Blocking circadian clock factor Rev-erbα inhibits growth plate chondrogenesis via up-regulating MAPK-ERK1/2 pathway

Abstract

Emerging evidence indicated circadian clock gene Rev-erbα was involved in cartilage metabolism, however the contribution of Rev-erbα to growth plate chondrogenesis remains unknown. Here, we found that Rev-erbα exhibited the spatiotemporal expression model in growth plate. Moreover, Rev-erbα antagonist SR8278 inhibited longitudinal elongation of metatarsal bone ex vivo. And morphological analysis exhibited SR8278 led to the reduced height of growth plate and hypertrophic zone. Furthermore, blocking Rev-erbα suppressed the proliferation and hypertrophic differentiation of chondrocytes in growth plate. Similarly, knock-down Rev-erbα inhibited the proliferation and differentiation of primary chondrocytes in vitro. The mechanistic study indicated that knock-down Rev-erbα up-regulated MAPK-ERK1/2 pathway in chondrocytes. However, restraint of MAPK-ERK1/2 pathway alleviated partially SR8278-inhibited longitudinal elongation of metatarsal bone and growth plate development. Therefore, our results provide evidence of the vital role of Rev-erbα on growth plate chondrogenesis.

Keywords: MAPK-ERK1/2; Rev-erbα; chondrogenesis; growth plate.

Figure 1.

The expression model of Rev-erbα in growth plate chondrocyte. (a) Immunofluorescence analysis of Rev-erbα in growth plate from 1, 2 and 4-week-old mice. Red boxed areas show higher magnification. Scale bars:100 μm. RZ: Resting zone, PZ: Proliferative zone, PHZ: Prehypertrophic zone, HZ: hypertrophic zone. (b) RT-qPCR analysis for the expression of Rev-erbα during primary chondrocyte differentiation (n = 3). Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 2.

Rev-erbα antagonist SR8278 inhibited longitudinal elongation of metatarsal bone. (a,b) Metatarsal bone was extracted from 0 day-old mice and cultured in serum-free MEM medium containing graded concentrations of Rev-erbα antagonist SR8278 (0–60 μM). Bone length was measured at the beginning and at the end of the 3-day culture using an eyepiece micrometer in a dissecting microscope (n = 10). (c) Toluidine blue staining of control and SR8278 metatarsal bone. Scale bars:100 μm. (d) The height of growth plate, resting zone, proliferative zone and hypertrophic zone from control and SR8278 metatarsal bone (n = 8). GP: Growth plate, RZ: Resting zone, PZ: Proliferative zone, HZ: hypertrophic zone. Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 3.

SR8278 resulted in inhibited proliferation and accumulation of chondrocyte in growth plate. After 3-day culture, Brdu was incorporated in the culture medium for 4 hours. (a) Immunofluorescence analysis of Brdu in growth plate from control and SR8278 metatarsal bone. Scale bars:100 μm. RZ: Resting zone, PZ: proliferative zone. (b) Quantification of Brdu-positive cells (green) in resting zone and proliferative zone of growth plate (n = 6). (c) Quantification of DAPI-positive cells (blue) in resting zone and proliferative zone of growth plate (n = 6). Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 4.

The effect of SR8278 on chondrocyte differentiation, apoptosis and mineralization in growth plate. (a) Immunohistochemistry analysis of Collagen X in hypertrophic zone of growth plate from control and SR8278 metatarsal bone. Scale bars:50 μm. (b). RNA was extracted from control and SR8278 metatarsal bone. And RT-qPCR analysis for the mRNA expression of Col2a1, Col10a1, Runx2, Sox9, Adamts5 and Mmp13 in control and SR8278 metatarsal bone (n = 3). (c) Immunofluorescence analysis of Tunel in growth plate from control and SR8278 metatarsal bone. Scale bars:200 μm. (d) Quantification of Tunel -positive cells (red) in growth plate from control and SR8278 metatarsal bone (n = 6). (e) The height of ossification center in the growth plate from control and SR8278 metatarsal bone (n = 8). (f) Von kossa for mineralization of ossification center in the growth plate from control and SR8278 metatarsal bone. Scale bars:100 μm. Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 5.

Knock-down Rev-erbα inhibited proliferation and differentiation of chondrocyte. (a) Primary chondrocyte was extracted from rib cartilage of <3-day-old mice. RT-qPCR analysis for the mRNA expression of Rev-erbα in primary chondrocyte infected with lentivirus-expressing control or shRNA Rev-erbα (n = 3). (b) RT-qPCR analysis for the mRNA expression of Col2a1, Col10a1, Runx2, Sox9, Adamts5 and Mmp13 in control and Rev-erbα shRNA chondrocyte (n = 3). (c) Western blot for the expression of Collagen II, Collagen X and MMP13 in control and Rev-erbα shRNA chondrocyte. Quantification is shown on the right using ImageJ software (n = 4). (d) Chondrocyte micro-masses were cultured in DMEM with 10% FBS for 6 days and were performed to alcian blue staining. The representative images of alcian blue of control and Rev-erbα shRNA micro-masses. (e) Edu was incorporated in the culture medium for 4 hours. Immunofluorescence analysis of Edu in control and Rev-erbα shRNA chondrocyte. Scale bars:100 μm. (f) Quantification of Edu-positive cells (red) in control and Rev-erbα shRNA chondrocyte (n = 6). Scale bars:100 μm. Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 6.

Knock-down Rev-erbα up-regulated MAPK-ERK1/2 signaling pathway. (a) Heatmap of RNA-seq analysis for differentially expressed genes between control and Rev-erbα shRNA chondrocyte. (b) Bar plot showing the top 10 enriched KEGG pathways in the differentially expressed genes between control and Rev-erbα shRNA chondrocyte. (c) Western blot for the protein expression of p-ERK1/2, ERK1/2, p-p38 and p38 in control and Rev-erbα shRNA chondrocyte. Quantification is shown on the right using ImageJ software (n = 4). (d) Immunohistochemistry analysis of p-ERK1/2 in hypertrophic zone of growth plate from control and SR8278 metatarsal bone. Green boxed areas show higher magnification in hypertrophic zone(HZ). Scale bars:100 μm. Data are presented as mean ± SD (Student t test; *P < 0.05, **P < 0.01).

Figure 7.

Restraint of MAPK-ERK1/2 abrogated SR8278-inhibited longitudinal elongation of metatarsal bone. Metatarsal bone was extracted from 0 day-old mice and cultured in serum-free MEM medium containing SR8278 or SCH772984 (SCH). (a,b) Bone length was measured at the beginning and at the end of the 3-day culture using an eyepiece micrometer in a dissecting microscope (n = 8). (c) Toluidine blue staining of control, SR8278 and SR8278+ SCH metatarsal bone. Scale bars:100 μm. (d) The height of resting zone, proliferative zone and hypertrophic zone from control, SR8278 and SR8278+ SCH metatarsal bone (n = 6). RZ: Resting zone, PZ: Proliferative zone, HZ: Hypertrophic zone. Data are presented as mean ± SD (One-way ANOVA; VS. control, *P < 0.05, **P < 0.01, VS. SR8278, ^#P < 0.05, ^##P < 0.01).