Differential proteomic analysis of tibial subchondral bone from male and female guinea pigs with spontaneous osteoarthritis

Abstract

A proteomic study on the tibial subchondral bone in guinea pigs with spontaneous osteoarthritis was performed to investigate the molecular alterations that occur in early osteoarthritis. A total of 132 healthy Hartley guinea pigs (aged 1 month; 66 female and 66 male) were randomly divided into 11 groups of six. Changes in articular cartilage and tibial subchondral bone were assessed using macroscopic examinations and micro-computed tomography. iTRAQ-integrated liquid chromatography-tandem mass spectrometry was used to identify differentially altered proteins in the tibial subchondral bone between 1- and 3-month-old guinea pigs, which were then validated using western blotting. A gradual progression of cartilage degeneration was observed in the knee joints of the subject animals from 5-11 months. With aging, the tibial subchondral trabecular bone acquired more plate-like and less anisotropic properties, with increased bone mineral density, bone volume, trabecular thickness and numbers. The proteomic study identified 138 and 113 proteins significantly differentially expressed between 3- and 1-month old guinea pigs in both the male and female animals, respectively. Western blotting confirmed the increased expression of osteoblast-associated protein S100 calcium-binding protein A8 (S100A8) and the deregulated expression of osteoclast-associated proteins coronin 1A (CORO1A) and T-cell immune regulator 1 (TCIRG1) in the 3-month old guinea pigs in comparison to the 1-month old guinea pigs. Spontaneous cartilage degeneration in the knee joints of male Hartley guinea pigs tended to be more serious compared with the females during the development of osteoarthritis. Together, the results suggest that osteoblast-associated protein S100A8 and osteoclast-associated proteins CORO1A and TCIRG1 are potentially key regulators of early osteoarthritic development in tibial subchondral bone.

Keywords: Hartley guinea pig; osteoarthritis; proteomics; subchondral bone.

Figure 1

Flow chart of the study design. (A) A flow chart of the design of the present study. (B) Comparison of body weights between male and female Hartley guinea pigs for each time point. Male animals showed significantly faster growth rates compared with the female guinea pigs. LC-MS/MS, liquid chromatography tandem mass spectrometry. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. female.

Figure 2

Macroscopic progression of cartilage degeneration in the knee joints of male and female Hartley guinea pigs aged 1-11 months. Articular cartilage damage initially (at 5 months of age) appeared in the area of the medial tibial plateau that was not covered by the meniscus in both male and female Hartley guinea pigs. Compared with females, the cartilage on the medial tibial plateau (the part not covered by the medial meniscus) of male guinea pigs underwent significant degeneration, which manifested as a rough cartilage surface, cartilage softening and erosion (red circle). The degree of articular cartilage degeneration increased with age, starting from 5 months in both male and female animals. At 11 months of age, cartilage degeneration was serious and had invaded the medial and lateral areas of the tibial plateau, femoral condyle and patellar fossa. The degree of cartilage degeneration of male guinea pigs was more severe than that of females (red circle). Representative images of femoral condyles, patellar fossas and tibial plateaus are shown.

Figure 3

Age and sex related micro-architectural alterations in the tibial subchondral trabecular bone in Hartley guinea pigs. Detailed microstructural changes in both the medial and lateral sides of subchondral trabecular bone in male and female guinea pigs are shown. (A) BMD. (B) BV/TV. (C) Tb. Th. (D) Tb. N. (E) SMI. (F) Tb. Pf. (G) DA. (H) Tb. Sp. Data are presented as the mean ± standard deviation. BV/TV, bone volume fraction; Tb. Th, trabecular thickness; Tb. N, trabecular number; SMI, structure model index; Tb. Pf, trabecular bone pattern factor; DA, degree of anisotropy; Tb. Sp, trabecular separation; BMD, the bone mineral density. ^#P<0.05 vs. female. ^*Significant differences were observed between ages in the same sex and detailed comparison results are available in Table SI and Table SII.

Figure 4

Heat maps of significantly altered proteins with definite identities detected in the subchondral bone of (A) male and (B) female Hartley guinea pigs. A total of 24 upregulated and 16 downregulated proteins were identified in male guinea pigs, and 18 upregulated and 13 downregulated proteins were identified in female guinea pigs. Upregulated and downregulated proteins are shown as red and green spots, respectively. Grey regions indicate that data is not available. 1MM, 1-month-old male guinea pig; 1MF, 1-month-old female guinea pig-3MM, 3-month-old male guinea pig; 3MF, 3-month-old female guinea pig.

Figure 5

Differentially expressed proteins with definite identities were grouped according to GO category. Numbers of differentially expressed proteins associated with GO terms in the GO categories (A) biological process, (B) molecular function and (C) cellular component GO. All data are presented on the basis of GO second-level terms. The numbers of upregulated and downregulated proteins for each category are shown. GO, Gene Ontology.

Figure 6

Validation of the three candidate differentially expressed proteins, CORO1A, S100A8 and TCIRG1, by western blotting. (A) Representative mass spectra of CORO1A, S100A8 and TCIRG1-derived peptides (AVDAAFTCPGSSQLYVTAGR, GGDDLDPNYVLSSR and AGLALPPPEGR, respectively) isolated from subchondral bone tissues from 3-month old guinea pigs. (B) Protein samples extracted from the subchondral bone tissues of the indicated age groups were subjected to western blotting. There was a continuous increase in the expression of CORO1A and S100A8 in the subchondral bone in both male and female guinea pigs from 1 to 6 months. Following an initial decrease, the abundance of TCIRG1 increased from month 3 to 6 in the female guinea pigs, whereas its expression levels remained relatively stable in the male guinea pigs. ACTB was used as the loading control. ACTB, β-actin; S100A8, S100 calcium-binding protein A8; CORO1A, coronin 1A; TCIRG1, T-cell immune regulator 1.