Targeted Overexpression of Osteoactivin in Cells of Osteoclastic Lineage Promotes Osteoclastic Resorption and Bone Loss in Mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption

Dachshund and Eyes Absent Proteins Form a Complex and Function Synergistically to Induce Ectopic Eye Development in Drosophila

AbstractThe eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Here we show that the dachshund and eyes absent genes act synergistically to induce ectopic retinal development and positively regulate the expression of each other. Moreover, we show that the Dachshund and Eyes Absent proteins can physically interact through conserved domains, suggesting a molecular basis for the genetic synergy observed and that a similar complex may function in mammals. We propose that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development

Comparison of static histomorphometric trabecular bone parameters at the secondary spongiosa of 4-week-old female <i>OA</i>-Tg mice with age- and sex-matched WT littermates (mean ± SEM).^*

*<p>Measurments were performed at a site that was 300 µm away from the growth plate.</p>**<p>N.S. = Not significant.</p>***<p>% (BV/TV), trabecular area per total tissue area in percentage; Total OC#, total number of TRAP positive multinucleated osteoclasts; <i>OC#.PM</i>, number of TRAP positive osteoclasts per bone surface length; TRAP.PM, TRAP-stained bone surface per total tissue area; Tb.N, trabecular number; Tb.Th, trabecular thickness; and Tb.Sp, trabecular spacing.</p

Schematic representation of the pGL3-TRAP-1B/C-<i>OA</i> expression plasmid.

<p>The pGL3-TRAP-1B/C-<i>OA</i> expression plasmid was generated by cloning the full-length mouse <i>OA</i> cDNA into the Hind3/XbaI restriction site of the pGL3-basic vector. The TRAP-1B/C promoter was then cloned into the Kpn1/Hind3 restriction sites.</p

Effects of siRNA-mediated <i>OA</i> suppression on average cell size (A), <i>in vitro</i> bone resorption activity (B), and expression of osteoclastic genes (C) in RAW264.7 cell-derived osteoclast-like cells.

<p>The dosage of <i>OA</i> siRNAs (29 pM) used in this experiment suppressed OA expression in RAW264.7 cells by greater than 70% (data not shown). RAW264.7 cells were treated with <i>OA</i> siRNAs or control siRNA in the presence of RANKL for 5 days. A shows the relative size of the derived TRAP positive, multinucleated osteoclast-like cells. Top is a representative photomicrograph of the derived osteoclast-like cells, and bottom summarizes the relative size (in relative percentage of the control siRNA-treated cells). B shows the bone resorption activity of the derived osteoclast-like cells determined by an <i>in vitro</i> resorption pit formation assay; and C summarizes the effects of OA siRNA on the relative expression levels of <i>MMP9</i>, <i>CALCR</i>, and <i>NFATc1</i> mRNA (determined by real-time RT-PCR and normalized by the respective expression level of β-actin). Results are shown as percentage of respective control siRNA-treated RAW264.7 cell-derived osteoclast-like cells and in mean ± SEM (n = 3 or 4 for each parameter). The dashed line represents the 100% of the control siRNA-treated controls.</p

Comparison of bone and pQCT parameters of 8 weeks old <i>OA</i> transgenic (Tg) mice with targeted <i>OA</i> overexpression in osteoclastic cells to those of 8 weeks old sex-matched WT littermates (mean±SEM).

*<p>Metaphysis parameters were measured at 22% in length down from the distal end of the femur;</p>#<p>Mid-diaphysis parameters were measured at the mid-shaft of the femur.</p

Effects of targeted overexpression of <i>OA</i> in osteoclastic cells on plasma levels of biomarkers of bone formation <i>in vivo</i>.

<p>In A, plasma levels of osteocalcin of female <i>OA</i>-Tg mice and WT littermates of 8 or 15.3 weeks of age were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated the number of mice per group. In B, plasma levels of pro-collagen type I N-terminal peptide (PINP) of both male and female 15.3-week-old <i>OA</i>-Tg mice and corresponding age- and sex-matched WT littermates were measured with a commercial ELISA kit. Results are shown as mean ± SEM with the indicated number of mice per group.</p

Age effect on the relative overexpression levels of <i>OA</i> mRNA in femurs of female <i>OA</i>-Tg mice.

<p>Total RNA was isolated from femurs of female <i>OA</i>-Tg mice of age of 4-, 8-, and 15.3-week-old and corresponding WT littermates (3–5 mice per group) and cDNA was prepared as described in Methods. The relative level of <i>OA</i> mRNA, determined by real-time RT-PCR and normalized against respective β-actin mRNA level), is reported as fold of that in corresponding age-matched WT osteoclasts (indicated by the dashed line) and shown as mean ± SEM (n = 3–5).</p

Effects of overexpression of <i>OA</i> on relative size of marrow-derived osteoclasts (A and B), and TRAP expression levels (C), size of resorption pits formed <i>in vitro</i> (D), expression levels of bone resorption genes (E), cellular Src tyrosine-527 phosphorylation level (F), and number of TRAP-expressing osteoclast-like cells formed in response to the RANKL and m-CSF treatment (G).

<p>In A–F, marrow-derived osteoclasts were generated from treatment of unattached marrow cells of 12-weeks-old male adult <i>OA</i>-Tg mice and corresponding WT littermates with RANKL and m-CSF for 7 days. In A, to identify the size of osteoclasts, actin rings were stained with FITC-Palloidin and visualized under a fluorescent microscope. Results are shown in mean ± SEM (n = 3 each for each parameter). In G, marrow-derived osteoclasts were generated by treating unattached marrow cells of five 4-weeks-old female <i>OA</i>-Tg mice or four 4-weeks-old female WT littermates. Results are shown as mean ± SEM.</p