DOI: 10.5061/dryad.msbcc2fw6
title: An unanticipated role for sphingosine kinase-2 in bone and in the anabolic effect of parathyroid hormone: Supplementary figures

description: Sphingosine-1-phosphate (S1P) is an anabolic clastokine. SPHK is the
 rate-limiting enzyme in S1P production and has two isoforms. To evaluate
 the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype
 of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and
 mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-).
 SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a
 14% lower spinal BMD (p&lt;0.01) and males a 22% lower BMD at the same
 site (p&lt;0.001). SPHK2-/- and control mice were subsequently treated
 either with daily PTH(1-34) or vehicle for 29 days. The response to PTH
 was significantly attenuated in the SPHK2-/-mice. The mean femoral BV/TV
 increased by 24.8% in the PTH-treated female control animals vs 10.6% in
 the vehicle-treated female controls (p &lt;0.01). In contrast, in the
 SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end
 of treatment was not significant (20.5 vs.13.3%, PTH vs. vehicle, p = NS).
 The anabolic response to PTH was significantly attenuated in the spine of
 male SPHK2-/- mice (29.7% vs. 23.1%, PTH vs. vehicle, in controls, p
 &lt;0.05; 26.9% vs. 19.5% PTH vs. vehicle in SPHK2-/- mice, p = NS).
 The spine responded normally in the SPHK2-/- female mice. Interestingly,
 suppression of Sost was blunted in the SPHK2-/- mice when those animals
 were treated with an anabolic PTH regimen. We conclude that SPHK2 has an
 important role in mediating both normal bone remodeling and the anabolic
 response to PTH.
description: <b>Micro-CT</b>:
 Mice in which SPHK1 was selectively deleted in osteoclasts
 (SPHK1-OC<sup>-/-</sup>) mice were generated by crossing
 cathepsin K<sup>cre+/</sup> mice with
 SPHK1<sup>flox/flox </sup>mice and then back crossing their
 cre+ offspring to the SPHK1<sup>flox/flox</sup> mice followed
 by appropriate genotyping. SPHK1<sup>flox/flox</sup>
 littermates lacking Cathepsin K<sup>cre+/</sup> matched for
 sex and weight were used controls. Femurs and spines were collected,
 femurs were stripped of soft tissue and both femur and spine were stored
 in 70% ethanol at 4 °C for subsequent microcomputed tomographic analyses
 (μCT). Data were acquired using a Scanco μCT-35 instrument (Scanco,
 Brutissellen, Switzerland) as previously described (13). Briefly, volumetric
 regions for trabecular analyses, selected within the endosteal borders of
 the distal femoral metaphysis to include the secondary spongiosa located 1
 mm from the growth plate and extending 1 mm proximally, were scanned at 12
 μm resolution. Cortical morphometry was quantified and averaged
 volumetrically through 50 serial cross-sections (600 μm) extending
 distally from the diaphyseal mid-point between proximal and distal growth
 plates. We used a customized thresholding technique (Scanco, Brutissellen,
 Switzerland) that provided the best segmentation of the bone tissue. Both
 2- and 3-D μCT data included bone volume to total volume fraction (BV/TV),
 trabecular number (Tb.N), thickness (Tb.Th), spacing (Tb.Sp), and
 connectivity density (Conn.D). Cortical thickness averaged for both
 cortices (Cor.Th) was also quantified.<i>
 </i> <b>PTH-induced changes in
 sCSF1 in SPHK2<sup>-/-‑
 </sup>animals: </b>Twenty SPHK2-/- animals were
 treated with either vehicle (n=10) or (1-34)hPTH (n=10) for 3 days with
 single daily injections of either vehicle or 80ng/g-bw h(1-34)PTH. At the
 end of the 3-day treatment, cortical bone was isolated as described above
 and expression of sCSF1 quantified using isoform-specific primers: F:
 ccaagaactgcaacaacagc; R: gggtggctttagggtacagg <b>Statistical
 Analyses</b>: Statistical analyses were performed using GraphPad
 Prism version 5.0c (GraphPad Software Inc., San Diego, CA, USA).
 Two-tailed <em>t</em> tests were used where appropriate.
 A <em>p</em> value &lt;0.05 was considered significant.
 Data are presented using bar graphs with standard errors of the
 mean.
description: <b>Figure S1. Baseline BMD
 Data in SPHK1-Oc<sup>-/-</sup></b>. In
 these 12 week-old male and female SPHK1-Oc-/- animals there were no
 significant differences in any trabecular bone parameters in the femur,
 spine and cortical bone compared to controls. (Female control n=9,
 SPHK1-Oc-/- n=9, male control n=3, SPHK1-Oc-/- n=7, NS=Not
 Significant) <b>Figure S2. An anabolic PTH
 regimen induces expression of the soluble isoform of CSF1 in cortical bone
 of SPHK2<sup>-/-</sup> mice</b>. Cortical bone was
 isolated from animals at the end of the anabolic PTH treatment regimen as
 described in the Results. Cortical bone RNA was prepared as described in
 the Methods and expression of the soluble isoform of CSF1 (sCSF1) was
 quantified by qPCR using the primers described in the Methods. PTH
 treatment induced significant expression of the soluble isoform of CSF1 in
 bone isolated from SPHK2<sup>-/-</sup> mice. All groups, n=5.
 ** p&lt;0.01; unless labelled with an asterisk, the differences
 observed were not significant.