Bone undergoes continuous remodeling, with osteoblasts mediating bone formation (Li et al. 2022). BMSCs in bone marrow stroma serve as the primary osteoblast source, capable of differentiating into multiple functional cell types under specific conditions (You et al. 2023). Accelerating BMSC osteogenic differentiation of is very important for the treatment of metabolic bone diseases such as osteoporosis (Guo, Y.c,, et al. 2020). However, further investigation is needed to explain specific mechanisms regulating osteogenic differentiation of BMSCs. In this work, we performed in vitro and in vivo assays to evaluate the potential effect of SENP3 in osteogenic differentiation and the pathogenesis of osteoporosis. Our data expounded addition of SENP3 accelerated osteogenic differentiation in vitro and alleviated osteoporosis in vivo by modulating the DLX2/SIRT3 axis.

SENP3, alters protein modifications by coupling target proteins. Under pathological conditions, changes in SENP3 levels affect the SUMOlyation of proteins, leading to abnormal cell activity and cellular responses, as well as the occurrence of human diseases, such as neurological disorders (Wang et al. 2017), cardiovascular and cerebrovascular diseases (Guo et al. 2013; Rawlings et al. 2019), and cancers (Wang et al. 2024). Moreover, SENP3 accelerated osteogenic differentiation by enhancing HOX level via de-SUMOylating RbBP5 in dental follicle stem cells (Nayak et al. 2014). SENP3 regulated osteogenic differentiation by regulating MLL1/MLL2 methyltransferase complexes (Nayak et al. 2014). However, there is little evidence to directly discuss the function of SENP3 in osteoporosis. This work revealed that SENP3 expression was declined in OVX mice. Besides, the study of Wang et al. illustrated that SENP3 addition alleviates bone loss in type II diabetes-caused osteoporotic rats (Wang et al. 2023). Similarly, this study elucidated that SENP3 prevents bone loss, alters bone metabolism, promotes bone formation in OVX mice. Moreover, SENP3 addition accelerated osteogenic differentiation of BMSCs, reflecting the stimulative effects on calcium deposit, ALP activity, and level of osteogenic genes.

Distal-less gene and its homologous gene Dlx, as homologous domain transcription factors, play an important role in distal limb development in animal kingdom (Hiruta et al. 2014). The Dlx gene family consists of six members (DLX1-6). Among them, it has been illustrated that DLX2 addition promotes early osteogenic differentiation by directly enhancing ALP, and promotes late osteogenic differentiation by directly increasing OCN (Zhang et al. 2019). Zeng et al. revealed that DLX2 facilitates BMSCs osteogenic differentiation via activating Wnt/β-catenin pathway (Zeng et al. 2020). There are similarities with previous studies. We observed that DLX2 depletion suppressed osteogenic differentiation, reflecting the repressive effects on calcium deposit, ALP activity, and expression of osteogenic genes. Moreover, DLX2 level was declined in OVX mice. Interestingly, Duverger et al. found that SUMOylation of DLX3 via SUMO1 enhances its transcriptional activity, which plays a key role in bone and tooth development (Duverger et al. 2011). In this work, we predicted from the SUMO website that DLX2 has SUMO sites. We first uncovered that SENP3 increased DLX2 stability by SUMO2/3. Addition of SENP3 increased DLX2 protein levels and has not change in mRNA level of DLX2. Moreover, DLX2 silencing did not alter the mRNA and protein levels of SENP3. Furthermore, DLX2 knockdown neutralized sh-SENP3-caused prohibitive influences on BMSCs osteogenic differentiation.

SIRT3, a member of the sirtuin family, has been confirmed as a positive factor in bone remodeling (Zheng et al. 2021). For instance, Huh et al. discovered that SIRT3 keeps bone homeostasis via modulating AMPK-PGC-1β signaling in mice (Ding et al. 2017). Zheng et al. elucidated that SIRT3 treatment can slow the bone destruction stimulated by titanium particles and enhance bone formation via GSK-3β/β-catenin signaling (Zheng et al. 2021). Liu et al. revealed that SIRT3 improves osteoporosis and combats BMSC aging by stabilizing mitochondrial homeostasis and heterochromatin. Similarly, we also revealed a beneficial effect of SIRT3 on osteoporosis. We found that SIRT3 was decreased in OVX mice in vivo and facilitated BMSC osteogenic differentiation in vitro. Moreover, we identified a novel mechanism by which SIRT3 is implicated in the progression of osteoporosis. Previous studies have pointed out DLX2 activates Wnt1 transcription promote osteogenic differentiation of hBMSCs (Zeng et al. 2020). Here, DLX2 served as a transcription factor of SIRT3 and accelerated SIRT3 transcription. SIRT3 addition neutralized sh-DLX2-induced suppressive effect on osteogenic differentiation.

In this study, hBMSCs were used for in vitro assays, whereas a mouse model was employed for in vivo validation. This experimental design was chosen based on both practical considerations and scientific rationale. hBMSCs possess well-characterized osteogenic differentiation potential and are widely used to explore osteogenesis-related molecular mechanisms due to their closer relevance to human bone biology. Using hBMSCs enhances the translational value of our findings and allows us to investigate the role of SENP3 in a context that better reflects potential clinical applications. On the other hand, the mouse model provides an essential in vivo platform to evaluate the systemic and physiological effects of SENP3 modulation on bone mass and architecture, which cannot be fully assessed in vitro. Although there are species-specific differences, the key regulatory pathways involved in osteogenic differentiation, including the SENP3–DLX2–SIRT3 axis and SUMOylation mechanisms, are evolutionarily conserved between humans and mice. Thus, we believe that this combined approach is appropriate and enables a comprehensive understanding of SENP3 function in both cellular and organismal contexts.

There are several limitations in this study. First, although we used human BMSCs for in vitro experiments and a mouse model for in vivo validation, interspecies variations could limit translational relevance. Second, while we demonstrated the regulatory role of SENP3 in osteogenic differentiation via the DLX2/SIRT3 axis and SUMO2/3-mediated stabilization, other downstream pathways or interacting proteins may also be involved and remain to be explored.

In summary, the present work illustrated that SENP3 mitigates osteoporosis through promoting SIRT3 transcription by increase of DLX2 stability via SUMO2/3. This work provides a new perspective for the treatment of osteoporosis.