Aging increases stromal/osteoblastic cell-induced osteoclastogenesis and alters the osteoclast precursor pool in the mouse.
Stromal/osteoblastic cell expression of RANKL and M-CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M-CSF expression, increased stromal/osteoblastic cell-induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age-related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone.


BACKGROUND
Bone mass is maintained through a balance between osteoblast and osteoclast activity. Osteoblasts regulate the number and activity of osteoclasts through expression of RANKL, osteoprotegerin (OPG), and macrophage-colony stimulation factor (M-CSF). To determine whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis and whether the osteoclast precursor pool changes with age, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo.


METHODS
Osteoclast precursors from male C57BL/6 mice of 6 weeks (young), 6 months (adult), and 24 months (old) of age were either co-cultured with stromal/osteoblastic cells from young, adult, or old mice or treated with M-CSF, RANKL, and/or OPG. Osteoclast precursor pool size was determined by fluorescence-activated cell sorting (FACS), and osteoclast formation was assessed by measuring the number of multinucleated TRACP(+) cells and pit formation. The levels of mRNA for RANKL, M-CSF, and OPG were determined by quantitative RT-PCR, and transcription was measured by PCR-based run-on assays. Osteoblast and osteoclast numbers in bone were measured by histomorphometry.


RESULTS
Osteoclast formation increased dramatically when stromal/osteoblastic cells from old compared with young donors were used to induce osteoclastogenesis. Regardless of the origin of the stromal/osteoblastic cells, the number of osteoclasts formed from the nonadherent population of cells increased with increasing age. Stromal/osteoblastic cell expression of RANKL and M-CSF increased, whereas OPG decreased with aging. Exogenously administered RANKL and M-CSF increased, dose-dependently, osteoclast formation from all donors, but the response was greater in cells from old donors. Osteoclast formation in vitro positively, and the ratio of osteoblasts to osteoclasts in vivo negatively, correlated with the ratio of RANKL to OPG expression in stromal/osteoblastic cells for all ages. The effects of RANKL-induced osteoclastogenesis in vitro were blocked by OPG, suggesting a causal relationship between RANKL expression and osteoclast-inducing potential. The osteoclast precursor pool and expression of RANK and c-fms increased with age.


CONCLUSIONS
Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool and alters the relationship between osteoblasts and osteoclasts in cancellous bone.
Cao
Jay J
JJ
Division of Endocrinology, Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Wronski
Thomas J
TJ
Iwaniec
Urszula
U
Phleger
Laura
L
Kurimoto
Pam
P
Boudignon
Benjamin
B
Halloran
Bernard P
BP
D000135:Acid Phosphatase / Q000494:pharmacology; D000367:Age Factors; D000375:Aging; D000818:Animals; D001862:Bone Resorption; D001842:Bone and Bones / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000378:metabolism; D018189:Immunomagnetic Separation; D007527:Isoenzymes / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000378:metabolism; D008264:Macrophages / Q000166:cytology; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D010024:Osteoporosis; D053244:Osteoprotegerin; D016133:Polymerase Chain Reaction; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology*; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors
D002352:Carrier Proteins; D006023:Glycoproteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D016173:Macrophage Colony-Stimulating Factor; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase