Abstract
NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Human patients with specific mutations in POR exhibit severe developmental malformations including disordered steroidogenesis, sexual ambiguities and various bone defects, similar to those seen in patients with Antley-Bixler syndrome (ABS). To probe the role of POR during bone development, we generated a conditional knockout mouse (CKO) by cross breeding Por lox/lox and Dermo1 Cre mice. CKO mice were smaller than their littermate controls and exhibited significant craniofacial and long bone abnormalities. Differential staining of the CKO mice skull bases shows premature fusion of the sphenooccipital and basioccipital-exoccipital synchondroses. Class III malocclusion was noted in adult knockout mice with an unusual overgrowth of the lower incisors. Shorter long bones were observed along with a reduction in the bone volume fraction, measured by microCT, in the Por-deleted mice compared to age- and sex-matched littermate controls. Concerted up- or down-regulation of proteins in the FGF signaling pathway observed by immunohistochemistry in the tibia samples of CKO mice compared to wild type controls shows a decrease in the FGF signaling pathway. To our knowledge, this is the first report of a mouse model that recapitulates both skull and long bone defects upon Por deletion, offering an approach to study the sequelae of POR mutations. This unique model demonstrates that P450 metabolism in bone itself is potentially important for proper bone development, and that an apparent link exists between the POR and FGF signaling pathways, begging the question of how an oxidation-reduction flavoprotein affects developmental and cellular signaling processes.
Highlights
NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for various endoplasmic reticulum (ER) resident proteins such as cytochromes P450 (CYPs), heme oxygenases (HOs), squalene monoxygenase, and fatty acid elongase [1]
Publications from other laboratories using Rosa26 reporter mice have shown that during endochondral ossification, Dermo1 Cre expression was observed in condensed mesenchyme and later in chondrocytes in growth plate cartilage and in osteoblasts in perichondrium, periosteum and endosteum
To ensure that any phenotype observed in these conditional knockout mouse (CKO) mice was not a consequence of lower POR levels in the liver, the major organ for cytochrome P450-mediated metabolism, liver microsomes were probed for POR protein levels (Figure 1B)
Summary
NADPH-cytochrome P450 oxidoreductase (POR) is the primary electron donor for various endoplasmic reticulum (ER) resident proteins such as cytochromes P450 (CYPs), heme oxygenases (HOs), squalene monoxygenase, and fatty acid elongase [1]. CYP51 (lanosterol 14α-demethylase) catalyzes the demethylation of lanosterol and 24,25-dihydrolanosterol in the cholesterol biosynthesis pathway, utilizing electrons from POR [4]. A key regulator of intracellular heme pools that converts toxic heme into biliverdin, ferrous iron (Fe+ 2) and carbon monoxide [8], has been shown to utilize electrons from POR for its activity [9]. Since POR is the only known electron donor for the CYPs and heme oxygenases, any alteration in its activity can be expected to have pleiotropic effects. Mutations in POR leading to bone deformities have been shown to segregate from fibroblast growth factor receptor II (FGFRII) mutations associated with ABS [12]
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