Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality.Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development

µCT analyses of tibial metaphyseal and trabecular bone phynotypes.

<p>N = 6, *P<0.05 <i>vs</i>. wild type.</p><p>µCT analyses of tibial metaphyseal and trabecular bone phynotypes.</p

Gene expression for two types of 1,25(OH)₂D₃ receptors in wild type and heterozygous mice.

<p>RNA was isolated from the livers of wild type and heterozygous mice at 5, 10, 15, and 30-weeks of age. The gene expression of <i>Pdia3</i> (A) and <i>nVdr</i> (B) were analyzed by real-time PCR and normalized to <i>S18</i>. N = 6, *<i>P</i><0.05 <i>vs</i>. wild type, ^#<i>P</i><0.05 vs. week 5.</p

Generation of <i>Pdia3</i> knockout mouse.

<p>A schematic representation of ablation of <i>Pdia3</i> gene using gene trap vector inserted into the intron 1 of mouse <i>Pdia3</i> gene. The exons are numbered and indicated by solid blue boxes. Primers used for genotyping are indicated as a, b and c (A). PKC activity of 1,25(OH)₂D₃ treated wild type and RST613 cells (B). *<i>P</i><0.05 vs. vehicle treatment, ^#<i>P</i><0.05 vs. wild type. The samples collected from E10.5 embryos were verified using genotyping (C), RT-PCR (D), western blot (E), and X-gal staining (F).</p

µCT and histological analyses of growth plates from <i>Pdia3</i> wild type and heterozygous mice.

<p>The left tibial growth plates were harvested at 5, 10, 15 and 30 weeks of age. Representative 2D cross section of proximal left tibia (A, C) and 3D reconstruction images of the growth plate (B, D) of 15-week of old wild type (A, B) and heterozygous mice (C, D). Tethers are indicated by a red box in 2D sections or an arrow in 3D images. The morphometric analyses of µCT scans showing cartilage volume (E) and tether volume (F). Trichrome staining of a cross section of the proximal left tibias of 10-week old wild type (G, I) and heterozygous mice (H, J). Histomorphometric analysis of growth plate thickness (K) and the percentage of hypertrophic cells per growth plate section (L). White bar represents wild type mice and black bar represents heterozygotes. N = 6, *<i>P</i><0.05 <i>vs</i>. wild type.</p

Reduced osteogenic differentiation capacity of bone marrow stem cells isolated from pdia3^+/− mice.

<p>The bone marrow stem cells (BMSCs) were isolated from wild type and heterozygous mice and cultured in osteogenic media for 14 and 28 days. The expression of collagen type I (<i>Col1</i>, A), alkaline phosphatase (<i>Alp</i>, B), Runt-related transcription factor 2 (<i>Runx2</i>, C), bone sialoprotein (<i>Bsp</i>, D), osteopontin (<i>Opn</i>, E) and osteoprotegerin (<i>Opg</i>, F) were evaluated by real-time PCR and normalized to housekeeping gene ribosomal protein s 18 (<i>S18</i>). Alkaline phosphatase activity (G) and the production of Osteocalcin (H) were measured in Day 28 cultures. Alizarin red and von Kossa staining were performed on Day 28 cultures to assess the mineralization and the positive staining areas were measured (I). White bar represents wild type culture and black bar represents heterozygous culture. N = 6, *<i>P</i><0.05 <i>vs</i>. wild type.</p

Disruption of pdia3 gene resulted in malformation during embryogenesis.

<p>The partially absorbed embryos were observed at E12.5 (A). E10.5 embryos were dissected and observed under stereomicroscopy (B, F–H) and confocal microscopy after acridine orange staining (C–E). The wild type embryos (C, F) and heterozygous embryos (D, G) were similar in size and larger than homozygous embryos. One homozygous embryo displayed hemorrhage around the heart area (B) and another one showed open neural tube (E, H). Scale bar = 1000 µm.</p