Abstract p75^NTR has emerged as a key regulator of skeletal development and bone homeostasis. To define its role, we characterized skeletal phenotypes in global and mesenchyme-specific p75^NTR knockout mouse models. Global deletion of p75^NTR resulted in postnatal growth retardation, decreased trabecular and cortical bone mass, and impaired growth plate architecture—hallmarks of an osteoporotic phenotype that persisted into adulthood. Conditional deletion of p75^NTR in mesenchymal progenitor cells using Prx1-Cre recapitulated these skeletal deficits, confirming a cell-autonomous role in bone development. In vitro, bone marrow stromal cells (BMSCs) derived from p75^NTR-deficient mouse exhibited diminished osteogenic differentiation capacity, reduced mineralization, and downregulation of key osteogenic genes. Transcriptomic profiling revealed significant suppression of the NGF-MAPK/AP-1 signaling axis in p75^NTR-deficient BMSCs. Functional studies demonstrated that loss of p75^NTR reduced JNK pathway activation and downstream epigenetic regulators, including Kdm4b and its target gene Dlx5. Overexpression of Kdm4b rescued mineralization defects and restored osteogenic gene expression in p75^NTR-deficient BMSCs, establishing a mechanistic link between p75^NTR signaling and osteoblast differentiation. These findings define the NGF–p75^NTR–JNK–KDM4B–Dlx5 axis as a central regulatory pathway in postnatal bone growth and osteogenesis. Given the critical role of p75^NTR in skeletal development and bone homeostasis, targeted modulation of this signaling cascade may represent a promising therapeutic approach for treating osteoporosis and other bone disorders. Keywords: p75^NTR, NGF, Mesenchymal progenitor cells, Osteogenic differentiation, Bone formation 1. Introduction Disruptions in bone homeostasis contribute to a wide spectrum of skeletal disorders, ranging from congenital conditions such as osteogenesis imperfecta and rickets to age-related osteoporosis. These diseases not only increase fracture risk but also substantially deteriorate patients' quality of life ([45]Rachner et al., 2011; [46]Kanis et al., 2013). Osteoporosis alone affects over 200 million individuals worldwide, with approximately one in three women and one in five men over the age of 50 experiencing an osteoporotic fracture in their lifetime ([47]Rachner et al., 2011; [48]Kanis et al., 2013). As the global population ages, the healthcare burden of bone disorders continues to rise, intensifying the need to understand the molecular mechanisms governing bone formation and to develop new therapeutic strategies. Bone homeostasis relies on a tightly regulated and dynamic balance between bone formation by osteoblasts and resorption by osteoclasts ([49]Harada and Rodan, 2003). Osteoblasts originate from mesenchymal stem cells (MSCs), and their proper differentiation is critical for maintaining bone mass and structural integrity ([50]Harada and Rodan, 2003; [51]Bianco and Robey, 2015). Several signaling pathways—including Bone Morphogenetic Proteins (BMPs), Transforming Growth Factor-β (TGF-β), and Wnt/β-catenin—have been extensively studied for their roles in regulating MSC proliferation and osteogenic commitment ([52]Harada and Rodan, 2003; [53]Bianco and Robey, 2015; [54]Chen et al., 2012). Emerging evidence suggests that the nervous system plays an active role in skeletal development via neurotrophins—growth factors traditionally known for their roles in neuronal survival and differentiation ([55]Su et al., 2018). Among these, nerve growth factor (NGF) has been shown to enhance osteoblast proliferation ([56]Akiyama et al., 2014), survival ([57]Mogi et al., 2000), and differentiation ([58]Liu et al., 2022; [59]Yada et al., 1994). In vivo, local NGF administration has been shown to promote bone formation during fracture healing and distraction osteogenesis ([60]Wang et al., 2006; [61]Grills et al., 1997), supporting its pro-osteogenic function. NGF signals through two distinct receptors: the high-affinity receptor TrkA and the low-affinity receptor p75^NTR ([62]Bartkowska et al., 2010). While NGF–TrkA signaling has been well-characterized in both neural tissues and bone ([63]Bartkowska et al., 2010; [64]Li et al., 2019; [65]Meyers et al., 2020; [66]Tomlinson et al., 2017; [67]Tomlinson et al., 2016; [68]Rivera et al., 2023; [69]Rivera et al., 2020; [70]Lee et al., 2021), the role of p75^NTR in the skeletal system is far less understood. p75^NTR, a transmembrane receptor that binds to all neurotrophins including as NGF, BDNF (Brain-derived neurotrophic factor), and NT(Neurotrophin)-3 and 4 ([71]Johnson et al., 1986; [72]Meeker and Williams, 2015), is known to mediate critical functions in the nervous system, including axonal growth, neuronal survival, and apoptosis through binding to NGF ([73]Dechant and Y-A., 2002; [74]Micera et al., 2007; [75]Roux and A., 2002). Outside of the nervous system, p75^NTR is expressed in osteocytes, chondrocytes, and MSCs ([76]Chartier et al., 2017; [77]Jones et al., 2010; [78]Cox et al., 2012; [79]Alexander et al., 2009) and has been suggested to play a key role in maintaining skeletal homeostasis ([80]Zhao et al., 2020). Notably, p75^NTR is also recognized as a marker for MSCs with high regenerative capacity ([81]Alvarez et al., 2015a; [82]Alvarez et al., 2015b; [83]Kuci et al., 2010). MSCs expressing p75^NTR display enhanced clonogenicity, proliferation, and differentiation potential, and they express elevated levels of genes involved in bone repair and skeletal remodeling ([84]Alvarez et al., 2015a; [85]Alvarez et al., 2015b; [86]Kuci et al., 2010; [87]Mabuchi et al., 2013; [88]Moscatelli et al., 2009; [89]Calabrese et al., 2015; [90]Cuthbert et al., 2015; [91]Churchman et al., 2012; [92]Zhao et al., 2019; [93]Yang et al., 2017). Functional studies further highlight that inhibition of p75^NTR impairs MSC proliferation and osteogenic differentiation ([94]Akiyama et al., 2014; [95]Mikami et al., 2012; [96]Li et al., 2018). However, the in vivo role of p75^NTR in mesenchymal progenitor cells and skeletal development remains poorly defined. Mechanistically, our previous work demonstrated that NGF promotes osteogenesis in human BMSCs and craniofacial regeneration through p75^NTR-mediated activation of the JNK pathway. This cascade involves the histone demethylase KDM4B, which binds to c-JUN to induce the expression of the master osteogenic transcription factor DLX5, thereby promoting MSC differentiation ([97]Liu et al., 2022). These findings suggest that NGF–p75^NTR–JNK signaling may constitute a novel regulatory axis in skeletal biology, but its functional relevance in vivo has not been established. Here, we hypothesized that p75^NTR signaling plays a critical role in postnatal bone growth and MSC-mediated osteogenesis through NGF-dependent activation of JNK signaling. To test this, we used both global and mesenchyme-specific p75^NTR knockout mouse models to examine skeletal development at early (4-week) and late (12-week) postnatal stages. We also evaluated NGF–p75^NTR–JNK signaling in BMSCs in vitro and identified key molecular mediators of osteogenic differentiation, including MAPK/AP-1 and epigenetic regulators such as Kdm4b and Dlx5. To our knowledge, this is the first comprehensive study to define the in vivo role of p75^NTR in regulating postnatal skeletal development and bone homeostasis using both global and mesenchyme-specific deletion models. Our findings establish p75^NTR as a critical regulator of osteoblast differentiation and postnatal bone mass, with potential therapeutic implications for bone disorders such as osteoporosis. 2. Materials and methods 2.1. Animals p75^NTR−/− (Stock No: 002213), p75^NTR flox (p75^NTRf/f) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines. All animal experiments were performed in accordance with protocols approved (AN195822) by the Animal Research Committee (ARC) of UCSF. The p75^NTR-knockout (p75^NTR−/−) and wild-type (p75^NTR+/+) littermates were generated by mating between heterozygous females and males. Genomic DNA was extracted from 1 mm tail tissue samples and genotyped using the polymerase chain reaction (PCR) protocol provided by the Jackson Laboratory. Male mice were included for all analyses. 2.2. Micro-computed tomography (μCT) analysis After being fixed in 4 % paraformaldehyde for 24 h, the femurs and skulls of the mice were subjected to μCT scanning. In brief, the microarchitecture of the proximal trabecular bone and midshaft cortical bone of the femur were measured by Skyscan 1275 μCT (Bruker, Kontich, Belgium), as previously described ([98]Hong et al., 2018). Bones were placed vertically in a scanning holder and scanned at the following settings: 10 μm resolution, 55 kVp energy, 145 μA intensity, and an integration time of 200 ms. Two-dimensional slices from each femur were combined using NRecon and CTAn/CTVol programs (Bruker) to form a three-dimensional reconstruction and quantify trabecular bone volume per tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, mm^−1), cortical bone per tissue volume (CtV/TV, %), cortical bone thickness (Ct. Th, mm), and femur length (mm). 2.3. Histological analysis After being fixed in 4 % paraformaldehyde, the tibias are decalcified with 10 % EDTA (Ethylenediaminetetraacetic acid)-based solution and embedded in paraffin and longitudinally sectioned into 5-μm-thick slices. After deparaffinized and rehydrated, Sections were stained using Alcian Blue (Sigma-Aldrich, St. Louis, MO, USA) and Nuclear Fast Red (Abcam, Cambridge, UK). The thickness of the Alcian blue-stained growth plates at the proximal tibia was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA). 2.4. Mouse BMSC harvest and culture Mouse BMSCs were isolated from 4 to 6 weeks old mice. Mice were sacrificed, and femurs and tibias were harvested. Bone marrow was flushed from the bones using 25-gauge needles and 3 ml syringes (BD Medical, Franklin Lakes, NJ, USA) and was pipetted into a homogeneous cell suspension and filtered through a 70-μm nylon mesh filter (BD Falcon, Franklin Lakes, NJ, USA) prior to plating. The cells were cultured in growth media (GM) consisting of MEM alpha with 10 % Fetal Bovine Serum (FBS), 1 % Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). 3 days after initial plating, the media containing any non-adherent cells was carefully removed and replaced with fresh growth media every 3–4 days. 2.5. Differentiation of mouse BMSCs into osteoblasts The potential of mouse BMSCs to differentiate into osteogenic lineages was examined using the following procedures. 1 × 10^5 cells were cultured in each well of a 24-well tissue culture plate for differentiation. When the cultures were 60–80 % confluent, the growth medium was replaced with osteogenic induction medium (OIM), which consists of MEM alpha medium containing 8 % FBS, 0.1 μM dexamethasone, 100 mM beta-glycerophosphate and 50 μg/ml ascorbic acid-2-phosphate. OIM was changed every 3 days to promote differentiation. On day 21 post-treatment, the cells were stained with 2 % Alizarin Red S (ARS) (Sigma-Aldrich, St. Louis, MO, USA) to evaluate osteogenic differentiation. For the control group, 1 × 10^5 cells were cultured with complete growth media, without differentiation agents. For JNK inhibitor (SP600125; Santa Cruz Biotechnology, TX, USA) treatment, cells were cultured with SP600125 (5 μM) and NGF (10 ng/ml). The lentiviral vector used to overexpress KDM4B, pLV[Exp]-EGFP:T2A:Puro-EF1A > mKdm4b (pLV-Kdm4b, ID: VB900020-9777kgb) and the empty lentiviral vector (pLV-empty) was constructed by VectorBuilder Inc. (Chicago, IL, USA). The cells were cultured with Vehicle (Saline) or NGF (10 ng/ml). Transfection of lentivirus vectors were performed according to the manufacturer's protocol before starting osteogenic differentiation. 2.6. Western blot of mouse BMSCs Cells were lysed in RIPA buffer containing protein inhibitors. Equal amounts of protein were resolved by SDS-PAGE and analyzed for the target proteins. Protein bands were visualized using an enhanced chemiluminescence assay (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions and scanned with a densitometer. The following antibodies were used: anti-phospho-JNK (Cell Signaling Technology, Danvers, MA, USA) and anti-phospho-c-Jun (Cell Signaling Technology, Danvers, MA, USA). 2.7. Quantitative real-time polymerase chain reaction (qRT-PCR) The total RNA from mouse BMSCs was extracted 7 days post culture using RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using SuperScript® III Reverse Transcriptase Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, qRT-PCR was performed using PowerUp™ SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol, and reactions were run on a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The sequences of the primers used for qRT-PCR were described in [99]Table S1. Primers were designed using Primer-Blast ([100]http://www.ncbi.nlm.nih.go/tools/primer-blast). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) served as control and the fold induction was calculated using the comparative ΔCt method and presented as relative transcript levels (2 − ΔΔCt). 2.8. Expression analysis of p75^NTR in p75^NTRf/f and Prx1-Cre; p75^NTR f/f tissues and cells Spleen, heart, liver, kidney, and bones were dissected from p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice. BMSCs were harvested as described above, and non-adherent cells were obtained as monocytes. Total RNAs of tissues and cells were harvested using RNeasy mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using SuperScript® III Reverse Transcriptase Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). PCR products amplified using p75^NTR and Gapdh primers were subjected to electrophoresis on a 1.5 % agarose gel. 2.9. BMSC transcriptome NanoString nCounter analysis The NanoString nCounter analysis in mouse BMSCs was conducted by the Core Center for Musculoskeletal Biology and Medicine (CCMBM) at UCSF. Mouse BMSCs used in the experiment were at passages 2 to 5 and were cultured with GM. Briefly, a second set of aliquoted RNA samples was used for NanoString comparison analysis. Each target gene of interest was detected using a pair of reporters and capture probes that target a continuous 100 nucleotide sequence. Hybridization between target mRNA and reporter-capture probe pairs was performed at 65 °C for 20 h using CT1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer's protocol. Post-hybridization processing was carried out on a fully automated nCounter Prep station liquid-handling robot. Excess probes were removed, and the probe/target complexes were aligned and immobilized in the nCounter cartridge, which was then placed in a digital analyzer for image acquisition and data processing (nCounter Digital Analyzer) as per the manufacturer's protocol. To assess a bunch of genes related to skeleton development, NanoString PanCancer IO360 Gene Expression panel was used. The expression level of a gene was measured by counting the number of times the specific barcode for that gene was detected, and the barcode counts were then tabulated in a comma-separated value (CSV) format. The raw digital count of expression was exported from nSolver v4.0 software for downstream analysis. Differential gene expression (DEG) analysis between p75^NTR+/+ and p75^NTR−/− groups was performed using the limma package in RStudio (Posit, PBC, MA, USA). Pathway enrichment analysis was performed using Enrichr ([101]https://maayanlab.cloud/Enrichr/) and WebGestalt ([102]https://www.webgestalt.org/). 2.10. Statistics All statistical analyses were performed using GraphPad Prism 10.4.1 (GraphPad Software, San Diego, CA, USA), except for the DEG analysis in the nCounter transcriptome assay. Normality of data was assessed using the Shapiro-Wilk test. For comparisons between two groups, unpaired two-tailed Student's t-tests were applied. For comparisons involving multiple groups, one-way or two-way ANOVA was performed, followed by Tukey's post hoc test for multiple comparisons. All in vitro experiments were independently repeated at least three times. Error bars in all figures represent the mean ± SEM. Statistical significance was indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. 3. Results 3.1. p75^NTR deficiency exhibits impaired skeletal growth and osteoporosis in vivo To investigate the role of p75^NTR in skeletal development, we examined mice with global deletion of p75^NTR (p75^NTR−/−) for postnatal phenotypic differences. p75^NTR−/− mice were born at expected Mendelian ratios with no evidence of perinatal lethality, indicating that p75^NTR deficiency does not impair embryonic survival. However, these mice exhibited progressive postnatal growth retardation, with significantly reduced body size and weight apparent by postnatal day 3 and persisting through 4 weeks of age ([103]Fig. 1A–B). Fig. 1. [104]Fig. 1 [105]Open in a new tab Global p75^NTR-deficient mice exhibited an osteoporotic phenotype. (A) Photos of 4 and 12-week-old p75^NTR+/+ and p75^NTR−/− mice. Scale bar: 4.0 cm. (B) Body weight curve of p75^NTR+/+ and p75^NTR−/− mice from P0 - P28. Three-dimensional Micro-CT images of 4 (C) and 12 (E) weeks old p75^NTR+/+ and p75^NTR−/− mice's whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th and length of femurs of 4 (D) and 12 (F) weeks old p75^NTR+/+ and p75^NTR−/− mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75^NTR+/+ (n = 3) and p75^NTR−/− (n = 4). Alcian blue staining images of the growth plate of 4 (G) and 12 (I) weeks old p75^NTR+/+ and p75^NTR−/− mice's tibias. The growth plate thickness (GP. Th) of 4 (H) and 12 (J) weeks old p75^NTR+/+ and p75^NTR−/− mice were quantified. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75^NTR+/+ (n = 6) and p75^NTR−/− (n = 5). Unpaired Student's t-test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed. At 4 weeks, p75^NTR−/− mice showed significant reductions in trabecular bone parameters—including BV/TV, Tb. Th, and Tb.N—compared to littermate controls ([106]Fig. 1C–D). Cortical bone measurements, including CtV/TV and Ct. Th, were also significantly decreased in p75^NTR−/− mice ([107]Fig. 1D). Consistent with these deficits, femur length was reduced by 23 % in p75^NTR−/− mice compared to controls ([108]Fig. 1D). To further assess growth plate morphology, we performed Alcian blue staining and found a significant reduction in growth plate thickness (GP·Th) in p75^NTR−/− mice at 4 weeks ([109]Fig. 1G–H). These findings suggest that p75^NTR deficiency impairs both trabecular and cortical bone formation, as well as growth plate development, during early postnatal skeletal growth. To assess whether p75^NTR deficiency also affects later stages of skeletal development and maturation, we analyzed adult mice at 12 weeks of age. Similar to the phenotype observed at 4 weeks, p75^NTR−/− mice at 12 weeks remained visibly smaller than their p75^NTR+/+ littermates ([110]Fig. 1A). femur length was significantly shorter, and both trabecular and cortical bone parameters were reduced in p75^NTR−/− mice compared to controls ([111]Fig. 1E–F). Growth plate architecture also remained disrupted, with reduced cartilage content and decreased thickness relative to littermate controls ([112]Fig. 1I–J). Phenotypic analysis of trabecular and cortical bone by Micro-CT was consistent with previous reports showing diminished bone parameters in 8-week-old p75^NTR−/− mice based on Micro-CT analysis ([113]Zhao et al., 2020). Taken together, these data indicate that global p75^NTR deficiency results in sustained postnatal growth impairment and compromised bone formation, affecting trabecular and cortical bone as well as growth plate integrity—hallmarks of an osteoporotic phenotype. 3.2. Specific deletion of p75^NTR in mesenchyme inhibits skeletal growth and bone mass regulation in vitro The phenotypes observed in p75^NTR−/− mice demonstrated that loss of p75^NTR is associated with decreased bone formation. However, these anti-osteogenic effects in p75^NTR−/− mice could be due to indirect systemic factors. To directly assess the role of p75^NTR in osteogenesis, we employed a conditional knockout approach using Prx1-Cre system crossed with a conditional allele of p75^NTR ([114]Logan et al., 2002). This strategy allowed for targeted deletion of p75^NTR specifically in mesenchymal progenitor cells. Specific deletion of p75^NTR expression was confirmed in bone tissues and BMSCs of Prx1-Cre; p75^NTR f/f mice, which had no change of p75^NTR expression as shown by PCR ([115]Fig. 2A and B). Fig. 2. [116]Fig. 2 [117]Open in a new tab MSC-specific p75^NTR-deficient mice exhibited an osteoporotic phenotype. (A) PCR results of p75^NTR in p75^NTRf/f and Prx1-Cre;p75^NTR f/f spleen, heart, liver, kidney, muscle, and bone. (B) PCR results of p75^NTR in p75^NTRf/f and Prx1-Cre; p75^NTR f/f monocyte and BMSC. p75^NTR expression was not detected in BMSCs. (C) Photos of 4 and 12-week-old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice. Scale bar: 4 cm. (D) Body weight of 4 and 12-week-old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice. For 4-week-old mice, p75^NTRf/f (n = 3) and Prx1-Cre; p75^NTR f/f (n = 4). For 12-week-old mice, p75^NTRf/f (n = 5) and Prx1-Cre; p75^NTR f/f (n = 4). Three-dimensional Micro-CT images of 4 (E) and 12 (G) weeks old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th, and length of femurs of 4 (F) and 12 (H) weeks old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, p75^NTRf/f (n = 4) and Prx1-Cre; p75^NTR f/f (n = 7). For 12-week-old mice, p75^NTRf/f (n = 6) and Prx1-Cre; p75^NTR f/f (n = 6). Alcian blue staining images of the growth plate of 4 (I) and 12 (K) weeks old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice's Tibias. The growth plate thickness (GP. Th) of 4 (J) and 12 (L) weeks old p75^NTRf/f and Prx1-Cre; p75^NTR f/f mice were quantified. For both ages, n = 5 for each group. Unpaired Student's t-test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed. Notably, Prx1-Cre; p75^NTR f/f mice exhibited growth patterns that parallel those observed in p75^NTR−/−. Prx1-Cre; p75^NTR f/f mice have consistently smaller body size and lower body weights compared to p75^NTRf/f littermates at both 4 weeks and 12 weeks of age ([118]Fig. 2C and D), suggesting p75^NTR signaling in mesenchymal-lineages may be critical for normal postnatal growth trajectories. To further characterize observed phenotypes, Micro-CT analysis of the femurs of Prx1-Cre; p75^NTR f/f mice was performed. At 4 weeks of age, Prx1-Cre; p75^NTR f/f mice had significant deficits in trabecular and cortical bone architecture compared to p75^NTRf/f littermates shown by BV/TV, Tb. Th, and CtV/TV volumetric analysis ([119]Fig. 2E and F). These deficits were only exacerbated by age. Adult 12-week-old Prx1-Cre; p75^NTR f/f mice exhibited greater reductions in BV/TV, Tb. Th, Tb. N, CtV/TV, Ct. Th and femur length when compared to age-matched p75^NTRf/f littermates ([120]Fig. 2G and H). Prx1-Cre; p75^NTR f/f mice also had demonstrably thinner growth plate thickness compared to p75^NTRf/f controls at both 4- and 12-weeks of age ([121]Fig. 2I-L). In both genotypes, growth plate thickness also decreased with age. These skeletal abnormalities all closely resembled the osteoporotic phenotypes observed in p75^NTR−/− mice. Collectively, these findings indicate that the osteoporotic phenotype associated with p75^NTR deficiency is primarily due to impaired mesenchyme-mediated bone formation. 3.3. p75^NTR directly regulates BMSC osteogenesis and bone formation in vitro Since the deletion of p75^NTR in mesenchyme resulted in an impaired bone phenotype in vivo, we investigated whether p75^NTR plays a cell-autonomous role in MSC osteoblast differentiation. To determine whether p75^NTR has a direct impact on MSC osteogenesis, bone marrow was isolated from the femur and tibia of p75^NTR+/+ and p75^NTR−/− mice ([122]Fig. 3A). BMSCs were subsequently cultured in osteogenic induction media (OIM) to assess their differentiation potential ([123]Fig. 3B). Fig. 3. [124]Fig. 3 [125]Open in a new tab p75^NTR mediates osteogenic differentiation of MSCs. (A) Schematic representation of the osteogenic differentiation process of BMSCs derived from p75^NTR+/+ and p75^NTR−/− mice. Created in BioRender. Kadota, C. (2025) [126]https://BioRender.com/a77c926 (B) Alizarin Red Staining (ARS) images of BMSCs harvested from p75^NTR+/+ and p75^NTR−/− mice cultured in GM and OIM for 21 days, along with quantification of Alizarin Red-positive areas. n = 4 for each group. (C) qRT-PCR analysis of osteoblast differentiation markers in BMSCs cultured in OIM. The expression of osteoblastic differentiation markers, including PTHrP, Runx2, Dlx5, Osx, and Alp, were compared, and p75^NTR−/− showed significant downregulation compared to p75^NTR+/+. n = 3 for each group. Unpaired Student's t-test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of experimental replicates. The loss of p75^NTR significantly impaired the mineralization capacity of BMSCs. Mineralized nodules, as evaluated by the percentage area of ARS staining, were markedly reduced in p75^NTR−/− BMSCs compared to p75^NTR+/+ ([127]Fig. 3B). To validate the mineralization data, expression analysis of key osteogenic gene markers was performed. Transcripts for Osterix (Osx), Runt-related transcription factor 2 (Runx2), Alkaline phosphatase (Alp), Parathyroid hormone-related protein (Pthrp), and Dlx5 were all suppressed during differentiation in p75^NTR−/− BMSCs ([128]Fig. 3C). These findings provide molecular evidence that p75^NTR is a critical regulator of BMSC osteogenesis. Overall, it was demonstrated that p75^NTR intrinsically regulates BMSC bone formation by promoting osteogenic differentiation and supporting the expression of essential osteogenic genes. 3.4. Transcriptomic analysis reveals downregulation of p75^NTR-NGF-MAPK/AP-1 Axis in p75^NTR−/− BMSCs To investigate the mechanisms responsible for the phenotypes observed in vivo and in vitro due to p75^NTR deficiency, we performed a targeted gene expression analysis using the NanoString nCounter platform on BMSCs isolated from 4-week-old p75^NTR+/+ and p75^NTR−/− littermates. A total of 637 genes were analyzed, and the genes with p < 0.05 were considered significant as shown in the volcano plot ([129]Fig. 4A). 15 genes were highlighted as significantly different between p75^NTR+/+ and p75^NTR−/− ([130]Fig. 4B); 14 were significantly downregulated (blue dots) and 1 was upregulated (red dots) in p75^NTR−/− BMSCs compared to p75^NTR+/+ ([131]Fig. 4A and [132]Table S2). To examine the signaling pathways and cellular functions associated with these 15 genes, we performed pathway analysis using Enrichr, a comprehensive gene set enrichment analysis tool that integrates multiple biological databases to identify relevant pathways, molecular functions, and biological processes associated with a given gene list. The analysis was performed using the BioPlanet 2019, ([133]Fig. 4C) BioCarta 2016, and ([134]Fig. 4D) Reactome pathways 2024 ([135]Fig. 4E). Notably, although p75^NTR is a low-affinity receptor for all neurotrophins including NGF, BDNF, NT-3, and NT-4, only the NGF signaling pathway was affected in p75^NTR−/− BMSCs, ranking as the most significantly downregulated pathways based on analyses using the BioPlanet 2019 and BioCarta 2016 databases. In addition, AP-1 (Activator Protein-1) and MAPK (Mitogen-Activated Protein Kinase) signaling pathways were also ranked among the top enriched pathways ([136]Fig. 4C and E). Importantly, similar pathway analysis results were observed even after excluding Ngfr, which was already knocked out in the model, using the remaining 14 genes ([137]Fig. S1). Even after performing GSEA (gene set enrichment analysis) using the top 100 genes ranked by ascending p-value, MAPK signaling-related pathways were listed as one of the down-regulated pathways among the top enriched pathways again ([138]Fig. S1D). Both the AP-1 and MAPK pathways are known downstream effectors of p75^NTR activation by NGF. Specifically, NGF binding to p75^NTR can initiate JNK (a member of the MAPK family) signaling, leading to the activation of AP-1, which in turn regulates a variety of cellular processes, including proliferation, differentiation, and apoptosis ([139]Meeker and Williams, 2015; [140]Coulson et al., 2004). Thus, these results indicate that the loss of p75^NTR impacts the NGF-p75^NTR-MAPK/AP-1 axis, resulting in impaired osteoblast differentiation from BMSCs. Fig. 4. [141]Fig. 4 [142]Open in a new tab p75^NTR-deficient BMSCs showed reduced NGF signaling and MAPK activation. (A) Volcano plot shows gene expression changes in bone marrow stem cells (BMSCs) derived from 4-week-old p75^NTR+/+ and p75^NTR−/− mice (n = 3 per group). Expression was profiled using the NanoString nCounter platform in a panel of 477 genes. The x-axis represents the log[2]fold change in p75^NTR−/− relative to p75^NTR+/+ and the y-axis is the –log[10](p-value). The dashed horizontal line indicates p = 0.05. Among the 637 genes tested, 15 were significantly downregulated (p < 0.05) in p75^NTR−/− BMSCs. Red dots indicate upregulated genes, while blue dots indicate downregulated genes in p75^NTR−/−. (B) Heatmap of the 15 differentially expressed genes. Red indicates higher expression, while blue indicates lower expression. (C)-(E) Bar graph of pathway enrichment analysis for the 15 differentially expressed genes. The analysis was performed using the BioPlanet 2019, (C) BioCarta 2016, and (E) Reactome pathways 2024. The y-axis lists the top 15 significantly enriched terms (p < 0.05), and the x-axis shows the –log[10](p-value). n represents the number of experimental replicates. Figure S1. Figure S1 [143]Open in a new tab Pathway analysis of p75^NTR-deficient BMSCs with significant 14 genes without Ngfr shows down-regulation of NGF and MAPK signaling pathways. Bar graph of pathway enrichment analysis for the 14 differentially expressed genes after excluding Ngfr. The analysis was performed using the (A) BioPlanet 2019, (B) BioCarta 2016, (C) Reactome pathways 2024. The y-axis lists the top 15 significantly enriched terms (p < 0.05), and the x-axis shows the –log[10](p-value). (D) GSEA using the top 100 genes ranked by ascending p-value. The y-axis shows the pathways, and the x-axis shows the Normalized Enrichment Score. 3.5. JNK signaling mediated p75^NTR-dependent osteogenic differentiation via KDM4B and Dlx5 is regulated by JNK signaling pathway Our previous study demonstrated that JNK is an essential mediator of the p75^NTR-NGF-MAPK/AP-1 axis and induces osteogenesis in human DMSC ([144]Liu et al., 2022). Therefore, the next step was to determine if perturbation of the JNK signaling pathway leads to the osteogenesis defects observed in this study. First, protein expression of the JNK pathway in BMSCs from p75^NTR+/+ and p75^NTR−/− was evaluated with Western blot analysis. Results showed that levels of phosphorylated JNK (p-JNK) and its downstream target phosphorylated c-JUN (p-c-JUN) were significantly reduced in BMSCs from p75^NTR−/− mice compared with those from p75^NTR+/+ litters ([145]Fig. 5A). In addition, treatment with SP600125, a JNK inhibitor, suppressed NGF-mediated osteogenic mineralization in vitro, confirming the critical role of JNK signaling in this pathway ([146]Fig. 5B). This suggests that the inhibition of JNK activity suppresses NGF-mediated osteoblast differentiation in mouse BMSCs. Furthermore, the activation of JNK signaling in NGF-induced osteoblast differentiation in human DMSCs involves epigenetic genes, including key histone modifiers such as Kdm4b, as downstream targets ([147]Liu et al., 2022). Based on this, we used qRT-PCR to examine the expression changes of epigenetic genes downstream of NGF-induced JNK signaling activation in BMSCs from p75^NTR+/+ and p75^NTR−/− mice. NGF treatment significantly upregulated the expression of several histone demethylases, including Kdm2b, Kdm4a, Kdm4b, Kdm4c, Kdm5a, Kdm6a, and Kdm6b, in p75^NTR+/+ BMSCs ([148]Fig. 5C). In p75^NTR+/+ mice, Kdm4b had the most demonstrable difference between the control and NGF-treated groups. NGF treatment led to a statistically significant increase in Kdm4b expression in p75^NTR+/+ BMSCs. A mild increase was also observed in p75^NTR deficient BMSCs, but this change was not statistically significant. At the same time, the expression of Kdm4b was significantly decreased in p75^NTR−/− BMSCs compared to p75^NTR+/+ BMSCs for both control and NGF-treated groups ([149]Fig. 5C). Taken together, this data suggests that the expression of Kdm4b is regulated by NGF-mediated p75^NTR signaling. Fig. 5. [150]Fig. 5 [151]Open in a new tab KDM4B regulates p75^NTR-mediated osteogenesis via JNK signaling pathway. (A)Western blot of JNK signaling pathway (p-JNK and p-c-Jun) in p75^NTR+/+ and p75^NTR−/− BMSCs. (B) ARS images and quantification after 21 days treatment in OIM with NGF (10 ng/ml) and/or SP600125 (5 μM). n = 4 for each group. (C) qRT-PCR analysis of epigenetic genes in p75^NTR+/+ and p75^NTR−/− BMSCs with or without NGF treatment at 2 h. n = 3 for each group. (D) ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV-Kdm4b, and NGF (10 ng/ml). n = 4 for each group. (E) Upper panel: ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV-Kdm4b in p75^NTR+/+ and p75^NTR−/− BMSCs. Lower panel: gene expression analysis of Kdm4b and Dlx5 after treating with pLV-empty or pLV-Kdm4b. Dlx5 expression increased along with the upregulation of Kdm4b. n = 4 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-way ANOVA with multiple comparison was used for c, d and e, followed by Tukey's post-hoc test. n represents the number of experimental replicates. To investigate the function of KDM4B on NGF-induced osteoblast differentiation, we overexpressed Kdm4b in mouse BMSCs and induced osteogenic differentiation in vitro. Lentiviral overexpression of Kdm4b in p75^NTR+/+ BMSCs significantly enhanced mineralization compared to empty vector-treated cells ([152]Fig. 5D-E). Notably, NGF treatment with Kdm4b-overexpressing p75^NTR+/+ BMSCs led to an even greater induction of mineralization ([153]Fig. 5D), suggesting that NGF enhances osteogenic differentiation with Kdm4b. Furthermore, we examined whether the overexpression of Kdm4b could rescue the impaired osteogenic differentiation phenotype in p75^NTR−/− cells. Kdm4b overexpression in p75^NTR-deficient BMSCs was able to rescue the reduced osteogenic differentiation, restoring their ability to mineralize ([154]Fig. 5E). Gene expression analysis by qRT-PCR revealed that the overexpression of Kdm4b increased its own expression while simultaneously upregulating Dlx5 ([155]Fig. 5E), which has been reported as a downstream target of KDM4B ([156]Liu et al., 2022). Dlx5 is a critical gene for osteoblast differentiation, which can stimulate master regulators of osteoblast differentiation such as Runx2 ([157]Kawane et al., 2014; [158]Holleville et al., 2007). These findings suggest that NGF-induced p75^NTR-mediated osteogenesis is regulated by the JNK signaling pathway, specifically targeting KDM4B and its downstream osteogenic gene, Dlx5. 4. Discussion Global deletion of p75^NTR exhibited delayed bone growth and osteoporotic bone mass reduction in the long bones of mice. These findings align with previous studies showing reduced bone mass in 8-week-old long bones ([159]Zhao et al., 2020) and in the alveolar bone of 1- and 4-month-old p75^NTR−/− mice ([160]Wang et al., 2020). However, as p75^NTR is expressed in both neural and non-neural tissues, the skeletal deficits observed in global knockouts could arise from indirect effects via nervous system dysfunction ([161]Wan et al., 2021). To address this, we generated Prx1-Cre; p75^NTR f/f mice where p75^NTR was selectively deleted in mesenchymal progenitor cells. These mice exhibited skeletal phenotypes nearly identical to those of global knockouts—including reduced femur length, diminished bone mass, and thinner growth plates—indicating that p75^NTR functions cell-autonomously within the mesenchymal lineage to regulate postnatal bone growth. These findings are supported by prior reports that p75^NTR deletion in PDGFRα-positive MSCs delays bone formation and impairs mesenchymal cell migration during bony defect regeneration ([162]Xu et al., 2022). Together, our data reinforces a critical role for p75^NTR in MSC-mediated osteoblast differentiation and matrix mineralization. Our in vitro findings further validate the in vivo phenotypes. p75^NTR−/− BMSCs showed a markedly reduced osteogenic differentiation capacity. The consistent impairment observed in both global and conditional knockout models, as well as in isolated BMSCs, underscores the intrinsic role of p75^NTR in promoting MSC-to-osteoblast commitment. Moreover, the reduction in femur length and growth plate thickness observed in both knockout models points to a potential role for p75^NTR in endochondral ossification, an area that remains underexplored. While the p75^NTR expression in cartilage has been reported ([163]Gigante et al., 2003; [164]Wen et al., 2012), its precise function in chondrocytes and growth plate maturation warrants further investigation. To gain mechanistic insights, we performed transcriptome analysis using the NanoString nCounter platform. Among 637 genes analyzed, 15 were significantly downregulated in p75^NTR−/− BMSCs compared to p75^NTR+/+ BMSCs, with enrichment analysis consistently highlighting disruption of the NGF signaling pathway. While p75^NTR binds multiple neurotrophins, our data suggests that NGF is the primary ligand mediating p75^NTR-dependent osteogenic signaling in BMSCs. Pathway analyses across multiple databases identified the NGF-p75^NTR-MAPK/AP-1 axis as significantly downregulated in p75^NTR deficient cells. The MAPK cascade, which includes JNK, p38, and ERK ([165]Kim and Choi, 2010), is known to promote osteoblast differentiation and bone formation ([166]Hayrapetyan et al., 2015; [167]Schindeler and Little, 2006). Although our study focused on JNK, it is likely that p75^NTR also interfaces with other MAPK branches to orchestrate osteogenesis. These findings highlight the broader complexity of MAPK signaling in skeletal development and suggest that p75^NTR may act as a central integrator of multiple signaling cues in the bone microenvironment. Previous work has shown that p75^NTR influences MSC proliferation, migration, and differentiation via both direct and paracrine mechanisms. For instance, Xu et al. reported that in a bone defect model of NGF^LysM mice, deletion of p75^NTR in myeloid cells led to reduced migration of PDGFRα-positive MSCs, likely via altered paracrine effects ([168]Xu et al., 2022). Similarly, Shan et al. found that ectomesenchymal stem cells from p75^NTR−/− mice had reduced proliferation via NF-κB signaling and impaired mineralization ([169]Shan et al., 2022). In our previous study, we demonstrated that NGF-p75^NTR signaling in human craniofacial MSCs activated JNK–KDM4B–DLX5 to promote osteogenesis ([170]Liu et al., 2022). Building on that, the current study provides direct evidence that loss of p75^NTR impairs JNK pathway activation and reduces the expression of its downstream effector, Kdm4b. Overexpression of Kdm4b restored osteoblast differentiation in p75^NTR−/− BMSCs and enhanced NGF-induced mineralization, suggesting that KDM4B is both necessary and sufficient for mediating NGF–p75^NTR–dependent bone formation. These findings position Kdm4b as an essential epigenetic mediator downstream of JNK, and they extend our understanding of how neurotrophin signaling regulates MSC fate. Importantly, in both control and p75^NTR−/− BMSCs, Kdm4b overexpression also increased the expression of Dlx5, a master osteogenic transcription factor and known target of KDM4B. Given that Dlx5 regulates key downstream effectors such as Runx2 and is indispensable for skeletal development ([171]Holleville et al., 2007), our findings suggest that disruption of the KDM4B–Dlx5 axis may be a major contributor to the impaired osteogenesis observed in p75^NTR-deficient mice. In the present study, while our findings establish a fundamental role for p75^NTR in skeletal development, the study has several limitations. Because bone is composed of many diverse and interacting bone-resident cell types, it is difficult to precisely attribute the observed phenotypes to specific cellular components. Future studies employing additional lineage-specific Cre drivers, combined with dynamic histomorphometry, will be essential for dissecting the contributions of distinct p75^NTR-expressing skeletal cell types to bone formation and remodeling. In addition, although we used targeted gene expression profiling, more comprehensive approaches such as bulk RNA-seq or single-cell RNA-seq will be critical for uncovering additional pathways and cellular heterogeneity involved in p75^NTR signaling. 5. Conclusion Overall, this study demonstrated that the function of p75^NTR in mesenchymal cells is crucial for bone formation in vivo. Additionally, we showed that p75^NTR may contribute to long bone growth and bone mass maintenance by promoting NGF-responsive osteoblast differentiation. Our findings suggest that p75^NTR regulates osteoblast differentiation in mouse BMSCs through the NGF-JNK signaling pathway by enhancing the expression of Kdm4b and Dlx5. These results contribute to the understanding of the pathogenesis of congenital bone disorders and age-related osteoporosis and are expected to serve as a foundation for the development of novel therapeutic strategies in the future. The following are the supplementary data related to this article. Table S1 Primers used for qPCR. [172]mmc2.docx^ (15.5KB, docx) Table S2 List of Significantly Differentially Expressed Genes in BMSC Identified by Nanostring nCounter Analysis (p < 0.05) [173]mmc3.docx^ (14.9KB, docx) CRediT authorship contribution statement Chiho Kadota-Watanabe: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jinsook Suh: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Zhenqing Liu: Writing – review & editing, Investigation, Formal analysis, Data curation. Eric Yin: Writing – review & editing, Visualization, Formal analysis. Kate Lindsey: Writing – review & editing, Investigation, Formal analysis. Isabelle Lao-Ngo: Writing – review & editing, Visualization, Formal analysis. Byron Zhao: Writing – review & editing, Investigation, Formal analysis. Jonathan H. Wu: Writing – review & editing, Investigation, Formal analysis. In Won Chang: Writing – review & editing, Investigation, Formal analysis, Data curation. Reuben H. Kim: Writing – review & editing, Supervision, Resources, Investigation, Formal analysis. Ophir D. Klein: Writing – review & editing, Supervision, Methodology, Formal analysis, Conceptualization. Christine Hong: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Conceptualization. Funding This work was supported by the Resource Allocation Program (RAP) grant, Core Center for Musculoskeletal Biology and Medicine (CCMBM) of UCSF. Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability The data that support the findings of this study are available on request from the corresponding author. References