Proteoglycan 4: a dynamic regulator of skeletogenesis and parathyroid hormone skeletal anabolism

Abstract

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure.

Fig. 1

PTH regulation of Prg4 mRNA. (A, B) Untreated 16-week-old C57BL6 wild-type mice were euthanized, and long bone, calvaria, bone marrow, and liver were harvested for gene expression analysis (n = 3/group). RNA was isolated and quantitative real-time PCR was performed to assess; (A) proteoglycan 4 (Prg4) mRNA, and (B) PTH/PTHrP receptor (PPR) mRNA expression (standardized to GAPDH levels). Relative quantification of data was determined using the standard curve method. (C–E) Eight- to 16-week-old C57BL6 wild-type mice were administered a single subcutaneous injection of PTH(1–34) (1 mg/g) or vehicle (VEH) (0.9% NaCl) control, euthanized 1, 4, 8, or 12 hours later, and whole liver, calvaria, and long bone were harvested for gene expression analysis (n ≥ 5/group). RNA was isolated and quantitative real-time PCR was performed to assess Prg4 mRNA expression (standardized to GAPDH levels) in (C) liver, (D) calvaria, and (E) long bone. Relative quantification of data generated was carried out using the comparative CT method. ^*p < 0.001; ^**p < 0.05 versus time-matched VEH. Data are expressed as mean ± SEM.

Fig. 2

Trabecular bone area and volume analysis. (A,B,D,E) Four-day-old Prg4 mutant (−/−) and wild-type (+/+) mice were administered intermittent PTH(1–34) (50 mg/kg) or vehicle (VEH) (0.9% NaCl) subcutaneous injection daily for 17 days (“young” mice). (B,C,E,F) Sixteen-week-old Prg4 −/− and +/+ mice were administered intermittent PTH(1–34) (50 mg/kg) or vehicle (VEH) (0.9% NaCl) control subcutaneous injection daily for 6 weeks (“adult” mice). Femur and tibia were harvested at time of euthanasia. (A–C) Histomorphometric analysis of trabecular bone area (BA/TA) in proximal tibia (secondary spongiosa) of young (n ≥ 11/group) and adult (n ≥ 13/group) mice. Representative images (4 × ) of H&E stained proximal tibial sections from, (A) young and (C) adult mice. (B) Trabecular BA/TA in the proximal tibia (secondary spongiosa). ^*p < 0.01 versus +/+ VEH; ^**p < 0.001 versus −/− VEH; ⁺p < 0.001 versus +/+ VEH; ⁺⁺p < 0.05 versus +/+ VEH; ⁺⁺⁺p < 0.01 versus −/− VEH and +/+ PTH. (D–F) Micro-CT analysis of distal femur trabecular bone volume fraction (BV/TV) in young (n ≥ 11/group) and adult (n = 8/group) mice. Representative reconstructed micro-CT cross-sectional images of distal femur from (D) young and (F) adult mice. Representative images were captured in the distal femur, extending 0.5 mm proximally from where analysis was initiated. (E) Distal femur trabecular BV/TV. ^*p < 0.001 versus +/+ VEH; ^**p < 0.05 versus +/+ VEH; ^***p < 0.001 versus −/− VEH; ⁺p < 0.01 versus +/+ VEH; ⁺⁺p < 0.01 versus +/+ VEH; ⁺⁺⁺p < 0.05 versus −/− VEH. For comparison of +/+ PTH versus −/− PTH samples, values were expressed as treatment over control prior to statistical analysis. Data are expressed as mean ± SEM.

Fig. 3

Tibial growth plate and femur length analysis in young mice. (A–E) Four-day-old Prg4 mutant (−/−) and wild-type (+/+) mice were administered intermittent PTH(1–34) (50 μg/kg) or vehicle (VEH) (0.9% NaCl) control subcutaneous injection daily for 17 days (“young” mice). (A–D) Tibial growth plate morphology and height were evaluated in H&E-stained proximal tibia sections from young Prg4 −/− versus +/+ mice (n ≥ 11/group). (A) Representative images (40 ×) of tibial growth plate from young Prg4 −/− versus +/+ mice. (B) Proliferative zone height. (C) Hypertrophic zone height. ^*p < 0.001: versus +/+ VEH; ^**p < 0.05: versus +/+ VEH; ^***p < 0.01: versus −/− VEH. (D) Total growth plate height. ^*p < 0.05: versus +/+ VEH; ^**p < 0.05: versus +/+ VEH; ^***p < 0.05: versus −/− VEH. (E) Femur length measurements performed via reconstructed micro-CT images of young Prg4 femurs (n ≥ 11/group).

Fig. 4

Bone marrow stromal cell (BMSC) in vitro osteoblastogenesis assays. (A–F) Untreated 16-week-old Prg4 mutant (−/−) and wild-type (+/+) mice were euthanized, femoral and tibial bone marrow was harvested, and BMSCs were isolated for in vitro osteoblastogenesis assays. (A) Cell numbers over time in BMSC cultures (n ≥ 6/group). (B) Collagen type II (col2) mRNA expression was assessed as a marker of chondrogenic potential in day 4 BMSC cultures (n ≥ 6/group). (C) Peroxisome proliferator-activated receptor-gamma2 (PPARg2) mRNA expression was assessed as a marker of adipogenic potential in day 4 BMSC cultures (n ≥ 6/group). (D) Osteocalcin (OCN) mRNA expression was assessed as a marker of osteoblast differentiation in day 9 BMSC cultures (n ≥ 10/group). RNA was standardized to GAPDH levels and relative quantification of data generated was determined using the standard curve method. (E) Representative images of day 14 BMSC von Kossa mineralization cultures. (F) Mineralization area per well in day 14 BMSC cultures (n ≥ 6/group). In vitro assays were carried out at least two times with similar results. Data are expressed as mean ± SEM.

Fig. 5

Proximal tibia bone cell numbers and activity. (A–H) Sixteen-week-old Prg4 mutant (−/−) and wild-type (+/+) mice were administered intermittent PTH(1–34) (50 mg/kg) or vehicle (VEH) (0.9% NaCl) control subcutaneous injection daily for 6 weeks (“adult” mice). Tibiae were isolated from adult Prg4 −/− and +/+ mice for histomorphometric analysis of bone cell numbers and activity. (A–C) Histomorphometric analysis of von Kossa–stained (with tetrachrome counterstain) proximal tibia sections was performed to assess osteoblast number per bone perimeter (N.Ob/B.Pm) and osteoid surface (OS/BS) in the secondary spongiosa. (A) Representative images (40 ×) of von Kossa–stained proximal tibia secondary spongiosa. (B) Osteoblast numbers (n ;:: 10/group). ^*p < 0.001 versus +/+ VEH; ^**p < 0.01 versus −/− VEH. (C) Osteoid surface (n ≥ 10/group). ^*p < 0.05 versus +/+ VEH; ^**p < 0.05 versus −/− VEH. (D,E) TRAP+ cell enumeration was carried out in proximal tibia sections to assess osteoclast numbers per bone perimeter in the secondary spongiosa. (D) Representative images (40 ×) of TRAP-stained proximal tibia secondary spongiosa. (E) TRAP+ cell number per bone perimeter (n ≥ 8/group). ^*p < 0.05 versus +/+ VEH; ^**p < 0.01 versus −/− VEH. (F–H) Dynamic histomorphometric analysis of calcein-labeled proximal tibia sections was performed to assess bone formation rates (BFR/BS) and mineral apposition rates (MAR) in the secondary spongiosa. (F) Representative images (40 ×) of dual calcein labels in proximal tibia secondary spongiosa. (G) Bone formation rate (n ≥ 8/group). ^*p < 0.01 versus +/+ VEH; ^**p < 0.01 versus −/− VEH. (H) Mineral apposition rate (n ≥ 8/group). ^*p < 0.01 versus +/+ VEH; ^**p < 0.01 versus −/− VEH. Data are expressed as mean ± SEM.

Fig. 6

Joint range of motion and animal mobility. (A–D) Sixteen-week-old Prg4 mutant (−/−) and wild-type (+/+) mice were administered intermittent PTH(1–34) (50 mg/kg) or vehicle (VEH) (0.9% NaCl) control subcutaneous injection daily for 6 weeks (“adult” mice). (A,B) Maximal hind paw extension was measured to assess joint range of motion. (A) Tibia was immobilized at 0 degrees and the maximal extension of the hind paw was measured. (B) Maximal hind paw extension (n ≥ 5/group). ^*p < 0.001 versus +/+ VEH. (C,D) Spontaneous exploratory behavior was assessed to evaluate animal mobility. Mice were placed in a 12-grid chamber for 1 minute; parameters assessed included number of grids crossed and number of hindlimb stands. (C) Number of grids crossed (n ≥ 5/group). ^*p < 0.01 versus +/+ VEH. (D) Number of hindlimb stands (n ≥ 5/group). ^*p < 0.05 versus +/+ VEH. Data are expressed as mean ± SEM.

Fig. 7

PTH regulation of bone marrow and liver gene expression. (A–F) Sixteen-week-old Prg4 mutant (−/−) and wild-type (+/+) mice were administered a single subcutaneous injection of PTH(1–34) (1 mg/g) or vehicle (VEH) (0.9% NaCl) control, euthanized 0 (no treatment control), 1, 4, 8, or 12 hours later, and long bone marrow (n ≥ 5/group) and whole liver (n ≥ 5/group) harvested for gene expression analysis. RNA was isolated and quantitative real-time PCR was performed to assess marrow; (A) PTH/PTHrP receptor (PPR), (B) insulin-like growth factor I (IGF-I), (C) basic fibroblast growth factor 2 (FGF-2), and liver; (D) PPR, (E) IGF-I, (F) FGF-2 mRNA expression (standardized to GAPDH levels). Relative quantification of data was determined using the comparative CT method. Line graphs represent PTH effects on mRNA expression over time. (A) Marrow PPR mRNA; ^*p < 0.05: +/+ PTH versus +/+ VEH; ^**p < 0.05: −/− PTH versus −/− VEH. (B) Marrow IGF-I mRNA; ^*p < 0.05: +/+ PTH versus +/+ VEH. (C) Marrow FGF-2 mRNA; ^*p < 0.05: +/+ PTH versus +/+ VEH; ^**p < 0.01: −/− PTH versus −/− VEH. (D) Liver PPR mRNA; ^*p < 0.05: −/− VEH versus +/+ VEH; ^**p < 0.05: +/+ PTH versus +/+ VEH; ^***p < 0.01: −/− PTH versus −/− VEH. (E) Liver IGF-I mRNA; ^*p < 0.05: −/− VEH versus +/+ VEH. (F) Liver FGF-2 mRNA; ^*p < 0.05: −/− VEH versus +/+ VEH; ^**p < 0.05: +/+ PTH versus +/+ VEH; ^***p < 0.05: −/− PTH versus −/− VEH. Data are expressed as mean ± SEM.