Histone deacetylase 4 deletion results in abnormal chondrocyte hypertrophy and premature ossification from collagen type 2α1‑expressing cells

Abstract

Histone deacetylase 4 (HDAC4) plays a vital role in chondrocyte hypertrophy and bone formation. To investigate the function of HDAC4 in postnatal skeletal development, the present study developed lineage‑specific HDAC4‑knockout mice [collagen type 2α1 (Col2α1)‑Cre, HDAC4d/d mice] by crossing transgenic mice expressing Cre recombinase. Thus, a specific ablation of HDAC4 was performed in Col2α1‑expressing mice cells. The knee joints of HDAC4fl/fl and Col2α1‑Cre, HDAC4d/d mice were analyzed at postnatal day (P)2‑P21 using an in vivo bromodeoxyuridine (BrdU) assay, and Safranin O, Von Kossa and whole‑body staining were used to evaluate the developmental growth plate, hypertrophic differentiation, mineralization and skeletal mineralization patterns. The trabecular bone was analyzed using microcomputed tomography. The expressions of BrdU, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)‑13, runt‑related transcription factor (Runx)‑2, osteoprotegerin (OPG), CD34, type X collagen (ColX), osteocalcin and Wnt5a were determined using immunohistochemistry, in situ hybridization (ISH) and reverse transcription‑quantitative (RT‑q)PCR. The results demonstrated that HDAC4‑null mice (HDAC4d/d mice) were severely runted; these mice had a shortened hypertrophic zone (histopathological evaluation), accelerated vascular invasion and articular mineralization (Von Kossa staining), elevated expressions of MMP‑13, Runx2, OPG and CD34 (RT‑qPCR and immunohistochemistry), downregulated expression of the proliferative marker BrdU and PCNA (immunohistochemistry), increased expression of ColX and decreased expression of Wnt5a (ISH). In conclusion, chondrocyte‑derived HDAC4 was responsible for regulating chondrocyte proliferation and differentiation as well as endochondral bone formation.

Figure 1.

Generation of HDAC4, Col2α1-Cre mice. (A) Mating of HDAC4^fl/−, Col2α1-Cre and HDAC4^fl/fl mice. (B) Representative PCR results of genomic DNA from wild-type and HDAC4 heterozygous and homozygous null animals. (C) Images of the HDAC4^fl/fl and HDAC4 ^d/d, Col2α1-Cre offspring at postnatal days 2, 4, 6 and 8. HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down.

Figure 2.

Safranin O/Fast green staining on paraffin sections of the femur and tibia tissues at P8 and P21. (A-C) Safranin O/Fast green staining showing femur and tibia tissue sections (magnification, ×4, ×10 and ×20 respectively) at P8. The HDAC4^d/d, Col2α1-Cre mice had fewer cartilage tissues in the hypertrophy zone at P8 compared with those in the HDAC4^fl/fl mice. Black arrows represent hypertrophic chondrocytes, and orange arrows represent chondrocytes degradation. Scar bars, 100 or 50 µm. (D) Safranin O/Fast green staining showing tibia tissue sections (magnification, ×4) at P21. The HDAC4^d/d, Col2α1-Cre mice had fewer proteoglycans but more ossification at P21 compared with the HDAC4^fl/fl mice. The yellow arrow represents zone of proteoglycans, the black arrow represents zone of ossification, Scale bars, 100 µm. P, postnatal day; HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down.

Figure 3.

Mineralization and vascular invasion of tissues at (A and B) P8 and (C and D) P14 using Von Kossa staining. (B1, the red circled regions) vascular invasion and (D1, the red circled regions) secondary center of ossification were observed earlier in the HDAC4^d/d, Col2α1-Cre mice compared with in HDAC4^fl/fl mice (A1 and C1, the red circled regions). The HDAC4^d/d, Col2α1-Cre mice had (B2 and D2) thicker cortical and (B3 and D3) cancellous bones compared with the (A2, C2, A3 and C3) HDAC4^fl/fl mice. Yellow circled areas represent growth plates. Black circled areas represent cortical bones. Green circled areas represent cancellous bones. Boxes indicate area magnified in adjacent figures. Scale bars, 100 µm. P, postnatal day; HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down.

Figure 4.

Premature ossification in HDAC4^d/d, Col2α1-Cre mice. (A-H) Alizarin red and Alcian blue stained skeletons of the HDAC4^fl/fl and HDAC4^d/d, Col2α1-Cre mice at P10, the red boxes and arrow represent xiphoid process and sternum, the green boxes and arrow represent lumbar vertebrae, black arrows represent tibia cartilage. (I and J) µCT analysis showing the bone image of tibial tissues from the HDAC4^fl/fl and the HDAC4^d/d, Col2α1-Cre mice at P8, yellow boxes and arrow represent cancellous bone. (K and L) Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. in the HDAC4^fl/fl and HDAC4^d/d, Col2α1-Cre mice were evaluated using µCT analysis. Each assay was performed in triplicates for each group. Scale bars, 200 µm. *P<0.05 vs. HDAC4^fl/fl mice. P, postnatal day; HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation.

Figure 5.

Decreased chondrocyte proliferation due to HDAC4 deletion. (A-L) Immunohistochemical staining of the tibial articular cartilage of HDAC4^fl/fl and HDAC4^d/d, Col2α1-Cre mice at P8 for determining the expressions of BrdU, PCNA, MMP-13, Runx2, OPG. and CD34 Positive signals (brown staining) are indicated by red arrows. Scale bars, 20 µm. (M-R) Semiquantitative analysis of positively expressed areas for BrdU, PCNA, MMP-13, Runx2, OPG and CD34. (S-Z) In situ hybridization on paraffin sections prepared for determining the expressions of Col X and Wnt5a in tibial tissues at P14. The HDAC4^d/d, Col2α1-Cre mice had decreased expressions of BrdU, PCNA and Wnt5a but increased expressions of MMP-13, Runx2, OPG, Col X and CD34. Boxed areas in S-V indicate the regions shown in W-Z, respectively. Scale bars, 50 µm. *P<0.05 vs. HDAC4^fl/fl mice. P, postnatal day; HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down; BrdU, bromodeoxyuridine; PCNA, proliferating cell nuclear antigen; MMP-13, matrix metalloproteinase-13; Runx2, runt-related transcription factor 2; Col X, type X collagen; OPG, osteoprotegerin.

Figure 6.

Reverse transcription quantitative-PCR was used to analyze the mRNA levels of (A) HDAC4, (B) PCNA, (C) MMP-13, (D) Runx2, (E) OPN and (F) osteocalcin in HDAC4^d/d, Col2α1-Cre and HDAC4fl^/fl mice from three independent experiments. n=3. *P<0.05 vs. control. HDAC4, histone deacetylase 4; Col2α1, collagen type 2α1; fl, floxed; d, down; PCNA, proliferating cell nuclear antigen; MMP-13, matrix metalloproteinase-13; Runx2, runt-related transcription factor 2; OPG, osteoprotegerin.