Osteoporosis and age-related skeletal fragility remain major public health challenges driven by an imbalance between bone formation and resorption that emerges with aging [1, 2]. Accumulating evidence implicates oxidative stress and cellular senescence as central, convergent mechanisms that impair osteoblast function, enhance osteoclastogenesis, and damage bone microarchitecture [3, 4, 5, 6]. In parallel, vitamin D signaling via the vitamin D receptor (VDR) contributes to skeletal health not only through mineral homeostasis but also by modulating redox balance, DNA damage responses, and inflammatory programs in bone cells [7, 8, 9, 10]. However, how insufficiency of active vitamin D (1,25(OH)₂D₃) integrates with oxidative stress to trigger p53–p21 and p16^{I⁢N⁢K⁢4⁢a} checkpoints, establish a stable senescence-associated secretory phenotype (SASP), and accelerate skeletal aging in vivo remains incompletely defined.

In the aging skeleton, reactive oxygen species (ROS) activate the DNA damage response and checkpoint pathways that can culminate in senescence, with p21 often marking an initial arrest and p16^{I⁢N⁢K⁢4⁢a} enforcing a more durable state linked to SASP-mediated paracrine effects on bone remodeling [3, 4, 5, 6, 11, 12]. Independent studies demonstrate that enhancing antioxidant defenses or targeting senescent cells improves bone mass and strength in preclinical models, underscoring the therapeutic potential of redox–senescence modulation [4, 13, 14]. Vitamin D/VDR signaling intersects with chromatin remodeling and cell-cycle regulators and has been reported to influence oxidative stress handling in multiple cell lineages, suggesting a plausible route by which active vitamin D sufficiency might restrain senescence initiation and maintenance in bone [9, 10, 15, 16, 17, 18, 19]. Yet, direct in vivo evidence connecting active vitamin D insufficiency to increased skeletal ROS, DDR activation, and a transition from p21- to p16-dominant senescence programs is limited.

Here, using genetic and pharmacological perturbations, we delineate a vitamin D–redox–senescence axis that controls skeletal remodeling in vivo. We show that active vitamin D insufficiency elevates ROS, suppresses nuclear factor erythroid 2-related factor 2 (Nrf2)/superoxide dismutase 1 (SOD1) defenses, and increases DNA damage in bone, leading to augmented p16-associated senescence and SASP, impaired osteoblastogenesis, and increased osteoclast activity. Antioxidant supplementation or 1,25(OH)₂D₃ repletion reverses these changes, and genetic reduction of p16 attenuates bone loss by rebalancing formation and resorption. These findings integrate and extend prior observations on redox and senescence in skeletal aging and position active vitamin D sufficiency as a modifiable upstream determinant of bone health in aging.

Our findings define a vitamin D–redox–senescence axis that governs skeletal remodeling during aging by linking active vitamin D insufficiency to heightened ROS, attenuated Nrf2/SOD1 defenses, and increased DNA damage, culminating in p53–p21 and p16 activation, SASP induction, suppressed osteoblastogenesis, elevated osteoclast activity, and trabecular deterioration [3, 5, 12, 25]. Both NAC and 1,25(OH)₂D₃ reversed these molecular and cellular abnormalities, and genetic attenuation of p16 restored bone mass by enhancing formation and reducing resorption while dampening SASP, underscoring senescence as a causal node in this pathway [4]. Mechanistically, our data support a model in which sufficient 1,25(OH)₂D₃ constrains oxidative and genotoxic stress to prevent sustained activation of senescence checkpoints and pro-osteoclastogenic SASP factors that impair osteoblast lineage commitment and promote osteoclastogenesis [3, 4, 5, 12, 25]. These results are consistent with literature linking ROS and DDR signaling to bone aging and demonstrating that bolstering antioxidant defenses curtails osteoclastogenesis and preserves osteoblast function [26, 27, 28, 29, 30]. They also converge with reports that VDR signaling interfaces with chromatin and cell-cycle regulators (for example, EZH2–p16) to restrain senescence programs in bone [31, 32]. At the tissue level, NAC and 1,25(OH)₂D₃ improved osteoblast indices, collagen deposition, and microarchitecture while reducing osteoclast surface, in line with studies showing that mitigating oxidative stress shifts the formation–resorption balance toward anabolism and that perturbation of redox sensors such as Nrf2 alters this balance [28, 29, 30, 33]. The reduction in osteoclast surface, together with vitamin D/VDR modulation of osteoclastogenic mediators, provides a plausible mechanism for antiresorptive benefit in aging bone [34, 35]. Collectively, these data highlight redox–senescence signaling as a tractable therapeutic target and suggest that maintaining sufficient active vitamin D or pharmacologically buffering ROS may mitigate skeletal aging by limiting p16-driven chronic senescence.

In parallel, in WT cohorts, long-term NAC (1 mg/mL in drinking water) and 1,25(OH)₂D₃ (0.1 µg/kg, s.c.) did not alter survival compared with untreated WT controls up to at least 600 days of age, indicating no lifespan extension under these regimens. Consistent with prior literature, neither intervention produced adverse skeletal phenotypes; NAC modestly improved redox status with trends toward preservation of age-sensitive trabecular microarchitecture [36], while 1,25(OH)₂D₃ maintained calcium homeostasis and attenuated age-related declines in bone formation indices, accompanied by activation of Nrf2- and VDR-dependent signaling [18]. These observations align with established evidence that vitamin D signaling mitigates skeletal aging and that antioxidant modulation of ROS can temper senescence-associated bone deterioration [37], providing contextual support for our mechanistic findings while explaining the omission of detailed WT control data from the main text.

It is important to acknowledge the inherent complexities of skeletal aging and to interpret our findings within this broader biological context. While the antioxidant NAC effectively rescued the bone phenotype in our model, supporting a key role for oxidative stress, its broad mechanism of action means that its benefits may extend beyond the direct quenching of reactive oxygen species to influence other redox-sensitive signaling pathways [38]. Likewise, while genetic deletion of p16^{I⁢n⁢k⁢4⁢a} provides strong evidence for its role as a key effector in this process, this intervention may have pleiotropic effects. Cellular senescence is a fundamentally bivalent process, with beneficial roles in tumor suppression and tissue repair that may be compromised by its systemic elimination [3, 4]. The long-term consequences of constitutive p16 deletion, including potential impacts on cancer incidence or wound healing, were not assessed in this study and represent an important area for future investigation [26]. Therefore, the vitamin D–redox–senescence axis identified here should be viewed as a critical contributor to skeletal aging, which likely interacts with other well-established regulators such as hormonal fluctuations, mechanical loading, and systemic inflammation to determine the final bone phenotype.

While both NAC and exogenous 1,25(OH)₂D₃ significantly rescued vitamin D deficiency-induced phenotypes, our study was not designed for direct therapeutic comparison. Notably, 1,25(OH)₂D₃ more completely normalized lifespan and senescence markers, suggesting more targeted pathway correction. Beyond its role as a glutathione precursor, NAC’s benefits likely involve pleiotropic mechanisms including direct ROS scavenging, NF-$\kappa$B modulation, and mitochondrial stabilization [39, 40]. Importantly, our data do not address toxicity profiles, optimal dosing, or long-term consequences of these interventions—particularly the tumor suppression trade-offs of constitutive p16 deletion [26]. Therefore, we emphasize physiological vitamin D replenishment (targeting 25(OH)D sufficiency) as a first-line approach supported by clinical guidelines [7], with intermittent active vitamin D regimens considered for targeted applications where hypercalcemia risks can be mitigated through close monitoring [41].

Genetic evidence from p16 deletion strengthens the causal link between senescence and osteopenia in the presence of vitamin D insufficiency. Removing p16 normalized bone mass, increased osteogenic programs, reduced osteoclast surface and RANKL/OPG ratio, and suppressed SASP. This mirrors evidence that targeting senescent cells prevents age-related bone loss and rejuvenates remodeling [26, 27] and supports p16 as a key effector coupling redox/genotoxic stress to skeletal aging [31, 32, 42]. It is important to contextualize the role of the Senescence-Associated Secretory Phenotype (SASP) as both a consequence of cellular stress and a driver of tissue pathology. The SASP is highly heterogeneous, but the components we identified as elevated in the bone of Cyp27b1^+⁣/- mice—including the pro-inflammatory cytokines TNF$\alpha$, IL-6, IL-1$\alpha$, and IL-1$\beta$, and the matrix metalloproteinase MMP13—are canonical factors with potent, well-established roles in skeletal remodeling. Specifically, these cytokines are known to robustly promote osteoclast differentiation and activity while inhibiting osteoblast function, thereby directly contributing to a net catabolic state in bone [43, 44]. While oxidative stress and DNA damage are the upstream instigators, our findings position the p16/SASP axis as a critical causal mediator rather than a passive bystander. The genetic deletion of p16 in Cyp27b1^+⁣/- mice not only suppressed the expression of these SASP factors but also rescued the bone loss phenotype entirely. This provides compelling evidence that the establishment of a p16-dependent senescent state, and the chronic paracrine signaling from its associated SASP, is a key mechanistic link between vitamin D insufficiency and the pathological disruption of bone homeostasis [45]. Therefore, the inflammation observed in our model appears to be an integral component of the senescence program, which perpetuates a pro-resorptive microenvironment.

Cellular senescence in bone reflects convergent yet distinct checkpoint programs governed by the p53–p21^{C⁢I⁢P⁢1} and p16^{I⁢N⁢K⁢4⁢a}–RB pathways. Acute oxidative stress commonly activates the DNA damage response (DDR), leading to p53 stabilization and transcriptional induction of p21, which enforces an initial, often reversible growth arrest to facilitate repair [5, 6, 12, 46]. In contrast, chronic or unresolvable stressors—including sustained ROS and mitochondrial dysfunction—favor engagement of p16^{I⁢N⁢K⁢4⁢a}, promoting a more durable RB-mediated arrest associated with a stable SASP [3, 5, 6, 12, 25]. In our model of active vitamin D insufficiency, we observed a rapid increase in p21 coincident with early ROS elevations, consistent with DDR activation, followed by a predominant and persistent upregulation of p16 that correlated with osteopenic remodeling and SASP enrichment. This temporal sequence supports a two-stage paradigm in osteolineage cells: p53–p21 predominates during the initiation phase, whereas p16 maintains long-term senescence and reinforces paracrine dysfunction affecting osteoclastogenesis and osteoblast differentiation [3, 5, 6, 12, 25]. Notably, antioxidant intervention attenuated p21 induction and blunted subsequent p16 accumulation, implicating oxidative stress as a shared upstream driver. These findings align with reports that p21-associated arrest can be reversible and less pro-inflammatory, whereas p16-driven senescence is more stable and SASP-potent, thereby exerting stronger deleterious effects on bone turnover [3, 4, 5, 6, 12, 25, 47]. Thus, while p16 appears to be the principal mediator of the sustained senescent phenotype underlying skeletal aging in our model, the p53–p21 axis likely acts as an early sentinel whose modulation may determine whether cells recover or transition to p16-dependent chronic senescence.

Beyond bone, lifespan extension with NAC and 1,25(OH)₂D₃ suggests organismal benefits from mitigating oxidative stress and senescence, echoing reports that senescence modulation extends healthspan and that systemic redox improvements benefit multiple tissues [14, 23, 26]. Translationally, maintaining vitamin D sufficiency may protect bone by preserving intracellular redox and genomic stability. Antioxidants and senescence-modulating approaches (senolytics/senomorphics) could complement vitamin D repletion in osteoporosis—particularly in vitamin D insufficiency—with careful attention to safety and dosing [14, 23, 26, 28]. Because pharmacologic 1,25(OH)₂D₃ can cause hypercalcemia, physiological vitamin D repletion and bone-targeted or intermittent regimens for active analogs warrant consideration [34].

Clinical implications emerge on several fronts. First, maintaining adequate vitamin D status may protect bone not only by optimizing calcium–phosphate balance but also by preserving intracellular redox and genomic stability in skeletal cells [4, 7, 9, 18, 19, 20, 48, 49, 50]. Second, antioxidants such as NAC and agents that activate Nrf2 could complement vitamin D repletion to dampen oxidative stress–driven bone aging, though dosing, duration, and offtarget effects require careful evaluation [37, 51]. Third, senescencetargeted strategies (senolytics/senomorphics) merit exploration in osteoporosis, particularly when vitamin D insufficiency is present, with attention to safety and tissue specificity [4, 14, 50]. Because pharmacologic 1,25(OH)₂D₃ can cause hypercalcemia, translational paths should prioritize physiological vitamin D repletion (cholecalciferol/25OHD) and consider bonetargeted or intermittent regimens for active analogs [7, 9, 18, 48, 49, 52].

The therapeutic potential of vitamin D in modulating senescence pathways suggests promising avenues for clinical intervention, particularly when combined with emerging senolytic agents. Preclinical evidence, including data presented in this study, indicates that vitamin D receptor activation can suppress senescence-associated secretory phenotype (SASP) and enhance clearance of senescent cells, potentially augmenting the efficacy of senolytics such as dasatinib and quercetin [14]. A combinatorial approach may allow for lower, safer doses of both agents, reducing the risk of adverse effects. However, the use of active vitamin D analogs, such as calcitriol, carries a well-documented risk of hypercalcemia, especially in older adults and individuals with impaired renal function [7]. To mitigate this, strategies including intermittent dosing regimens, close monitoring of serum calcium and parathyroid hormone levels, and patient selection based on vitamin D deficiency status may be employed [41]. Furthermore, the use of less calcemic vitamin D analogs or prodrug formulations currently under investigation could offer a safer profile for long-term use in aging populations [53]. Future clinical trials should evaluate the safety and efficacy of such combination therapies, including active vitamin D or analogs with anti-oxidants and/or senolytics to ameliorate age-related conditions such as osteoporosis.

Active vitamin D insufficiency orchestrates a ROS $\to$ DNA damage $\to$ p16/SASP cascade that disrupts bone remodeling by suppressing osteoblastogenesis and enhancing osteoclastogenesis, culminating in trabecular deterioration and reduced bone mineral density. Restoring redox homeostasis with N-acetylcysteine, repleting 1,25-dihydroxyvitamin D₃, or genetically disabling p16 each reversed oxidative damage, dampened senescence, and normalized formation–resorption balance, thereby rescuing bone mass and microarchitecture. These findings establish a causal vitamin D–redox–senescence axis in skeletal aging and provide a preclinical rationale for investigating redox modulation and senescence control as potential therapeutic avenues. From a clinical perspective, ensuring physiological vitamin D sufficiency remains the foundational and proven approach. Our work suggests that targeting the downstream redox-senescence axis could, in the future, offer a complementary strategy; however, we emphasize that this is a long-term prospect. The successful translation of such approaches would require robust clinical investigation and must first overcome significant known challenges, including target tissue-specific delivery, the determination of optimal dosage and duration, and rigorous assessment of long-term safety and off-target effects. Future studies should define cell type–specific mechanisms, integrate redox metabolomics with single-cell senescence mapping, and test combination interventions in primary and secondary osteoporosis models.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

DM conceived and supervised the project, secured funding, provided research resources and facilities, validated findings, and led manuscript writing and revision. WQ performed primary experiments, analyzed data, and developed experimental methodologies. MH and LC conducted supporting experiments and assisted with data analysis and methodology development. DG made subbstantial contributions to the conception or design of the work and made critical revision to the article for important intellectual content. All authors approved the final version of this manuscript. All authors contributed to editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The use of animals in this study was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (Approval number: IACUC-1802007), ensuring compliance with China’s ethical guidelines for laboratory animal care and use.

Not applicable.

This work was supported by grants from the National Natural Science Foundation of China (81730066 to DM).

The authors declare no conflict of interest.