Osteoclasts and Microgravity

Abstract

Astronauts are at risk of losing 1.0% to 1.5% of their bone mass for every month they spend in space despite their adherence to diets and exercise regimens designed to protect their musculoskeletal systems. This loss is the result of microgravity-related impairment of osteocyte and osteoblast function and the consequent upregulation of osteoclast-mediated bone resorption. This review describes the ontogeny of osteoclast hematopoietic stem cells and the contributions macrophage colony stimulating factor, receptor activator of the nuclear factor-kappa B ligand, and the calcineurin pathways make in osteoclast differentiation and provides details of bone formation, the osteoclast cytoskeleton, the immune regulation of osteoclasts, and osteoclast mechanotransduction on Earth, in space, and under conditions of simulated microgravity. The article discusses the need to better understand how osteoclasts are able to function in zero gravity and reviews current and prospective therapies that may be used to treat osteoclast-mediated bone disease.

Keywords: M-CSF; RANKL; bone; cytokines; microgravity; osteoblasts; osteoclasts; osteocytes; spaceflight.

Figure 1

The cycle of bone formation. Hematopoietic stem cell ligation of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) initiates their differentiation into mature bone resorbing osteoclasts. Osteoclast-mediated bone resorption releases osteoblast growth factors transforming growth factor (TGF)-β, bone morphogenetic proteins (BMPs), and insulin-like growth factor (IGF)-1 resulting in osteoblast differentiation from mesenchymal stem cells, with subsequent bone formation and mineralization. Osteoblasts trapped in bone matrix evolve into osteocytes, the most abundant cells in bone and the primary regulators of bone homeostasis.

Figure 2

Bone marrow hematopoietic stem cells (HSCs) (colored blue) expressing type-2 receptor for sphingosine-1-phosphate (S1PR2) enter the circulation by binding S1P, a chemotactic lysophospholipid normally present in high concentrations in blood. Circulating CXCR4-expressing HSCs are attracted to bone surfaces by gradients of stromal-cell-derived factor-1 (SDF-1) (CXCL12) secreted by CXCR4⁺ RANK⁺ CD45⁻ stromal marrow cells (colored yellow). HSCs may then be recycled to the bone marrow by binding S1P to S1PR1 or stay at bone surfaces where they evolve into mature, bone-resorbing osteoclasts by binding M-CSF and RANKL produced primarily by osteoblasts and osteocytes. OB, osteoblast; RBC, red blood cell; VEC, vascular endothelial cell.

Figure 3

Binding of RANKL to RANK activates TNF receptor activating factors (TRAFs) 1, 2, 3, 5, and 6. TRAF 6 recruits and activates a kinase cascade, which includes extracellular regulated kinase (ERK), c-jun, N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38), phosphatidylinositol-3 kinase (PI3K), IkB, and AkT. This cascade initiates the transcription of AP-1, c-Fos, NF-κB, and nuclear factor of activated T cells cytoplasmic calcineurin-dependent 1 (NFATc1), with consequent induction of the osteoclast specific genes, TRAP, cathepsin K, DC-STAMP, and ATP6v0d2. C-IAP1/2, cellular amino-terminal inhibitor of apoptosis 1 and cellular amino-terminal inhibitor of apoptosis 2; AKT, a serine protein kinase from the protein kinase AGC subfamily;

Figure 4

Membrane expressed osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells-2 (TREM2) pair with adaptor molecules, Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12 k-DA (DAP12) to activate immunoreceptor tyrosine-based activation motif (ITAM). ITAM activates spleen tyrosine kinase (SyK), Burton’s tyrosine kinase (BtK), and phospholipase C gamma (PLCγ) to induce calcium signaling which is required to activate cAMP response element-binding protein (CREB) and the calcineurin pathway, a key costimulatory pathway of NFATc1 and an important signaling component of a number of immune cell receptors.

Figure 5

Circulating HSCs binding S1P to S1PR2 are attracted to bone surfaces by chemokines such as SDF-1. Here, they differentiate into committed myeloid precursors expressing proviral integrin 1 (PU.1), microphthalmia-associated transcription factor (MITF), and transcription factor E3 (TfE3), transcription factors that induce the expression of C-Fms, the receptor for M-CSF. Binding of M-CSF to C-Fms results in the expression of transcription factors activator protein 1 (AP-1), NF-κB, NFATc1, and RANK, the receptor for RANKL. Binding of M-CSF and RANKL to their cognate receptors promotes further differentiation into osteoclast precursors expressing transcription factors NF-κB, NFATc1, and dendritic cell-specific transmembrane protein (DC-STAMP). These cells fuse, forming mature TRAP-, cathepsin K-positive osteoclasts.

Figure 6

Top view tracing of a photomicrograph of an adherent osteoclast showing the sealing zone’s podosomes (small circles). Microtubules (dotted lines) extend from perinuclear regions to the podosome belt. When they reach the belt, they can stop, bend back toward the cell center, cross the belt, or form a circular network above the belt. Hydrochloric acid and proteases are secreted inside the sealing zone to resorb bone. The typical lifespan of an osteocyte is two weeks. N, nucleus; MT, microtubules; LP, lamellipodia.

Figure 7

Comparison of bone homeostasis in Earth’s gravity (G), in the microgravity of space (MG), and under conditions of simulated microgravity (SMG). The darker colors and thicker lines indicate increased bone structure or enhanced activity. Bone mineral densities and cortical and trabecular microstructures are decreased and osteocyte secretion of sclerostin is increased in MG and SMG. Sclerostin inhibits osteoblastogenesis and bone formation by blocking Wnt/β-catenin signaling in osteoblast stem cells and enhances osteoclastogenesis by reducing osteoblast production of RANKL-binding osteoprotegerin (OPG). Osteoclast differentiation and fusion, bone resorption, expression of regulatory genes, and production of osteoclast-specific proteins are also upregulated in MG and SMG.