The sirtuin SIRT6, a NAD+-dependent histone deacetylase and mono-ADP-ribosyltransferase with established anti-ageing functions, is required for skeletal integrity: SIRT6-null mice lose roughly 30% of their bone mineral density and die at about four weeks of age, and this phenotype has been used as a model of senile osteoporosis. The source study (Zhang De-Mao et al., 2016) characterized this phenotype — weak bone formation, impaired matrix mineralization, decreased serum osteocalcin (a bone-formation marker), increased TRAP5b (tartrate-resistant acid phosphatase 5b, a bone-resorption marker), and a distinctive osteoclast abnormality (fewer mature giant osteoclasts and more, smaller TRAP+ cells) that the authors proposed reflects disrupted late-stage osteoclast fusion — and posed the question of how SIRT6 deficiency mechanistically produces this bone loss. Later work has resolved major branches: SIRT6 in osteoblasts/osteocytes restrains osteoclastogenesis through NF-kappaB, RANKL and osteoprotegerin; SIRT6 in preosteoclasts stabilizes estrogen receptor alpha to limit age-related resorption; SIRT6 loss in osteocytes triggers a senescence-PAI-1 (plasminogen activator inhibitor-1) axis. However, the molecular basis of this osteoclast abnormality — including whether late-stage fusion/maturation is genuinely disrupted, since the same group's 2018 follow-up reinterpreted it as more numerous, smaller, already-multinucleated osteoclasts driven by precursor proliferation and osteoblast-paracrine signaling — and the integration of SIRT6's chromatin and glucolipid-metabolic functions in bone-lineage cells with the systemic ageing milieu, needed to reconcile conflicting cell-type-specific findings into one account of the in vivo phenotype, remain open; the field's 2023 review states that the specific mechanisms by which SIRT6 regulates glucolipid metabolism in bone cells remain unclear.

Background

SIRT6 is a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes; it deacetylates histone H3 at lysines 9 and 56 at gene promoters and also acts as a mono-ADP-ribosyltransferase, thereby repressing or activating specific transcriptional programs. It is best known for its anti-ageing role: mice lacking SIRT6 develop a lethal premature-ageing (progeroid) syndrome with severe genomic instability, metabolic derangement (hypoglycemia, low insulin-like growth factor levels), spinal abnormalities and early-onset bone loss, dying at roughly four weeks of age (Mostoslavsky et al., 2006). Bone is maintained by continuous remodeling in which osteoblasts (bone-forming cells derived from bone marrow mesenchymal stem/stromal cells, BMSCs) and osteoclasts (bone-resorbing cells derived from hematopoietic precursors, which fuse into multinucleated giant cells during terminal differentiation) are coupled, in part through the osteoblast-expressed ligand RANKL, which promotes osteoclastogenesis, and its decoy receptor osteoprotegerin (OPG), which blocks it. Osteocytes, the cells embedded in mineralized matrix, regulate both remodeling and phosphate homeostasis. In osteoporosis this balance shifts toward bone loss; in senile (age-related) osteoporosis (SOP), declining bone formation and altered resorption both contribute. Key molecular players include the osteoblast transcription factors Runx2 and osterix (Sp7), the inflammatory transcription factor NF-kappaB (whose target genes promote osteoclast differentiation), and the senescence-associated secretory program (the pro-inflammatory secretome that aged, senescent cells release). Against this background, Zhang De-Mao et al. (2016) characterized SIRT6-null mice as a model of SOP: they confirmed severely reduced trabecular and cortical bone, a low bone formation rate, decreased serum osteocalcin (a marker of mature osteoblast activity), and increased serum TRAP5b (tartrate-resistant acid phosphatase 5b, a marker of osteoclast number and activity). In vitro, BMSCs from knockout mice initially showed increased alkaline phosphatase and collagen reporter activity at day 7 but weakened collagen deposition and calcification at late stages, while osteoclast cultures showed fewer mature multinucleated giant osteoclasts and more individual TRAP+ cells, leading the authors to propose that late-stage osteoclast fusion was disrupted by SIRT6 deficiency. Prior work had already established parts of the mechanism: SIRT6 knockdown impairs rat BMSC osteogenesis partly through NF-kappaB activation (Sun et al., 2014), and Sugatani et al. (2015) showed that SIRT6 physically interacts with Runx2 and osterix, deacetylating H3K9 at their promoters, with resulting Runx2/osterix elevation, Dkk1 and OPG up-regulation, and low-turnover osteopenia. SIRT6 was also reported to promote mesenchymal-stem-cell osteogenic differentiation through BMP (bone morphogenetic protein) signaling in a manner dependent on the transcriptional cofactor PCAF (p300/CBP-associated factor, a histone acetyltransferase) rather than on SIRT6's own deacetylase activity (Zhang Ping et al., 2017). The source paper nonetheless stated that how SIRT6 regulates bone metabolism and induces bone loss during ageing remained unknown, and declared the aim of obtaining more detailed mechanistic evidence.

Problem Statement

By what molecular and cellular mechanisms does SIRT6 deficiency produce the bone-loss phenotype observed in SIRT6-null mice and used as a model of senile osteoporosis? The source study posed this question in general terms — it "remains unknown how SIRT6 regulates the progress of bone metabolism and how SIRT6 induces bone loss during the ageing progress" — and declared the aim of obtaining more detailed mechanistic evidence on the mechanism of senile osteoporosis with SIRT6 deficiency. Later work has resolved several cell-type-specific branches (osteoblast/osteocyte NF-kappaB–RANKL–OPG control, preosteoclast estrogen receptor alpha stabilization, the osteocyte senescence-PAI-1 axis, BMP/PCAF- and VDR-dependent signaling), yet a full mechanistic account is still lacking, and the question remains open in the following three respects, which are jointly required: a resolution must supply an explicit mechanism for each and integrate them into a single coherent causal account of the in vivo bone phenotype, so that an answer covering only one respect does not resolve the task. The chromatin-level and metabolic pathways through which SIRT6 governs late-stage osteoblast maturation and matrix mineralization in bone-lineage cells in vivo are established only in fragmentary and partly contradictory form (NF-kappaB repression, BMP/PCAF signaling, Runx2/osterix and H3K9/H3K56 target-gene control). The molecular explanation of the osteoclast abnormality is unsettled: the source paper proposed that cell fusion at the late stage of osteoclast formation "might have been disrupted by SIRT6 deficiency, thereby inhibiting the maturation process", but the same group's 2018 follow-up reinterpreted the observation — the more numerous, smaller TRAP+ osteoclasts were multinucleated (already fused) yet lacked vacuole-like morphology, and their increase was attributed to enhanced precursor proliferation and osteoblast-paracrine NF-kappaB signaling — and no specific fusion/maturation machinery downstream of SIRT6 has been identified. And the cell-type-specific mechanisms, which conflict in sign across models (OPG up versus down, Sirt6 pro- versus anti-osteoclastogenic, low- versus high-turnover phenotypes), have not been integrated with each other, with SIRT6's chromatin and glucolipid-metabolic functions in bone-lineage cells, or with the systemic metabolic and inflammatory derangement of the premature-ageing milieu, into a coherent causal account of how SIRT6 loss produces the in vivo bone phenotype.

References

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