Table 1.
List of primary antibodies used in this study.
Table 2.
List of secondary antibodies used in this study.
Table 3.
List of primer pairs for semi-quantitative RT-PCR.
Table 4.
List of primer pairs for quantitative RT-PCR.
Fig 1.
The highest abundance of peroxisomes was detected in osteoblast and osteoclast cells as examples for intramembranous and endochondral ossification.
(A-B) Paraffin sections of the calvaria (A) and mandible (B) of newborn mice (P0.5) were stained with hematoxylin and eosin to give an overview on bone architecture and localization of osteoblasts (OB), osteocytes (OC) and osteoclasts (bold arrows). (C-H) Immunofluorescence stainings for PEX14 (C, E, G) and catalase (D, F, H) were performed in paraffin sections from the calvaria (C, D) and mandible (E, F) of newborn mouse and vertebrae of adult mice (G, H) Bold arrows in E and F indicate cathepsin K (CTSK)-positive osteoclasts. Please note the higher abundance of peroxisomes in osteoblasts and osteoclasts than in osteocytes as well as in adult (P40) compared to newborn mice (P0.5).
Fig 2.
Hypertrophic chondrocytes contained the highest numerical abundance of peroxisomes compared to proliferative chondrocytes as examples for endochondral ossification.
(A-H) Immunofluorescence stainings of the peroxisomal membrane and matrix proteins PEX14 (A, B, G), catalase (C, D, H) and ABCD3 (E, F) were performed in paraffin-sections from the cartilage (A-D: vertebrae; E, F: femur growth plate; G, H: ribs) of 40-day (A-F) and P0.5 newborn (G, H) mice. Nuclei were stained with Hoechst 33342 (Hoechst) or TOTO-3-iodide (TOTO-3); ALP immunoreactivity (B, D) was used as a marker for skeletal tissue. HC = hypertrophic chondrocytes.
Fig 3.
The distribution of Pex11ß cRNA revealed the strongest expression in the mineralization areas of the cartilage and in osteoblasts of the calvaria of newborn mice.
(A-H) Higher magnifications of in situ hybridization preparations of Pex11ß cRNA in vertebrae (A), the calvaria (B), ribs (C, D), femur (F, G), and the mandible (H) are shown. The corresponding negative controls were hybridized with the complementary Pex11ß mRNA strand (I). Please note that the expression of Pex11ß cRNA in the calvariae showed a higher level in osteoblasts than in osteocytes. OB: osteoblasts.
Fig 4.
The peroxisome numerical abundance increased during osteoblast differentiation.
(A) In primary osteoblast cultures (day 12) from the calvariae of newborn mice, more than 95% of the cells were positively stained for the early osteoblast marker OPN. (B) The formation of calcium nodules increased in primary osteoblasts during culture as shown by Alizarin red staining of the Petri dishes. (C-L) Immunofluorescence stainings of osteoblasts at different time points during culture detecting cell proliferation using anti-Ki67 antibodies (C-G) and peroxisome numerical abundance using anti-PEX14 antibodies (H-L). Nuclei were visualized with Hoechst 33342 (Hoechst, C-F). Representative images (C-F, H-K) and quantitative analysis (G, L) are shown.
Fig 5.
Osteoblast differentiation is accompanied by increases in the expression of peroxisome-related genes and proteins.
(A-D) Semiquantitative RT-PCR of genes involved in osteoblast differentiation (A), peroxisome biogenesis (B) as well as of peroxisomal enzymes and transporters (C) and Ppars (D). The mRNA level of the housekeeping gene Gapdh is included in (A). G-H. Increases in the protein level of peroxisomal membrane and matrix proteins during osteoblast differentiation were confirmed by Western blot analyses of organelle fractions from primary osteoblasts. (F) Osteoblasts after 15 days in culture were collected and subjected to differential centrifugation to obtain enriched organelle fractions (S2, P2, S3, and P3: all the details can be found in Methods, chapter 11). Fractionation quality is demonstrated by Western blots for the peroxisomal marker protein PEX14, mitochondrial marker protein UQCRC2, and the cytosolic, extracellular and vesicular marker protein OPN. (G-H) Time-dependent changes in the protein levels of mitochondrial proteins SOD2, UQCRC2 (G), the bone maturation marker OPN (G) and of peroxisomal membrane (PEX13, PEX14, ABCD3) and matrix (Thiolase, Catalase) proteins (H) are shown.
Fig 6.
Stimulation of all three PPARs increased the peroxisome number and metabolic function in calvarial osteoblasts.
Osteoblasts were exposed to agonists and antagonists of PPARɑ (Cip, GW6471), of PPARß (GW0742, GSK0660), and PPARɣ (Tro, GW9662). Cell homogenates were analyzed for the protein level of catalase, PEX14, and PEX13 using ɑ-tubulin as housekeeping protein to ensure equal protein loading on the gel. Semiquantitative analysis of the integrated optical signal intensities of the proteins related to ɑ-tubulin with controls set to 1 are shown in numbers directly below the bands.
Fig 7.
PPARɑ/ß/ɣ activation induced peroxisome proliferation in calvarial osteoblasts.
(A-H) Primary osteoblasts were treated with vehicle (Vh, A, B) ciprofibrate (Cip, C), GW6471 (D), GW0742 (E), GSK0660 (F), troglitazone (Tro, G) and GW9662 (H) and were stained for PEX14. The strong immunoreactivity and homogenous distribution of PEX14 in individual peroxisomes indicates an increase in the peroxisome number (peroxisome proliferation) and not in PEX14 protein in each individual peroxisome.
Fig 8.
In calvarial osteoblasts, predominantly expressing PPARß, PPAR-modulating drugs affect not only the mRNA level of their own receptor, but indirectly impact the expression of the other Ppars as well.
(A) Comparative analysis of the mRNA levels of Pparɑ, Pparß and Pparɣ in calvarial osteoblasts after 10 days in culture. (B-D) Quantitative RT-PCR analysis of the Ppar mRNA levels in osteoblasts after treatment with agonists and antagonists of PPARɑ (B), PPARß (C), and PPARɣ (D). Significant differences in comparison to non-treated controls were given as *p<0.05; **p<0.01 and ***p<0.001 using ANOVA-1 followed by post-hoc Scheffé-test.
Fig 9.
Activation of PPARß increased the peroxisome number and metabolic function in MC3T3-E1 cells.
(A) Comparative analysis of the mRNA levels (qRT-PCR) of Pparɑ, Pparß and Pparɣin MC3T3-E1 cells. (B) MC3T3-E1 cells were treated with the six PPAR-modulating drugs. PPRE-activity was measured using the Dual Luciferase Reporter Gene Assay. Significant differences in comparison to untreated controls were given as *p≤0.05; **p≤0.01 and ***p≤0.001 using ANOVA-1 followed by post-hoc Scheffé-test. (C-H) Treatment of MC3T3-E1 cells with the PPARß agonist GW0742 (D, G) increased the number of peroxisomes as detected by immunofluorescence stainings for PEX14 (C-E) and PEX13 (F-H) in comparison to cells treated with vehicle (control; C, F) and the PPARß antagonist GSK0660 (E, H). G. Semiquantitative RT-PCR analysis of genes regulating peroxisome number (Pex11) as well as peroxisome biogenesis (Pex13) and metabolic function (Cat, Acox1) after treatment of MC3T3-E1 cells with GW0742.
Table 5.
Activation of PPARß induced the expression of genes related to peroxisome proliferation and metabolic function.
Primary calvarial osteoblasts and MC3T3-E1 cells were treated with the PPARß agonist GW0742 (30 μM) and the PPARß antagonist GSK0660 (150 nM) and we analyzed the mRNA levels (qRT-PCR) of the indicated genes. Significant differences between the means ± SD (n = 4) of non-treated and drug-treated cells were given as: *p≤0.05; ***p≤0.001.
Fig 10.
Activation of PPARß accelerated osteoblast differentiation and maturation.
(A) Gene expression profile of early (Alp, Col1ɑ1), middle and late stage (Opn, Oc) markers as well as of Runx2 which is expressed at all stages of osteoblast differentiation. (B-G) Evaluation of cell proliferation (by detecting ki67, B-D) and maturation (by detecting osteocalcin, E-G) of osteoblasts treated with the PPARß agonist GW0742 and the PPARß antagonist GSK0660.