Lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development

Abstract SH2‐containing inositol‐5′‐phosphatase‐1 (SHIP‐1) controls the phosphatidylinositol‐3′‐kinase (PI3K) initiated signaling pathway by limiting cell membrane recruitment and activation of Akt. Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the PI3K signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis. Here, we report that the lipid phosphatase SHIP‐1 regulates chondrocyte hypertrophy and skeletal development through its expression in osteochondroprogenitor cells. Global SHIP‐1 knockout led to accelerated chondrocyte hypertrophy and premature formation of the secondary ossification center in the bones of postnatal mice. Drastically higher vascularization and greater number of c‐kit + progenitors associated with sinusoids in the bone marrow also indicated more advanced chondrocyte hypertrophic differentiation in SHIP‐1 knockout mice than in wild‐type mice. In corroboration with the in vivo phenotype, SHIP‐1 deficient PDGFRα + Sca‐1 + osteochondroprogenitor cells exhibited rapid differentiation into hypertrophic chondrocytes under chondrogenic culture conditions in vitro. Furthermore, SHIP‐1 deficiency inhibited hypoxia‐induced cellular activation of Akt and extracellular‐signal‐regulated kinase (Erk) and suppressed hypoxia‐induced cell proliferation. These results suggest that SHIP‐1 is required for hypoxia‐induced growth signaling under physiological hypoxia in the bone marrow. In conclusion, the lipid phosphatase SHIP‐1 regulates skeletal development by modulating chondrogenesis and the hypoxia response of the osteochondroprogenitors during endochondral bone formation.


| INTRODUCTION
Skeletal development begins in the embryo when multipotent mesenchymal cells arise from the ectoderm and mesoderm, migrate to specific sites and commit to a skeletal fate. While most skeletogenic progenitors will differentiate into chondrocytes to form cartilage and later into osteoblasts to form bone, some remain as mesenchymal stem cells (MSCs) in the bone marrow (BM) throughout life (Kobayashi & Kronenberg, 2014). The primary skeleton, which is entirely cartilaginous, will progressively transform into bone during fetal and postnatal growth through a developmental process known as endochondral ossification (Mackie, Ahmed, Tatarczuch, Chen, & Mirams, 2008). During endochondral bone formation, chondrocytes undergo proliferation, hypertrophic differentiation and apoptosis (Stevens & Williams, 1999). Blood vessel invasion is accompanied by an influx of osteoblastic progenitors from the perichondrium resulting in the replacement of cartilage with bone (Kronenberg, 2003;Long & Ornitz, 2013;Maes et al., 2010). A corollary of the complexity of the skeletal development is a high incidence of osteochondrodysplasia at a young age, which often constitutes high risk for skeleton degenerative diseases such as osteoarthritis and osteoporosis later in life (Lefebvre & Bhattaram, 2010;Long & Ornitz, 2013). Thus, elucidating the biological processes that control endochondral bone development is critical for understanding human skeletal dysplasia and adult abnormal fracture healing.
Despite the fact that many of the growth factors important to cartilage development and functions are able to activate the phosphatidylinositol-3′-kinase (PI3K) signal transduction pathway, little is known about the role of PI3K signaling in chondrocyte biology and its contribution to mammalian skeletogenesis (Guntur & Rosen, 2013;Kita, Kimura, Nakamura, Yoshikawa, & Nakano, 2008;Ulici et al., 2009).

| Mouse maintenance and genotyping
Animal procedures followed protocols approved by the Institutional Animal Care and Use Committees at Rhode Island Hospital. The SHIP-1 KO mice (Inpp5d < tm1Dmt > /J, Stock Nr. 003534) of a mixed genetic background (129/C57BL/6) were purchased from the Jackson Laboratory (Bar Harbor, ME) and backcrossed with C57BL/6 mice (C57BL/6 J, Stock Nr. 000664) for multiple generations. Mouse genotyping was carried out following protocols from the Jackson Laboratory.

| Histology and immunohistochemistry
For histological analyses, the hind limbs of the postnatal mice were dissected, fixed in 10% formalin solution in neutral buffer. Paraffin sections were prepared following standard procedures. For morphological evaluation, the sections were stained with Safranin-O/Fast Green using 1% Safranin-O solution with counterstaining by hematoxylin and 0.2% Fast Green. For collagen type X (Col X) immunohistochemical (IHC) staining, the sections were deparaffinized with xylene and rehydrated in a descending series of ethanol concentrations. Heatmediated antigen retrieval was performed using 1 x citrate buffer (pH 6.0). The deparaffinized and rehydrated sections were blocked and incubated with 1:150 dilution of the anti-collagen type X antibody (purified mouse monoclonal X53, 14-9771-82, ThermoFisher, Waltham, MA) overnight at 4°C. Slides were then incubated with the EnVision Dual Link System-HRP solution (Dako, Santa Clara, CA) containing goat anti-mouse and anti-rabbit immunoglobulins conjugated to the peroxidase-labeled polymer. Following chromogenic development, the slides were counterstained with Harri's hematoxylin. Images were taken by using a Nikon Eclipse E800 microscope equipped with a camera and SPOT software (SPOT Imaging, Sterling Heights, MI).
2.3 | Immunofluorescent staining and laser-scanning cytometry of BM sections Laser-scanning cytometry was performed as described earlier . Briefly, the mice were perfused post-mortem with 10 ml paraformaldehyde-lysine-periodate (PLP) fixative through the vena cava to achieve rapid in situ fixation and optimal preservation of the BM tissue. Femoral bones were isolated, fixed in PLP for 4-8 hr, rehydrated in 30% sucrose/phosphate-

| PDGFRα + Sca-1 + (PαS) MSC isolation and cultivation
Murine BM PαS MSCs from WT and SHIP-1 KO mice (6-week old) were isolated and cultured using reagents and methods as previously described by Morikawa et al. (2009). Briefly, femurs and tibias from WT or SHIP-1 KO mice were dissected and crushed with mortar and pestle. The bone fragments were gently washed once in HBSS supplemented with 2% fetal bovine serum (FBS), 10 mM Hepes, and 1% penicillin/streptomycin (P/S). The cell suspension filtered through a 70 μm cell strainer (BD Falcon) was discarded. The bone fragments were incubated for 1 hr at 37°C in 20 ml DMEM (Invitrogen) containing 0.2% collagenase (Wako Chemicals), 10 mM Hepes, and 1% P/S. The cell suspension was filtered through a cell strainer to remove debris and bone pieces, and cells were collected by centrifugation at 280g for 7 min at 4°C. The pellet was immersed in 1 ml water for 5-10 s to burst the red blood cells, after which 1 ml of 2 × PBS containing 4% FBS was added, and the suspension was filtered through a cell strainer. The cells were suspended in ice-cold HBSS containing supplements as above at 1-5 × 10 7 cell/ml, and (PαS) cells were allowed to adhere to the plastic surface of a 25 cm 2 tissue culture flask (Falcon 3081) for 48 hr without disturbance in α-MEM medium (Invitrogen) supplemented with 10% nonheatinactivated FBS (Hyclone), 10% horse serum (Sigma), 1x L-Glutamine (Invitrogen) and 1% P/S (Peister et al., 2004).

| PαS MSC proliferation assay
Proliferation of PαS MSCs was measured using a 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Cayman Chemical) according to the manufacturer's instruction. In brief, the cells were seeded at a density of 5 × 10 3 per well in a 96-well plate in 100 μl of complete medium in a regular CO 2 incubator or in a hypoxia chamber. At the indicated time points, 100 μl MTT reagent was added into each well, and then formazan crystals were extracted by crystal dissolving solution. Absorbance was measured with a microplate reader at 570 nm (Molecular Devices). and phospho-Erk (Thr202/Tyr204) (Cell Signaling). Anti-β-actin antibody (Novus) was used to detect β-actin as loading controls. anti-mouse collagen type II (Col II) polyclonal antibody (AF3615; R&D Systems), which was then visualized by using a NorthernLights 557conjugated Donkey Anti-Sheep Secondary Antibody (NL010; R&D Systems). Hypertrophic chondrocyte differentiation was verified by using a mouse anti-collagen type X (Col X) monoclonal antibody (X53) conjugated with eFluor 570, (41-9771-82; Thermo Fisher Scientific).

| Chondrogenic differentiation of PαS MSCs
The nuclei were counterstained with DAPI (Biolegend). Images were taken with a Nikon Eclipse E800 fluorescence microscope equipped with a camera and SPOT software.

| Gene expression analysis of SHIP-1 KO PαS MSCs
To determine the gene expression pattern of WT and SHIP-1 KO PαS MSCs, we obtained the mouse MSC RT 2 Profiler PCR Array (Qiagen), which allows simultaneous analysis of 84 key genes involved in maintenance, self-renewal and differentiation of MSCs. Briefly, total RNA was prepared from WT and SHIP-1 KO PαS MSCs after 48 hr culture in hypoxia of 2% oxygen. Each cDNA was synthesized and mixed with RT 2 SYBR Green qPCR mastermix (Qiagen) and an equal volume was added into each well of a 96-well array plate. Real-time PCR was performed using the CFX 96 Real-Time System (Bio-Rad).
The C t values for all wells were exported to a Microsoft Excel spreadsheet for use with the PCR Array Data Analysis software (Qiagen, www.SABiosciences.com/pcrarraydataanalysis.php) according to the manufacturer's instruction. The resulting data were shown by fold change that represents relative mRNA expression for SHIP-1 KO PαS MSCs compared with WT cells.

| Statistical analysis
Data are shown as the mean values ± SEM. The significance of difference in the mean values was calculated with an unpaired twotailed Student's t test using Microsoft Excel. P values of < .05 were considered to be statistically significant.

| SHIP-1 is required for hypoxia-induced growth signaling in MSCs
It has been reported that SHIP-1 activity is required to suppress Akt and Erk phosphorylation were well-established as a proliferative response to growth factors and stresses in many cell types (Zhang & Liu, 2002). These results suggest that reduced stability of HIF-1α combined with impaired activation of both Akt MSCs after chondrogenic induction was quantified by using the NIH ImageJ program and indicated as ImageJ Area Units. Middle and right panels, gene expression levels of Col2a1 and Col10a1 were evaluated by quantitative real-time PCR. GAPDH expression was assessed as an internal reference for quantification. Gene expression levels were expressed as relative fold increase over the internal control. Student's t test was performed and p < .05 is considered significant. Col II, collagen type II; Col X, collagen type X; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; IHC, immunohistochemical; MSC, mesenchymal stem cell; PCR, polymerase chain reaction; SHIP-1, SH2-containing inositol-5′-phosphatase-1; WT, wild-type SO ET AL.

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expression and high-level Col10a1 gene expression, indicating intrinsic hypertrophic differentiation potential.
Since chondrocyte hypertrophic differentiation precedes blood vessel invasion and osteoprogenitor influx (Maes et al., 2010;Sacchetti et al., 2007), accelerated differentiation into hypertrophic chondrocytes by SHIP-1 KO MSCs likely contributes to the greater vessel density and increased number of progenitor cells in the BM. We believe that osteoclasts may also play a role in the observed phenotype, which warrants further studies. We further speculate that excessive vessel invasion may deplete the osteoblastic progenitor pool of the perichondrium, resulting in thinner cortical bones, and augmented and disorganized trabeculae formation in the BM. The nature of the cellular and molecular mechanisms responsible for coupling angiogenesis and skeletal development remains poorly understood, but a primary driving force is tissue hypoxia (Schipani et al., 2001).