An unanticipated role for sphingosine kinase-2 in bone and in the anabolic effect of parathyroid hormone: Supplementary figures

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<0.01) and males a 22% lower BMD at the same site (p<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 <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 <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.

Methods
Micro-CT: Mice in which SPHK1 was selectively deleted in osteoclasts (SPHK1-OC-/-) mice were generated by crossing cathepsin Kcre+/ mice with SPHK1flox/flox mice and then back crossing their cre+ offspring to the SPHK1flox/flox mice followed by appropriate genotyping. SPHK1flox/flox littermates lacking Cathepsin Kcre+/ 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.

PTH-induced changes in sCSF1 in SPHK2-/-‑ animals: 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

Statistical Analyses: Statistical analyses were performed using GraphPad Prism version 5.0c (GraphPad Software Inc., San Diego, CA, USA). Two-tailed t tests were used where appropriate. A p value <0.05 was considered significant. Data are presented using bar graphs with standard errors of the mean.