A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization

Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA

Deletion of CRTAC1 inhibits pain of post-traumatic OA in female mice.

<p><b>A</b>, Gait analyses of right hind limb (surgical) of wild type and <i>Crtac1</i>^<i>-/-</i> female mice 16 weeks after DMM surgery displaying decreased brake time and paw angle, and increased paw area and midline distance after ablation of <i>Crtac1</i>. <b>B</b>, Gait analyses showing no significant difference about these parameters in contralateral left hind limb of wild type and <i>Crtac1</i>^<i>-/-</i> female mice 16 weeks after DMM surgery. All data are means ± SDs. Two-tailed <i>t-tests</i> were performed.</p

Similar histologic changes of OA in wild type and <i>Crtac1</i>^<i>-/-</i> mice 8 weeks after DMM surgery.

<p><b>A</b> and <b>B</b>, Safranin-O/fast green stain of right knee joints of wild type and <i>Crtac1</i>^<i>-/-</i> mice showing changes of articular cartilage (arrowheads) and osteophyte formation (<b>B</b>, arrows) 8 weeks after DMM surgery. <b>C</b> and <b>D</b>, The maximum and summed OA histologic scores for articular cartilage (<b>C</b>) and osteophytes (<b>D</b>) in wild type and <i>Crtac1</i>^<i>-/-</i> mice 8 weeks after DMM surgery. Data were pooled from male (5 wild type, 3 <i>Crtac1</i>^<i>-/-</i>) and female (3 wild type, 3 <i>Crtac1</i>^<i>-/-</i>) animals. The maximum cartilage scores for male mice: 5 WT: 1, 1, 2, 2, 3; 3 <i>Crtac1</i>^<i>-/-</i>: 0.5, 1, 3; and for females: 3 WT: 1, 1, 3; 3 <i>Crtac1</i>^<i>-/-</i>: 0.5, 1, 1. The summed cartilage scores for male mice: 5 WT: 3.5, 4.5, 5, 5.5, 10; 3 <i>Crtac1</i>^<i>-/-</i>: 2.5, 5.5, 7; and for females: 3 WT: 3.5, 4, 9.5; 3 <i>Crtac1</i>^<i>-/-</i>: 2.5, 3.5, 5. The maximum osteophyte scores for male mice: 5 WT: 1, 1, 1, 1, 2; 3 <i>Crtac1</i>^<i>-/-</i>: 1, 1, 2; and for females: 3 WT: 1, 1, 1; 3 <i>Crtac1</i>^<i>-/-</i>: 1, 1, 2. The summed osteophyte scores for male mice: 5 WT: 3, 3, 5, 6, 6; 3 <i>Crtac1</i>^<i>-/-</i>: 3, 4, 8; and for females: 3 WT: 4, 5, 5; 3 <i>Crtac1</i>^<i>-/-</i>: 4, 5, 6. All data are means ± SDs. Nonparametric Mann-Whitney tests were performed. Scale bars: 200 μm (<b>A</b>), 400 μm (<b>B</b>).</p

Ablation of CRTAC1 inhibits post-traumatic OA in female mice.

<p><b>A</b>, Safranin-O/fast green stain of knee articular cartilage of wild type and <i>Crtac1</i>^<i>-/-</i> female mice 16 weeks after DMM surgery showing decreased structural destruction of articular cartilage after deletion of <i>Crtac1</i>. <b>B</b>, The maximum and summed OA histologic scores for articular cartilage 16 weeks after DMM surgery in wild type and <i>Crtac1</i>^<i>-/-</i> female mice. <b>C</b>, Safranin-O/fast green stain of right knee joints showing that ablation of <i>Crtac1</i> reduce osteophyte formation at the margin of articular cartilage (arrows) 16 weeks after DMM surgery. <b>D</b>, Maximum and summed histologic scores of osteophytes in wild type and <i>Crtac1</i>^<i>-/-</i> female mice 16 weeks after DMM surgery. Scale bars: 200 μm (A), 400 μm (C). All data are means ± SDs. Nonparametric Mann-Whitney tests were performed.</p

RANK-Independent Osteoclast Formation and Bone Erosion in Inflammatory Arthritis

OBJECTIVE: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. METHODS: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures. RESULTS: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice. CONCLUSION: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints

IL-1β and TNF-α stimulate CRTAC1 expression in human articular chondrocytes and synovial fibroblasts.

<p><b>A</b>, Real-time PCR analysis of <i>CRTAC1</i> expression in human articular chondrocytes isolated from macroscopically normal parts of OA cartilage treated by IL-1β for 24 hrs. Two-tailed <i>t-test</i> was performed. <b>B</b>, Real-time PCR analysis of <i>CRTAC1</i> expression in human OA synovial fibroblasts after treatment with IL-1β for indicated times. <b>C</b>, Real-time PCR analysis of <i>CRTAC1</i> in human OA synovial fibroblasts treated with TNF-α for indicated times. Two-Way ANOVA followed by Sidak’s test for multiple comparisons was performed for <b>B</b> and <b>C</b>. All data are presented as means ± SDs.</p

<i>Crtac1</i> knockout mice demonstrate normal bone and cartilage development at 12 weeks of age.

<p><b>A</b>, Micro-CT (μCT) analyses of trabecular (BV/TV, Tb.N, Tb.Th, Tb.Sp) and cortical (Cortical Th., Cortical BV/TV) bone parameters in female wild type and <i>Crtac1</i>^<i>-/-</i> mice of 12 weeks age. All data are means ± SDs. Two-tailed <i>t-tests</i> were performed. <b>B</b>, Three-D μCT images (upper panels) and safranin-O/fast green stain (middle and lower panels) of mouse knee joints showing bone or articular cartilage of wild type and <i>Crtac1</i>^<i>-/-</i> female mice at 12 weeks of age. The local lack of safranin-O staining in articular cartilage of the femoral condyle of <i>Crtac1</i>^<i>-/-</i> mouse is an artifact of cutting/staining, not a real lesion. The artifact is also observed in the femoral condyle of WT mouse in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159157#pone.0159157.s003" target="_blank">S3B Fig</a>. Scale bars, 1 mm (upper panels), 800 μm (middle panels), 200 μm (lower panels).</p