < 0.01, significantly different from the value obtained in samples isolated at ZT4. NS, not significant.

FIGURE 7.

Phenotypes in Bmal1_cho^−/− mice. A, several tissues including bone, cartilage, brain, intestine, kidney, liver, and lung were isolated from Bmal1_cho^−/− mice followed by extracting DNA and subsequent PCR genotyping for Δallele. B, costal chondrocytes were prepared from the ribs followed by culture for 7 days for determination of BMAL1 expression by Western blotting. C, general appearance of 4-week-old male α1(II)-collagen-Cre;Bmal1^+/+ and Bmal1_cho^−/− mice. D–F, body weight (D), body length (E), and bone length (F) in 4-week-old male Bmal1^+/+, Bmal1^fl/fl, α1(II)-collagen-Cre;Bmal1^+/+ and Bmal1_cho^−/− mice. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in Bmal1^fl/fl or α1(II)-collagen-Cre;Bmal1^+/+ mice. G, rib growth plates were isolated from Bmal1_cho^−/− and control mice grouped by the random mixture of Bmal1^+/+, Bmal1^fl/fl, and α1(II)-collagen-Cre;Bmal1^+/+ followed by determination of expression levels of Ihh by real time-based RT-PCR. *, p < 0.05, **, p < 0.01, significantly different from each control value obtained in control mice.

FIGURE 8.

Schematic representation of proposal. In the growth plate, chondrogenic differentiation would be under the oscillatory regulation by IHH expressed with circadian rhythmicity mediated by a mechanism relevant to the chondrocytic clock genes toward the diurnal and nocturnal longitudinal bone growth during skeletogenesis.