Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages

Apoptosis occurs at an extraordinary rate in the human body and the effective clearance of dead cells (efferocytosis) is necessary to maintain homeostasis and promote healing, yet the contribution and impact of this process in bone is unclear. Bone formation requires that bone marrow stromal cells (BMSCs) differentiate into osteoblasts which direct matrix formation and either become osteocytes, bone lining cells, or undergo apoptosis. A series of experiments were performed to identify the regulators and consequences of macrophage efferocytosis of apoptotic BMSCs (apBMSCs). Bone marrow derived macrophages treated with the anti‐inflammatory cytokine interleukin‐10 (IL‐10) exhibited increased efferocytosis of apBMSCs compared to vehicle treated macrophages. Additionally, IL‐10 increased anti‐inflammatory M2‐like macrophages (CD206+), and further enhanced efferocytosis within the CD206+ population. Stattic, an inhibitor of STAT3 phosphorylation, reduced the IL‐10‐mediated shift in M2 macrophage polarization and diminished IL‐10‐directed efferocytosis of apBMSCs by macrophages implicating the STAT3 signaling pathway. Cell culture supernatants and RNA from macrophages co‐cultured with apoptotic bone cells showed increased secretion of monocyte chemotactic protein 1/chemokine (C‐C motif) ligand 2 (MCP‐1/CCL2) and transforming growth factor beta 1 (TGF‐β1) and increased ccl2 gene expression. In conclusion, IL‐10 increases M2 macrophage polarization and enhances macrophage‐mediated engulfment of apBMSCs in a STAT3 phosphorylation‐dependent manner. After engulfment of apoptotic bone cells, macrophages secrete TGF‐β1 and MCP‐1/CCL2, factors which fuel the remodeling process. A better understanding of the role of macrophage efferocytosis as it relates to normal and abnormal bone turnover will provide vital information for future therapeutic approaches to treat bone related diseases. J. Cell. Biochem. 117: 2697–2706, 2016. © 2016 Wiley Periodicals, Inc.The process of efferocytosis (clearance of apoptotic cells) has been characterized in various tissues but the role of efferocytosis in the bone microenvironment is unclear. Bone marrow macrophage efferocytosis of apoptotic osteoblastic cells was enhanced by interleukin‐10 in a STAT‐3 dependent manner and resulted in increased production of TGF‐β1 and CCL‐2. The process of efferocytosis is likely important in bone remodeling and osseous wound healing.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/1/jcb25567.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134491/2/jcb25567_am.pd

Inflammatory bone loss associated with MFG‐E8 deficiency is rescued by teriparatide

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/1/fsb2fj201701238r-sup-0002.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/2/fsb2fj201701238r.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154457/3/fsb2fj201701238r-sup-0001.pd

Impact of proteoglycan‐4 and parathyroid hormone on articular cartilage

Proteoglycan‐4 ( Prg4 ) protects synovial joints from arthropathic changes by mechanisms that are incompletely understood. Parathyroid hormone (PTH), known for its anabolic actions in bone, increases Prg4 expression and has been reported to inhibit articular cartilage degeneration in arthropathic joints. To investigate the effect of Prg4 and PTH on articular cartilage, 16‐week‐old Prg4 mutant and wild‐type mice were treated with intermittent PTH (1–34) or vehicle control daily for six weeks. Analyses included histology of the knee joint, micro‐CT of the distal femur, and serum biochemical analysis of type II collagen fragments (CTX‐II). Compared to wild‐type littermates, Prg4 mutant mice had an acellular layer of material lining the surfaces of the articular cartilage and menisci, increased articular cartilage degradation, increased serum CTX‐II concentrations, decreased articular chondrocyte apoptosis, increased synovium SDF‐1 expression, and irregularly contoured subchondral bone. PTH‐treated Prg4 mutant mice developed a secondary deposit overlaying the acellular layer of material lining the joint surfaces, but PTH‐treatment did not alter signs of articular cartilage degeneration in Prg4 mutant mice. The increased joint SDF‐1 levels and irregular subchondral bone found in Prg4 mutant mice introduce novel candidate mechanisms by which Prg4 protects articular cartilage. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 183–190, 2013Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94686/1/22207_ftp.pd

Parathyroid Hormone Mediates Hematopoietic Cell Expansion through Interleukin-6

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45+ and CD11b+ cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin- Sca-1+c-Kit+ (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

The Past, Present, and Future of Genetically Engineered Mouse Models for Skeletal Biology

The ability to create genetically engineered mouse models (GEMMs) has exponentially increased our understanding of many areas of biology. Musculoskeletal biology is no exception. In this review, we will first discuss the historical development of GEMMs and how these developments have influenced musculoskeletal disease research. This review will also update our 2008 review that appeared in BONEKey, a journal that is no longer readily available online. We will first review the historical development of GEMMs in general, followed by a particular emphasis on the ability to perform tissue-specific (conditional) knockouts focusing on musculoskeletal tissues. We will then discuss how the development of CRISPR/Cas-based technologies during the last decade has revolutionized the generation of GEMMs

Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6.

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+) and CD11b(+) cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(-) Sca-1(+)c-Kit(+) (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion

Working Model: IL-6 mediates the PTH anti-apoptotic effect on cells of the hematopoietic lineage.

<p>PTH induces IL-6 expression in stromal cells. Flt-3L increases hematopoietic cell proliferation. IL-6 acts in conjunction with Flt-3L reducing apoptosis in the hematopoietic cell compartment.</p

PTH failed to increase LSK cells in IL-6 deficient mice.

<p>A) Bone marrow was isolated from four-day-old wild-type (WT) and IL-6-deficient mice IL-6 KO) (n≥9/group) and flow cytometric analyses of Lin^- Sca-1⁺ c-Kit⁺ (LSK) cells were performed. Graph represents the percentage of LSK cells out of total bone marrow cells. B) Bone marrow was isolated from 26-day-old wild-type and IL-6 deficient mice (n≥7/group) and flow cytometric analyses of Lin^- Sca-1⁺ c-Kit⁺ (LSK) cells were performed. Graph represents the percentage of LSK cells per total bone marrow cells. C) Four-day-old wild-type and IL-6-deficient mice (n≥6/group) were treated daily with 50 µg/kg PTH or vehicle for 3 weeks. Bone marrow was isolated and flow cytometric analyses of Lin^- Sca-1⁺ c-Kit⁺ (LSK) cells were performed. Fold induction of LSK cells measured as treatment over vehicle (control) of the respective phenotype, *<i>p</i><0.05 versus wildtype vehicle.</p

PTH augments Flt-3L cell expansion.

<p>Whole bone marrow was isolated from C57B6 mice and seeded at 1.8×10⁵ cells/cm² and treated with vehicle, Flt-3L (100 ng/ml), PTH (10 nM), or a combination of Flt-3L and PTH. Non-adherent (A), and adherent (B), cell populations were harvested at days 2, 4, 6, and 8, then enumerated using trypan blue exclusion. Data shown are mean ± SEM of 2 experiments performed in duplicate. Error bars are present on all data points. * <i>p</i><0.05 versus vehicle <i>** p</i><0.05 versus all other groups.</p