Deletion of clock gene Bmal1 impaired the chondrocyte function due to disruption of the HIF1α-VEGF signaling pathway

Abstract

Several studies have demonstrated the core circadian rhythm gene Bmal1 could regulate the clock control genes (CCGs) expression and maintain the integrity in cartilage tissue. In addition, its abnormal expression is connected with the occurrence and development of several diseases including osteoarthritis (OA). However, the relationship between Bmal1 and cartilage development still needs to be fully elucidated. Here, we bred tamoxifen-induced cartilage-specific knockout mice to learn the effects of Bmal1 on the cartilage development and its underlying mechanisms at specific time points. We observed that Bmal1 ablated mice showed growth retardation during puberty, and the length of whole growth plate and the proliferation zone were both shorter than those in the control group. Deletion of Bmal1 significantly inhibited the chondrocytes proliferation and activated cells apoptosis in the growth plate. Meanwhile, knockout of Bmal1 attenuated the expression of VEGF and HIF1α and enhanced the level of MMP13 and Runx2 in the growth plate chondrocytes. Consistent with these findings in vivo, ablation of Bmal1 could also lead to decrease chondrocytes proliferation, the expression of HIF1α and VEGF and elevate apoptosis in cultured chondrocytes. These findings suggest that Bmal1 plays a pivotal role in cartilage development by regulating the HIF1α-VEGF signaling pathway.

Keywords: Bmal1; Circadian rhythm; HIF; VEGF; endochondral ossification; growth plate chondrocytes.

Figure 1.

Deletion of Bmal1 in the postnatal stage. (a) The genomic DNA extracted from different tissues of BMAL1CKO mice and control littermates was analyzed by PCR as described in “Materials and Methods”. (b) The total RNA was extracted from the growth plate and liver of BMAL1^flox/flox and BMAL1CKO mice at 4 weeks and the relative expression of Bmal1 mRNA was detected by PCR (n= 5/group). Results are presented as gene expression levels in all groups normalized to controls. (c) The total protein was extracted from the BMAL1^flox/flox and BMAL1CKO mice growth plate at 4 weeks and tested by Western blot (n = 3/group). A representative blot from three independent experiments was presented and quantification of protein expression of BMAL1 was shown on the right. BMAL1 protein level was normalized to GAPDH. (d) Immunohistochemistry was used to analyze the expression of BMAL1 in tibia growth plate of BMAL1^flox/flox and BMAL1CKO mice at 6 weeks. Scale bars = 50μm. Data are expressed as means ± SE in each bar graph. *p < 0.05, **p < 0.01.

Figure 2.

Cartilage-specific ablation of bmal1 disrupted molecular rhythms and several genes associated with normal cartilage development. (a) The total RNA was extracted from the growth plate of BMAL1^flox/flox and BMAL1CKO mice at different time points and the relative expression of mRNA was detected by qPCR (n= 3 for each time point and group). Relative expressions were normalized to GAPDH. (b) Western blot was performed to analyze the clock genes and cartilage-related factors expression in growth plate from the BMAL1CKO mice and the BMAL1^flox/flox littermates at 4-h intervals for 24 h and quantification of protein expression were also shown. The clock and cartilage-related protein level was normalized to GAPDH. Data are expressed as means ± SE in each bar graph. *p < 0.05.

Figure 3.

Deletion of Bmal1 in the postnatal stage affected the endochondral bone formation in BMAL1CKO mice. The ^TamCartBmal1^flox/flox mice were generated and in which Bmal1 gene was conditional ablated in growth plate chondrocytes by injection of tamoxifen intraperitoneally. (a) A schematic model depicting tamoxifen administration schedule postnatally. The blue arrows indicated the time points of tamoxifen administration at P5, P7, P9 and P11. The red arrows indicated the harvest time at P0, P14, P28 and P42. (b) Femur and tibia from BMAL1^flox/flox and BMAL1CKO mice at 4 or 6 weeks of age. (c–e) The body length, femur length and tibia length were analyzed at indicated time points and the effect of Bmal1 ablation in the postnatal stage was observed at 4-week-old and 6-week-old BMAL1CKO mice (n= 10/group). Histological examination of tibia growth plate of BMAL1^flox/flox and BMAL1CKO mice at 4weeks and 6 weeks of age (f) and the growth plate RZ, PZ and HZ heights were quantitatively analyzed, respectively (g and h) (n= 10/group). Scale bars = 100μm. (i) Percentage of the length of RZ (red), PZ (green) and HZ (blue) over the total length of tibia growth plate of the BMAL1^flox/flox and BMAL1CKO mice at 4weeks and 6 weeks of age. Data are expressed as means ± SE in each bar graph. *p < 0.05. RZ = resting zone; PZ = proliferative zone; HZ = hypertrophic zone.

Figure 4.

Bmal1 ablation affected chondrocytes proliferation, differentiation and apoptosis. Representative images of BrdU immunohistochemical analysis of tibia growth plate from BMAL1^flox/flox and BMAL1CKO mice at 4-week-old after 2 h (a) and 48 h (b) of BrdU assay. BrdU-positive cells were stained by brown color (indicated by the arrow). Scale bars = 100μm. Boxed areas on the right represent higher magnification. Scale bars = 50μm. (c) The number of BrdU positive cells quantitatively (n= 5/group). (d) Immunohistochemical analysis of Col10a expression in BMAL1^flox/flox and BMAL1CKO mice tibia tissue at 4-week and 6-week (n= 5/group). Scale bars = 100μm. (e) Col10a mRNA in growth plate cartilage of BMAL1^flox/flox and BMAL1CKO mice was tested by qPCR (n= 5/group). (f) Representative images of the TUNEL-positive cells in tibial growth plate at 4-week-old and 6-week-old BMAL1^flox/flox and BMAL1CKO mice. TUNEL positive cells were labeled red fluorescence. Scale bars = 100μm. (g) Quantitatively analyzed the number of TUNEL-positive cells, respectively (n= 5/group). (h) Western blot analysis showing the expression of Bcl-2 and Caspase-3 in tibia growth plate of BMAL1^flox/flox and BMAL1CKO mice (n= 3/group). Data are expressed as means ± SE in each bar graph. *p < 0.05.

Figure 5.

Deletion of Bmal1 Led to the alteration of VEGF, HIF1α and Several Factors Expression in the Growth Plate Cartilage. (a) VEGF immunofluorescence in longitudinal sections of BMAL1^flox/flox and BMAL1CKO mice tibias at 4-week-old and 6-week-old. VEGF, green; DNA, blue. Scale bars = 100μm. (b) Quantitatively analyzed the number of VEGF-positive cells, respectively (n= 4/group). (c) Immunohistochemical analysis of MMP13, Runx2, VEGF and HIF1α expression in BMAL1^flox/flox and BMAL1CKO mice tibia tissue at 4-week age mice (n= 5/group). Scale bars = 100μm. (d) Quantitatively analyzed the number of IHC-positive cells, respectively (n= 4/group). (e) Western blot analyzed the protein expression of Runx2, MMP13, VEGF and HIF1α in cartilage tissues harvested from 4-week-old and 6-week-old BMAL1^flox/flox and BMAL1CKO mice. (f) Relative expression of Runx2, MMP13, VEGF and HIF1α mRNA in the growth plate of BMAL1^flox/flox and BMAL1CKO mice at 4-week-old and 6-week-old was detected by qPCR (n= 5/group). Results were presented as gene expression levels in all groups normalized to controls. Data are expressed as means ± SE in each bar graph. *p < 0.05.

Figure 6.

Effect of Bmal1 ablation on chondrocyte function and HIF1α, HIF2α and VEGF Expression in vitro (a and b) BrdU labeling of primary chondrocytes derived from BMAL1^flox/flox and BMAL1CKO mice. The arrows showed the representative BrdU-positive cells. The labeled cells were also quantitatively analyzed (n= 4/group). (c) Col10a mRNA expression in cultured chondrocytes from control and BMAL1CKO mice was tested by qPCR (n= 5/group). (d and e) Deletion of Bmal1 increased the number of TUNEL-positive cells in cultured chondrocytes. The representative TUNEL positive cells were labeled by red fluorescence. The result was also quantitatively analyzed (n= 5/group). (f and g) Western blot and qPCR analysis were used to show the expression of Bmal1, HIF1α, HIF2α and VEGF in primary chondrocytes derived from BMAL1CKO and control mice under normoxia and hypoxia (n= 5/group). (h and i) Western blot and qPCR analysis showing the expression of Bmal1 and other factors in chondrocytes transfected with Bmal1 siRNA under normal oxygen and low oxygen (n= 3/group). Data are expressed as means ± SE in each bar graph. *p< 0.05. **p < 0.01.

Figure 7.

Recovery of Bmal1 expression improved the chondrocytes function. (a) For the rescue experiment, chondrocytes from BMAL1CKO mice were transfected with overexpressed Bmal1 plasmid, the expression of Bmal1, HIF1α, HIF2α and VEGF were detected by Western blot. (b) Proliferation and differentiation of the BMAL1CKO mice chondrocytes were partly recovered by transfection of overexpression plasmid for Bmal1. (c) Relative mRNA expression level of several factors for proliferation, differentiation and apoptosis in BMAL1CKO chondrocytes transfected with Bmal1 overexpression plasmid was detected by qPCR test (n= 3/group). Results were presented as gene expression levels in all groups normalized to controls. (d) Overexpression of Bmal1 decreased the number of TUNEL-positive cells in cultured BMAL1CKO mice chondrocytes. The representative TUNEL positive cells were labeled by red fluorescence (n= 3/group). Data are expressed as means ± SE in each bar graph. *p < 0.05.

Figure 8.

Schematic diagram of proposed model to explain the molecular mechanism of Bmal1 deficiency influenced the HIF1α-VEGF axis in the chondrocytes. The BMAL1/CLOCK complex could not only trigger the circadian rhythms by binding to DNA elements E-box in the promoters of target genes, for example, Cry1 and Per1 but also regulate the HIF1α activity in chondrocytes. Then, the affected HIF1α by ablation of BMAL1 in chondrocytes inhibited the downstream factor, for example, VEGF and finally induced a disordered endochondral ossification afterbirth.