Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species

Authors:

Hongkang Zhou, America B. Newnum, Joseph R. Martin, Ping Li, Mark T. Nelson, Akira Moh, Xin-Yuan Fu, Hiroki Yokota and Jiliang Li

Abstract:

Signal transducers and activators of transcription 3 (Stat3) is a transcription factor expressed in many cell types including osteoblasts, osteocytes, and osteoclasts. STAT3 mutations cause a rare human immunodeficiency disease that presents reduced bone mineral density and recurrent pathological fractures. To investigate the role of Stat3 in load-driven bone metabolism, two strains of osteoblast/osteocyte-selective Stat3 knockout (KO) mice were generated. Compared to age-matched littermate controls, this selective inactivation of Stat3 significantly lowered bone mineral density (7–12%, p < 0.05) as well as ultimate force (21–34%, p < 0.01). In ulna loading (2.50–2.75 N with 120 cycles/day at 2 Hz for 3 consecutive days), Stat3 KO mice were less responsive than littermate controls as indicated by reduction in relative mineralizing surface (rMS/BS, 47–59%, p < 0.05) and relative bone formation rate (rBFR/BS, 64–75%, p < 0.001). Furthermore, inactivation of Stat3 suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Taken together, the results support the notion that the loss-of-function mutation of Stat3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.

Link to Article:

http://dx.doi.org/10.1016/j.bone.2011.04.020

Crebbp haploinsufficiency in mice alters the bone marrow microenvironment leading to loss of stem cells and excessive myelopoiesis

Authors

Stephanie N. Zimmer, Qing Zhou, Ting Zhou, Ziming Cheng, Sherry L. Abboud-Werner, Diane Horn, Michael Lecocke, Ruth White, Andrei V. Krivtsov, Scott A. Armstrong, Andrew L. Kung, David M. Livingston, and Vivienne I. Rebel

Abstract

CREB binding protein (CREBBP) is important for cell-autonomous regulation of hematopoiesis, including the stem cell compartment. Here we describe that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the bone marrow (BM) microenvironment of Crebbp+/- mice was unable to properly maintain the immature stem - and progenitor pools. Instead, it stimulates myeloid differentiation that progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone, predominantly caused by increased osteoclastogenesis. Although colony-forming unit-fibroblast and the total osteoblast numbers were decreased, the bone formation rate was similar to that found in wild-type mice. At a molecular level, we found that the known hematopoietic modulators MMP9 and KITL were decreased with heterozygous levels of Crebbp. Lastly, potentially important regulatory proteins (ESAM1 and CDH5) were increased on Crebbp+/- endothelial cells. Together, our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and prevent excessive myeloproliferation.

Link to Article

http://dx.doi.org/10.1182/blood-2010-09-307942

Effect of paricalcitol and cinacalcet on serum phosphate, FGF-23, and bone in rats with chronic kidney disease

Authors

Jane L. Finch, Masanori Tokumoto, Hironori Nakamura, Wei Yao, Mohammad Shahnazari, Nancy Lane, and Eduardo Slatopolsky

Abstract

Calcimimetics activate the calcium-sensing receptor (CaR) and reduce parathyroid hormone (PTH) by increasing the sensitivity of the parathyroid CaR to ambient calcium. The calcimimetic, cinacalcet, is effective in treating secondary hyperparathyroidism in dialysis patients [chronic kidney disease (CKD 5)], but little is known about its effects on stage 3–4 CKD patients. We compared cinacalcet and paricalcitol in uremic rats with creatinine clearances “equivalent” to patients with CKD 3–4. Uremia was induced in anesthetized rats using the 5/6th nephrectomy model. Groups were 1) uremic control, 2) uremic + cinacalcet (U+Cin; 15 mg·kg−1·day−1 po for 6 wk), 3) uremic + paricalcitol (U+Par; 0.16 μg/kg, 3 × wk, ip for 6 wk), and 4) normal. Unlike U+Par animals, cinacalcet promoted hypocalcemia and marked hyperphosphatemia. The Ca × P in U+Cin rats was twice that of U+Par rats. Both compounds suppressed PTH. Serum 1,25-(OH)2D3 was decreased in both U+Par and U+Cin rats. Serum FGF-23 was increased in U+Par but not in U+Cin, where it tended to decrease. Analysis of tibiae showed that U+Cin, but not U+Par, rats had reduced bone volume. U+Cin rats had similar bone formation and reduced osteoid surface, but higher bone resorption. Hypocalcemia, hyperphosphatemia, low 1,25-(OH)2D3, and cinacalcet itself may play a role in the detrimental effects on bone seen in U+Cin rats. This requires further investigation. In conclusion, due to its effects on bone and to the hypocalcemia and severe hyperphosphatemia it induces, we believe that cinacalcet should not be used in patients with CKD without further detailed studies.

Link to Article

http://ajprenal.physiology.org/content/298/6/F1315.abstract

Characterization of Ex Vivo–Generated Bovine and Human Cartilage by Immunohistochemical, Biochemical, and Magnetic Resonance Imaging Analyses

Authors

Ashleigh E. Nugent, David A. Reiter, Kenneth W. Fishbein, Denise L. McBurney, Travis Murray, Dorota Bartusik, Sharan Ramaswamy, Richard G. Spencer, Walter E. Horton

Abstract

Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 μL spot cultures with 5 × 106 each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo–formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo–formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.

Link to Article

http://dx.doi.org/10.1089/ten.tea.2009.0717

Lengthening of mouse hindlimbs with joint loading

Authors

Ping Zhang, Kazunori Hamamura, Charles H. Turner and Hiroki Yokota

Abstract

For devising clinical approaches to treating limb length discrepancies, strategies that will generate differential longitudinal growth need to be improved. This report addresses the following question: does knee loading increase bone length of the loaded hindlimb? Knee loading has been shown to induce anabolic responses on the periosteal and endosteal surfaces, but its effects on longitudinal bone growth have not yet been examined. In the present studies, loads were applied to the left hindlimb (5-min bouts at 0.5 N) of C57/BL/6 mice (21 mice, ~8 weeks old). Compared to the contralateral and age-matched control groups, knee loading increased the length of the femur by 2.3 and 3.5%, together with the tibia by 2.3 and 3.7% (all P < 0.001), respectively. In accordance with the length measurements, knee loading elevated BMD and BMC in both the femur and the tibia. Histological analysis of the proximal tibia revealed that the loaded growth plate elevated its height by 19.5% (P < 0.001) and the cross-sectional area by 30.7% (P < 0.05). Particularly in the hypertrophic zone, knee loading increased the number of chondrocytes (P < 0.01) as well as their cellular height (P < 0.001) along the length of the tibia. Taken together, this study demonstrates for the first time the potential effectiveness of knee loading in adjusting limb length discrepancy.

Link to Article

http://dx.doi.org/10.1007/s00774-009-0135-x

c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis

Authors

Ricardo T Paniagua, Anna Chang, Melissa M Mariano, Emily A Stein, Qian Wang, Tamsin M Lindstrom, Orr Sharpe, Claire Roscow, Peggy P Ho, David M Lee, and William H Robinson

Abstract

Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis. We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA. GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid. These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.

Link to Article

http://dx.doi.org/10.1186/ar2940