Abstract
Ossification defects leading to craniofacial dysmorphism or rhizomelia are typical phenotypes in patients and corresponding knockout mouse models with distinct peroxisomal disorders. Despite these obvious skeletal pathologies, to date no careful analysis exists on the distribution and function of peroxisomes in skeletal tissues and their alterations during ossification. Therefore, we analyzed the peroxisomal compartment in different cell types of mouse cartilage and bone as well as in primary cultures of calvarial osteoblasts. The peroxisome number and metabolism strongly increased in chondrocytes during endochondral ossification from the reserve to the hypertrophic zone, whereas in bone, metabolically active osteoblasts contained a higher numerical abundance of this organelle than osteocytes. The high abundance of peroxisomes in these skeletal cell types is reflected by high levels of Pex11β gene expression. During culture, calvarial pre-osteoblasts differentiated into secretory osteoblasts accompanied by peroxisome proliferation and increased levels of peroxisomal genes and proteins. Since many peroxisomal genes contain a PPAR-responsive element, we analyzed the gene expression of PPARɑ/ß/ɣ in calvarial osteoblasts and MC3T3-E1 cells, revealing higher levels for PPARß than for PPARɑ and PPARɣ. Treatment with different PPAR agonists and antagonists not only changed the peroxisomal compartment and associated gene expression, but also induced complex alterations of the gene expression patterns of the other PPAR family members. Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPRE-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat), whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels. In the same way, treatment of calvarial osteoblasts with GW0742 increased in peroxisome number and related gene expression and accelerated osteoblast differentiation. Taken together, our results suggest that PPARß regulates the numerical abundance and metabolic function of peroxisomes via Pex11ß in parallel to osteoblast differentiation.
Highlights
Peroxisomes are ubiquitous organelles in eukaryotic cells that play a central role in lipid and reactive oxygen species metabolism
Studies in M3CT3-E1 cells showed that the PPARß agonist GW0742 activated the PPAR-response element (PPRE)-mediated luciferase expression and up-regulated peroxisomal gene transcription (Pex11, Pex13, Pex14, Acox1 and Cat), whereas the PPARß antagonist GSK0660 led to repression of the PPRE and a decrease of the corresponding mRNA levels
Since the peroxisomal biogenesis protein PEX14 is an ideal marker for the detection of peroxisomes independent of their metabolic activity [31], immunofluorescence preparations for PEX14 in comparison to the metabolic proteins catalase and ABCD3 were used to analyze the distribution of peroxisomes in the distinct cell types during intramembranous (Fig 1) and endochondral (Fig 2) ossification
Summary
Peroxisomes are ubiquitous organelles in eukaryotic cells that play a central role in lipid and reactive oxygen species metabolism (reviewed by [1]). Peroxisome biogenesis is mediated by more than 32 PEX genes and their corresponding gene products, the peroxins. Peroxins are responsible for the synthesis of the peroxisomal membrane (e.g. PEX3, PEX19), the matrix import (e.g. PEX2, PEX5, PEX7, PEX13 and PEX14) and proliferation of peroxisomes (e.g. PEX11 family) [2]. The importance of these organelles for the development of the skeleton is best demonstrated in patients suffering from peroxisomal biogenesis disorders (PBDs) leading to a complete disruption of peroxisomal metabolic function. Most corresponding knockout mouse models (e.g. for Pex5 [8]; for Pex11ß [9]; for Pex13 [10]) showed a general growth retardation. In a hypomorphic Pex7neo/neo mouse, exhibiting less Pex gene transcripts, a delayed endochondral ossification was noted already at postnatal day 1 and the adult animals (10 weeks of age) were petite [13]
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