Upregulation of Parathyroid Hormone Receptor 1 (PTH1R) in Non-Mechanostimulated Osteocytes Under High-Glucose Conditions Promotes a Macrophage Pro-Inflammatory and Osteoclastogenic Phenotype via IL-6 Secretion

TL;DR

High glucose levels in bone cells increase PTH1R, leading to inflammation and bone breakdown via IL-6, which could be a new treatment target for diabetic bone disease.

Contribution

Identifies PTH1R upregulation in osteocytes under high glucose as a novel driver of macrophage inflammation and osteoclastogenesis via IL-6 secretion.

Findings

High glucose in non-mechanostimulated osteocytes increases PTH1R expression and IL-6 secretion.

IL-6 promotes macrophage M1 polarization and upregulates osteoclastogenic markers like TRAP and RANK.

Blocking IL-6 reduces macrophage inflammation, confirming its role in the osteocyte–macrophage signaling axis.

Abstract

Diabetes mellitus disrupts bone homeostasis, inducing bone fragility, through mechanisms involving chronic inflammation and altered cellular signaling. Osteocytes, the primary mechanosensory cells in bone, play a pivotal role in regulating bone remodeling via the secretion of factors that influence both osteoclast and osteoblast activity. We investigated the impact of high glucose on osteocytic parathyroid hormone receptor type 1 (PTH1R) expression and its downstream effects on interleukin-6 (IL-6) secretion, macrophage polarization, and osteoclastogenesis. Using both in vitro and ex vivo bone models, we demonstrate that elevated glucose levels in static conditions without mechanical stimulation induce the overexpression of PTH1R in osteocytes. PTH1R upregulation in turn enhances osteocytic IL-6 secretion associated with the promotion of a pro-inflammatory macrophage M1 phenotype (increased tumor necrosis factor (TNF)-α/CD206 and inducible nitric oxide synthase (iNOS)/CD206 ratios) and the upregulation of the pro-osteoclastogenic markers tartrate-resistant acid phosphatase (TRAP) and receptor activator of nuclear factor kappa-Β (RANK). Neutralization of IL-6 in the osteocytic secretome attenuated macrophage inflammatory gene overexpression, underscoring IL-6’s critical role in this regulatory axis. Our findings reveal that a high-glucose environment triggers osteocytic dysregulation of PTH1R-mediated signaling pathways, amplifying inflammatory and osteoclastogenic activity in bone via IL-6. This osteocyte–macrophage crosstalk may contribute to the increased bone resorption and impaired regeneration observed in diabetic bone disease. Targeting PTH1R upregulation and the IL-6 signaling pathway in osteocytes could represent a novel therapeutic approach to mitigating bone complications associated with diabetes.

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Full text

1. Introduction

Diabetes mellitus (DM) is one of the most prevalent metabolic diseases worldwide, characterized by alterations in glucose metabolism that transcend simple glycemic control, affecting multiple organ systems, including bone tissue [1]. Chronic hyperglycemia and metabolic impairment may arise from either the autoimmune destruction of pancreatic $[eqn]$ -cells, which leads to insufficient insulin production in type 1 diabetes mellitus (T1D) [2], or from the coexistence of $[eqn]$ -cell dysfunction and insulin resistance in peripheral tissues, as occurs in type 2 diabetes mellitus (T2D) [3]. In childhood-onset T1D, impaired bone development leads to reduced strength and a markedly higher fracture risk [4], whereas in T2D, fracture susceptibility is also elevated despite normal or even increased BMD [5,6]. In patients with diabetes, elevated glucose and oxidative stress contribute to increasing the levels of advanced glycation end products (AGEs), which are thought to play a significant role in bone fragility [7,8]. Engagement of the receptor for AGEs (RAGE) in bone-derived cells promotes the release of inflammatory cytokines and reactive oxygen species (ROS), fostering persistent inflammation and enhancing bone resorption [9]. The accumulation of these deleterious factors also impairs bone quality and formation by disrupting collagen, osteocalcin and osteoblast mineralization [10].

Osteocytes, the predominant cell type within bone tissue, sense mechanical stimuli and accordingly adjust the secretion of signaling molecules that influence both osteoclast and osteoblast activity [11,12]. In response to mechanical loading and unloading, osteocytes release specific signaling molecules, targeting surrounding cells in the bone microenvironment, primarily through the RANKL/OPG pathways [13]. Osteocytes represent a major source of Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL), which is essential for osteoclast differentiation [14]. Conversely, they also produce osteoprotegerin (OPG), a soluble receptor that binds RANKL and prevents osteoclast formation. In this way, osteocytes can promote bone resorption by increasing RANKL and reducing OPG or alternatively inhibit it by shifting the balance in favor of OPG [15]. In fact, lack of mechanical stimulation induces osteocytes to produce RANKL and promote osteoclastogenesis [16]. Interestingly, high glucose has been shown to affect osteocyte function by inducing osteocyte apoptosis, inhibiting the ERK/PI3K/Akt signaling pathway, suppressing the Wnt/ $[eqn]$ -catenin pathway, and compromising mechanotransduction [17,18]. However, the putative concomitant actions of a high-glucose environment and static non-mechanostimulated conditions on osteocyte communication with other bone cells has not yet been extensively explored.

Type 1 parathyroid hormone receptor (PTH1R) is a key component in regulating bone homeostasis through its expression in osteocytes. PTH1R is stimulated by its agonists parathyroid hormone (PTH) and PTH-related protein (PTHrP) or by a mechanical and ligand-independent mechanism in these cells, triggering signaling cascades that influence both bone formation and resorption [19,20,21]. Under physiological conditions, activation of PTH1R in osteocytes rapidly suppresses the expression of sclerostin (SOST), which translates into greater bone formation. At the same time, PTH1R enhances the expression of RANKL in osteocytes, promoting osteoclast differentiation and activation [22,23]. In a diabetic context, actions of PTH1R are markedly impaired in osteocytes, which display a decreased inhibition of SOST expression leading to elevated levels of sclerostin, a factor that limits bone formation [1,19].

Within the bone marrow cavity, bone cells and immune cells coexist and interact through common regulatory factors. The immune system also contributes significantly to tissue repair and regeneration, influencing the overall regenerative potential of bone [24]. Acute inflammation promotes the proliferation and differentiation of osteoblast cells [25], while chronic inflammation suppresses factors that activate these cells, inhibiting the formation of new bone and promoting osteolytic lesions [26]. For this reason, the ability of osteocytes to communicate with the surrounding immunologic microenvironment in bone is essential, with particular emphasis on macrophages [27]. Macrophages in bone tissue can polarize according to different stimuli from their environment, grouping into two main types: M1 or classically activated macrophages, which have pro-inflammatory functions, and M2 or alternatively activated macrophages, which play anti-inflammatory roles in immune regulation and tissue repair or remodeling [28]. M1 macrophages are activated by inflammatory cytokines (i.e., IFN- $[eqn]$ and TNF- $[eqn]$ ) and bacterial lipopolysaccharide (LPS), producing numerous pro-inflammatory cytokines such as TNF- $[eqn]$ , IL-1 $[eqn]$ , and IL-6, among others. The primary function of M1 macrophages is to eliminate pathogens via the generation of ROS and nitric oxide (a compound synthesized by the inducible nitric oxide synthase [iNOS]), which can cause tissue damage and hinder tissue repair [29,30]. In bone, M1 macrophages promote resorption by serving as osteoclast precursors and releasing cytokines that stimulate osteoclastogenesis, inhibit osteoblasts, and enhance RANKL-mediated osteoclast differentiation [31]. Anti-inflammatory M2 macrophages polarize in response to cytokines such as IL-4 and IL-13 and display an anti-inflammatory cytokine profile, characterized by the production of IL-10 and transforming growth factor-beta (TGF- $[eqn]$ ) and expression of endocytic receptors such as CD163 and CD206 that act as M2 markers [32]. M2 macrophages promote osteogenesis by secreting osteoinductive factors such as IL-4, which enhances mesenchymal stem cell differentiation and upregulates osteogenic genes while simultaneously inhibiting osteoclastogenesis via RANKL downregulation and OPG upregulation [31]. Moreover, M2 macrophages recruit regulatory immune cells through the secretion of chemokines, possess a strong phagocytic capacity, and promote tissue repair, wound healing, angiogenesis, and fibrosis [33,34].

In the bone microenvironment, diabetes onset induces a profound metabolic reprogramming in macrophages, characterized by a shift toward glycolysis that results in increased production of ROS and the activation of pro-inflammatory pathways favoring polarization towards the M1 phenotype, preventing the proper transition from M1 to M2 that is essential for successful bone regeneration [35,36]. Subsequent chronic inflammation not only compromises osteogenic differentiation and promotes osteoblastic apoptosis but also stimulates osteoclastogenesis, resulting in a net imbalance toward bone resorption that characterizes diabetic bone alterations [37].

Based on these observations, we hypothesize that diabetes and static non-mechanostimulated conditions impact PTH1R in osteocytes leading to altered osteocytic secreted factors that modulate the deleterious actions of macrophages in bone in the absence of mechanical forces. Herein, we aim to determine whether high glucose induces PTH1R modulation in non-mechanostimulated osteocytes and whether these actions could alter the secretion of the factor IL-6, which regulates macrophage polarization and osteoclastogenesis.

2. Results

Diabetes profoundly affects bone by different mechanisms, including inflammation, which is associated in this setting with decreased bone formation and number of osteoblasts and increased number of osteoclasts and risk of fracture [38]. There is growing evidence that macrophages play an important role during bone inflammatory processes that might impact bone formation and repair [39].

Herein, we aimed to investigate whether osteocytes, master regulators of bone cell resorption and formation, might also modulate macrophage behavior and responses in high-glucose environments and the putative mechanism underlying this regulatory process. We first observed that RAW 264.7 macrophages exposed to a high-glucose culture medium and stimulated with the secretome of MLO-Y4 osteocytes incubated in high-glucose conditions (HG-osteocytes) overexpressed the pro-inflammatory cytokine and M1 marker TNF-α [40] and the osteoclastogenic markers RANK and TRAP [41] compared to macrophages stimulated with the secretome of osteocytes cultured in low-glucose medium (Figure 1A–H). No significant differences were observed in either the expression of the M1 marker iNOS and the M2 marker CD206 [32] or the key transcription regulator of osteoclast differentiation nuclear factor of activated T-cells 1 (NFATc1) [42] (Figure 1A–H). Remarkably, macrophage stimulation with high glucose in the absence of the osteocyte secretome did not significantly affect the expression of any of the genes studied (Figure 1A–H). Moreover, the production of reactive oxygen species (ROS), a feature associated with pro-inflammatory M1 macrophage polarization [43], was increased in RAW 264.7 cells stimulated with the secretome of osteocytes cultured in high-glucose medium compared to control cells (Figure 1I). The ROS assay provides functional support for the pro-inflammatory M1-like phenotype induced by the secretome of high-glucose-cultured osteocytes.

These findings suggest that in a high-glucose environment the secretome from osteocytes influences macrophages, potentially enhancing their M1 pro-inflammatory responses and promoting some pro-osteoclast differentiation features, according to the observed gene overexpression of TRAP and RANK.

Next, we evaluated whether the secretome of osteocytes could also modulate macrophage polarization or the osteoclastogenic response under M1- and M2-inducing conditions in a high-glucose environment. Our experiments corroborated that LPS induces gene expression changes associated with polarization towards an M1 phenotype, as evidenced by a significant increase in the levels of TNF- $[eqn]$ and iNOS M1 markers and downregulated expression of the M2 marker CD206 in RAW 264.7 macrophages (Figure 2A–C). In contrast, no significant effects of LPS were observed on the expression of the pro-osteoclastogenic genes RANK, TRAP and NFATc1 in the period of study (Figure 2F–H). Notably, macrophage exposure to glucose reduced LPS-dependent overexpression of TNF- $[eqn]$ and iNOS without changing CD206 levels (Figure 2A–C). Interestingly, the secretome from osteocytes further increased TNF-α expression, overcoming glucose inhibitory actions on the expression of this gene (Figure 2A), and induced upregulation of the levels of the osteoclastogenic gene TRAP in RAW 264.7 macrophages (Figure 2G).

These observations imply that the secretome from osteocytes cultured under high glucose might exacerbate some LPS-induced pro-inflammatory responses of macrophages.

We next confirmed that IL-4 induces opposite actions on macrophage M1 and M2 markers, thus decreasing TNF- $[eqn]$ and iNOS expression and overexpressing CD206 in RAW 264.7 macrophages (Figure 3A–C). In addition, the exposure of macrophages to high levels of glucose decreased IL-4-induced overexpression of CD206 in these cells without further affecting TNF- $[eqn]$ and iNOS decreased levels (Figure 3A–C). Unlike what we observed in LPS-treated cells, the secretome of HG osteocytes did not have an additive effect on the actions of IL-4 (Figure 3A–C). In fact, the secretome of HG osteocytes caused TNF- $[eqn]$ and iNOS overexpression, therefore reversing the actions of high glucose exposure alone in RAW 264.7 cells (Figure 3A,B). Additionally, regarding the osteoclastogenic genes, we observed that IL-4-stimulated macrophages only displayed upregulated TRAP levels regardless of their stimulation with high glucose or the secretome of osteocytes (Figure 3G,H). This upregulation was further enhanced by the secretome of osteocytes cultured in control (normal) glucose conditions (Figure 3G). These results suggest a pro-inflammatory effect on macrophages caused by the secretome of HG osteocytes.

Our findings mentioned above suggest that osteocytes secrete soluble factors under high-glucose culture conditions that might modulate macrophage polarization and osteoclastogenesis. We and others have previously described that osteocytic parathyroid hormone type I receptor (PTH1R) regulates the secretion of immunomodulatory and pro-osteoclastogenic factors such as IL-6 and RANKL, respectively [44,45]. Thus, we tested whether high-glucose conditions might modulate the expression of PTH1R using different cell models and ex vivo animal models. In this regard, mRNA upregulation of PTH1R was observed in MLO-Y4 and OCY-454 osteocytes and in ex vivo bones cultured in high-glucose conditions (Figure 4A–C). PTH1R protein overexpression was also confirmed in MLO-Y4 osteocytes (Figure 4D).

To evaluate the impact of high-glucose-induced upregulation of PTH1R on the expression of pro-immunomodulatory and pro-osteoclastogenic factors in osteocytes, we transfected a PTH1R-containing plasmid vector to overexpress the receptor in high- or normal-glucose conditions in these cells. Transfection with the PTH1R plasmid vector markedly increased PTH1R levels in MLO-Y4 osteocytes, whereas incubation with high glucose did not further upregulate these levels (Figure 5A). Both IL-6 and RANKL expression and the levels of the RANKL-soluble decoy receptor OPG were substantially enhanced in high-glucose-incubated MLO-Y4 cells (Figure 5A–D). In contrast, PTH1R-overexpressing cells showed IL-6 upregulation without affecting RANKL expression (Figure 5B,C), suggesting that increased levels of PTH1R primarily affect IL-6 expression while high glucose modulates both IL-6 and RANKL expression in osteocytes. Of note, PTH1R and high glucose synergistically enhanced IL-6 expression (Figure 5B). The RANKL/OPG ratio, an indicator of the remodeling signals triggered by osteocytes [15], was only altered in PTH1R-overexpressing osteocytes under high-glucose conditions, where it was shown to be significantly reduced compared to other experimental conditions of the study (Figure 5D). Therefore, a high-glucose environment seems to induce at least two different mechanisms that control IL-6 expression in osteocytes: one PTH1R and high-glucose-dependent and another PTH1R-independent and high-glucose-dependent. On the other hand, a glucose-dependent mechanism is preferentially suggested to trigger RANKL overexpression in MLO-Y4 osteocytes.

We next aimed to evaluate the role of the HG osteocytic-produced IL-6 on the polarization and osteoclastogenic markers of macrophages. For this purpose, the secretome of MLO-Y4 osteocytes was incubated or not with an IL-6 blocking antibody prior to the stimulation of macrophages. TNF- $[eqn]$ overexpression and an increased TNF- $[eqn]$ ratio induced by the secretome of HG osteocytes was significantly inhibited by the IL-6 blocking antibody (Figure 6A,D), supporting the role of IL-6 as an osteocytic secreted factor that modulates pro-inflammatory markers likely driving M1 polarization in macrophages. Overexpression of iNOS, RANK and TRAP markers after incubation with the HG osteocyte secretome showed a non-significant decrease with IL-6 neutralization (Figure 6B,F,G), suggesting that the levels of these macrophage markers might also be regulated by osteocytic IL-6.

Next, we investigated whether HG osteocyte-derived IL-6 could modulate osteoclast formation. With this aim, the secretome of MLO-Y4 osteocytes was incubated or not with an IL-6 blocking antibody prior to the stimulation of macrophages. The number of osteoclasts, assessed as larger cells with three or more nuclei, increased in RAW 264.7 cells incubated with the secretome of control or HG-cultured osteocytes (Figure 7A,B). In contrast, the percentage of osteoclastic cells was significantly lower in RAW 264.7 cell cultures incubated with the secretome treated with the IL-6 blocking antibody, suggesting that osteoclastogenesis is mediated by IL-6 secretion in these experimental conditions.

Next, we used ex vivo bone cultures to corroborate our previous findings. The analysis of genes linked to bone resorption, inflammation, and osteoclastogenesis revealed changes in TNF- $[eqn]$ and TRAP expression without changes in the expression of other genes (Figure 8A–F). The IL-6 neutralizing antibody reduced the expression of these markers under high-glucose conditions, showing a significant reduction in TRAP expression levels (Figure 8E). These results further support that IL-6 mediates osteoclastogenic responses in high-glucose conditions in bone tissue.

3. Discussion

High glucose levels in diabetes mellitus impair several processes that modulate bone remodeling, cell communication and formation. For years, osteocytes have awakened much interest due to their mechanosensing capacities and role as master regulators of osteoblast and osteoclast actions. However, osteocyte communication and regulation of other cells of the immune system that form part of the skeletal environment, such as macrophages, has been less well studied, particularly in models that simulate diabetes. Herein, we report the deleterious actions of a high-glucose environment on the communication of non-mechanically stimulated osteocytes with macrophages. We describe a mechanism whereby high glucose induces PTH1R overexpression in static osteocytes, resulting in increased secretion of IL-6, which leads macrophages to an M1 pro-inflammatory polarization phenotype and enhanced osteoclastogenic potential.

Our results show that macrophages exposed to a high-glucose culture medium do not acquire a pro-inflammatory phenotype, based on upregulated TNF-α/CD206 and iNOS/CD206 ratios and ROS production, unless these cells are incubated with the secretome of high-glucose-cultured static osteocytes. In this regard, previous studies have shown that incubation of bone-marrow-derived macrophages from non-diabetic or diabetic mice with a high-glucose dose equivalent to that used in our current experiments does not induce the secretion of pro-inflammatory cytokines, including TNF- $[eqn]$ , Il-1 $[eqn]$ and IL-6 [46]. Thus, these and our observations suggest that exposure to high glucose alone is not sufficient to trigger an M1 pro-inflammatory phenotype in bone macrophages. Interestingly, these observations contrast with previous studies performed on circulating monocytes, in which type 2 diabetes in humans or murine models fed a high-fat diet and exhibiting impaired glucose tolerance has been associated with increased levels of pro-inflammatory M1 monocytes [47,48]. These findings may suggest that bone-marrow-derived monocytes could behave differently from their circulating counterparts. Alternatively, differences in the pattern of exposure to high glucose—such as variations in concentration, duration, and cellular context—across experimental approaches may account for the divergent results observed regarding M1 polarization. In contrast to what was observed with high-glucose stimulation alone, priming high-glucose conditions with other stimuli such as LPS [46] or with the secretome of high-glucose-cultured static osteocytes, as performed in our study, promotes a substantial pro-inflammatory response in RAW 264.7 macrophages. Furthermore, we have also observed upregulated levels of pro-osteoclastogenic markers in RAW 264.7 macrophages exposed to the secretome of high-glucose-cultured static osteocytes that were not apparent in RAW 264.7 cells cultured in a high-glucose environment without the osteocyte secretome [49]. The actions of high glucose on osteoclast differentiation have proven to be controversial. Some in vivo and in vitro studies show enhanced osteoclastogenesis and bone resorption in diabetic models [50]. In contrast, other in vitro and in vivo models and analyses in human patients describe no significant effects of high glucose on osteoclast activity [51]. Furthermore, additional reports suggest that high glucose can even inhibit RANKL-induced osteoclast differentiation [49,52] and decrease resorption, in association with reduced serum levels of bone resorption markers [53,54]. These contradictory findings may reflect the complexity of interactions between factors and cells in bone that probably vary in the different evaluated in vitro and in vivo models. Nonetheless, the secretome of static osteocytes seems to play an important role regulating both pro-inflammatory and osteoclastogenic markers of macrophages under high-glucose conditions. These effects were even visible when macrophages were exposed to pro-inflammatory (LPS) or anti-inflammatory (IL-4) factors.

Osteocytes exposed to high-glucose environments overexpress inflammatory cytokines (e.g., IL-6 and TNF-α) and the osteoclastogenic factor RANKL, as revealed in studies performed in OCY454-12H osteocytic cells [55]. However, prior to our study there was no mechanism that described the role of PTH1R as a factor linking osteocyte altered secretion of pro-inflammatory cytokines with macrophage function in high-glucose conditions. In our study, we induced PTH1R overexpression via plasmid transfection to demonstrate that elevated receptor levels are sufficient to induce IL-6 overexpression in osteocytes, supporting a direct role for PTH1R signaling in this context. Notably, high-glucose conditions elicited a greater increase in IL-6 expression than PTH1R overexpression alone, suggesting that additional PTH1R-independent mechanisms contribute to IL-6 regulation under high-glucose stress. Thus, our results suggest that high glucose alters the expression of pro-inflammatory and pro-osteoclastogenic factors in osteocytes by at least two different mechanisms: one based on high-glucose induction of PTH1R overexpression, subsequently leading to IL-6 upregulation, and another relying on high-glucose stimulation of IL-6 and RANKL in a PTH1R- independent manner. Future studies are required to determine the potential contribution of alternative pathways to the effects of hyperglycemia beyond PTH1R signaling.

Despite being highly mechanosensitive cells, osteocytes cultured in vitro in medium with low levels of glucose and exposed to static conditions or mechanical stimulation by fluid flow did not display significant changes in IL-6 secretion or PTH1R mRNA expression [45]. Therefore, under these experimental conditions, upregulation of IL-6 levels and PTH1R overexpression in osteocytes seems to be mainly driven by the exposure of these cells to high glucose rather than to mechanical stimulation.

In addition to our current results, upregulation of PTH1R expression in cells exposed to high glucose has also been observed in MC3T3-E1 osteoblasts [56] and in diabetic models not related to bone, such as renal tubuloepithelial cells cultured with high glucose and in the kidneys of mice with streptozotocin-induced diabetes [57]. Our previous studies associated PTH1R expression or stimulation with the modulation of IL-6 secretion by osteocytes [45,58]. In fact, stimulation of osteoblastic cells with PTH-related protein (PTHrP), an agonist of PTH1R that is secreted by osteoblasts acting in a paracrine fashion [59], increases IL-6 levels by a mechanism dependent on Nuclear Factor-κB activation [60]. Moreover, infusion with the PTH1R agonist PTH has proved to increase IL-6 serum levels in association with the upregulation of bone resorption markers [61]. Therefore, it is plausible that glucose-dependent overexpression of PTH1R in osteocytes might increase the pool of receptor that can be stimulated by secreted PTHrP, resulting in IL-6 upregulated levels. Furthermore, it is well known that alterations in the expression of osteocytic PTH1R modulate bone remodeling signals such as RANKL and OPG, affecting bone functions and finally leading to bone diseases [62,63]. Our data reveal that although RANKL expression is not modified with PTH1R upregulation in control MLO-Y4 osteocytes, a synergistic upregulation of this factor is observed in PTH1R-overexpressing osteocytes exposed to high glucose levels. These observations suggest that both high glucose and PTH1R overexpression might act independently on osteocytic RANKL expression. PTH1R-dependent regulation of RANKL expression has been attributed in primary mouse calvaria osteoblasts to a cascade of signals including salt-inducible kinases 2 and 3 and protein phosphatases 1,2,4 and 5, which ultimately regulate the nuclear translocation of CREB-regulated transcriptional coactivator 2 (CRTC2) [64]. Even though CRTC2 has been described as a key transcription factor related to glucose homeostasis, a possible association of this factor or others linking high glucose and RANKL expression has not been elucidated yet.

Our in vitro IL-6 neutralizing experiments show that IL-6 seems to be involved in macrophage polarization to an M1 pro-inflammatory phenotype and in osteoclast differentiation without significant changes in osteoclast marker expression, and the ex vivo data suggest IL-6 positive involvement in osteoclastogenesis. In this regard, previous studies have shown that IL-6 increases osteocyte-mediated differentiation of osteoclasts by inducing RANKL [65] and that IL-6-promoted osteoclastogenesis depends mostly on IL-6 receptors present in cells of the osteoblastic lineage but not on osteoclast progenitors [66]. Moreover, in vitro exposure of osteoclast precursors to IL-6 has been associated with impaired osteoclastogenesis and the promotion of pro-inflammatory responses [67]. In contrast, IL-6 neutralization in the ex vivo model may impair IL-6-dependent osteocyte-mediated osteoclastogenesis, as shown by a tendency toward a decrease in the RANKL/OPG ratio in the presence of an IL-6 blocking antibody and high-glucose conditions. In our hands, IL-6 neutralization in vitro impacts the macrophage M1-related markers directly without significantly affecting osteoclastogenic markers that are likely overexpressed in high-glucose conditions due to osteocyte-dependent upregulation of RANKL. However, osteoclast formation driven by the secretome of osteocytes is indeed inhibited by incubation with the IL-6 antibody. These contrasting results may be due to different experimental conditions for each test. An evaluation of osteoclastic markers was performed just in the presence of the osteocytic secretome, thus potentially triggering a milder pro-osteoclastogenic response in which IL-6 neutralization, although showing inhibition tendencies, had no significant actions. However, the osteoclastogenesis test included RANKL exogenous stimulation to trigger a viewable osteoclast differentiation. In these conditions of high RANKL concentrations, IL-6 blocking seems to be crucial for osteoclast differentiation.

Based on the aforementioned observations, we propose that PTH1R upregulation in non-mechanically stimulated osteocytes plays a paramount role as a driver of pro-inflammatory and pro-osteoclastogenic factor secretion that mediates osteocyte–macrophage crosstalk in high-glucose conditions (proposed model shown in Figure 9). Although in vivo validation represents an important future direction to corroborate our in vitro and ex vivo findings, targeting altered PTH1R and IL-6 levels in osteocytes could represent a novel therapeutic approach to ameliorate inflammatory processes and increased resorption in diabetes.

4. Materials and Methods

4.1. Cell Culture

Mouse osteocytic MLO-Y4 cells, originally developed by Dr. L. Bonewald (University of Missouri, Kansas City, MO, USA), were cultured using minimum essential medium ( $[eqn]$ -MEM) (Thermo Fisher Scientific, Waltham, MA, USA, EE.UU.) supplemented with 2.5% fetal bovine serum (FBS) and 2.5% calf serum (FCS), 2 mM L-glutamine, and antibiotics (100 units/mL penicillin and 100 $[eqn]$ g/mL streptomycin). The cells were maintained at 37 °C in a humidified environment containing 5% CO_2_. They were seeded at a density of 25,000 cells/cm^2^ on type I collagen-coated (C8919-20 ML, Sigma Aldrich, St. Louis, MO, USA, EE.UU.) plates or glass slides and cultured until near confluence. The following day, the medium was switched to $[eqn]$ -MEM without phenol red and supplemented with 1% FBS and elevated D-glucose (25 mM) to mimic diabetic conditions for 24 h. Afterwards, cells were incubated for 18 h in $[eqn]$ -MEM without phenol red and serum, after which conditioned media (CM) were collected.

OCY-454 mouse osteocytic cells were kindly provided by Dr. P. Divieti (Boston University, Boston, MA, USA), who developed this cell line. The cells were cultured in $[eqn]$ -MEM medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 $[eqn]$ g/mL streptomycin. Culturing was carried out in a humidified atmosphere containing 5% CO_2_ at 33 °C for proliferation and at 37 °C for differentiation. For proliferation, OCY-454 cells require a surface coated with type I collagen (C8919-20ML, Sigma Aldrich, St. Louis, MO, USA, EE.UU.) and must be maintained below full confluence. At the start of the experiments, cells were seeded at a density of 55,000 cm^2^ in standard culture plates. During the first three days, the cells proliferated at 33 °C; then, the culture medium was renewed every three days to induce differentiation under humid conditions with 5% CO_2_ at 37 °C. After 10 days of differentiation, treatment with D-glucose (25 mM) was conducted for 48 h.

RAW 264.7 murine macrophages (TIB-71™; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, maintained in a humidified incubator with 5% CO_2_ at 37 °C. To investigate the impact of high glucose (25 mM D-glucose) on M1 and M2 macrophage polarization, cells were plated at a density of 50,000 cells/cm^2^ in standard culture plates. On the day following seeding, the culture medium was supplemented with 25 mM D-glucose to mimic hyperglycemic conditions. After 24 h, 20% conditioned medium obtained from MLO-Y4 osteocytes was added. Then, 24 h later, macrophages were stimulated towards M1 or M2 phenotypes using 100 ng/mL LPS (L4391-1MG, Merck (Rahway, NJ, USA)) or 20 ng/mL IL-4 (ab259406, Abcam (Cambridge, UK)), respectively. After an additional 24 h incubation, RNA was isolated using TRIzol (Ambion, Life Technologies, Carlsbad, CA, USA).

4.2. ROS Assay

The ROS assays were performed using 35 mm ibidi^®^ culture plates (ibidi GmbH; Planegg, Germany), seeding RAW 264.7 cells at a density of 15,000 cells/cm^2^ in their corresponding culture medium. The following day, cells were treated for 48 h with the CM obtained from murine osteocytes MLO-Y4 and, 24 h later, 25 mM D-glucose or 25 mM L-glucose (vehicle) was added. After the incubation period, the culture medium was replaced with α-MEM without phenol red to avoid autofluorescence, and the cells were treated for 10 min with the 2′,7′-Dichlorofluorescein (DCF) reagent (20 $[eqn]$ M). Afterwards, the cells were washed three times with PBS, and α-MEM without phenol red was added again to perform the assays. Fluorescence intensity basal values were measured using a Leica DMI8 confocal microscope.

4.3. Western Blot

Cell lysis of MLO-Y4 cells was performed using a Subcellular Protein Fractionation Kit (78840, Thermo Fisher Scientific) to isolate the membrane protein fraction following the manufacturer’s instructions. Protein quantification was performed using a BCA assay (Thermo Fisher Scientific) with bovine serum albumin (BSA) as a standard. Subsequently, aliquots containing 20 μg of total protein were prepared and separated by electrophoresis on 12.5% polyacrylamide gels (SDS-PAGE) under reducing conditions (β-mercaptoethanol) in the presence of sodium dodecyl sulfate (SDS). Each well of the gel was loaded with 20 μg of protein, and a molecular weight marker (Precision Plus Protein™ Kaleidoscope™, BIO-RAD, Hercules, CA, USA) was included to estimate protein size. Proteins were then transferred onto activated nitrocellulose membranes (0.2 μm, BIO-RAD) at a constant voltage of 100 V for 1 h. After transfer, membranes were blocked with 5% milk in a blocking solution for 1 h under gentle agitation. They were then incubated overnight at 4 °C with rabbit polyclonal primary antibodies against PTH1R (PA5-77689, Invitrogen, Carlsbad, CA, USA) and actin (AB1801, Abcam), each at a 1:1000 dilution. Following several washes with 1× TBS-T (20 mM Tris base, 140 mM NaCl, 0.1% Tween-20, pH 7.6), the membranes were incubated for 1 h at room temperature with a goat anti-rabbit secondary antibody (AB6721, Abcam) under gentle agitation. Finally, the membranes were washed with 1× TBS-T, and protein bands were visualized by chemiluminescence using the Pierce™ ECL Plus Western Blotting Substrate (BIO-RAD) according to the manufacturer’s instructions and imaged with a ChemiDoc™ Touch Imaging System (BIO-RAD).

4.4. Cell Transfection

Cells were plated at a density of 25,000 cells/cm^2^ on culture dishes pre-coated with type I collagen (Sigma Aldrich, St. Louis, MO, USA). MLO-Y4 cells were then transfected with 1 $[eqn]$ g of a human ^GFP^-PTH1R plasmid, generously provided by Dr. Peter Friedman, utilizing Lipofectamine 3000 (L3000008, Life Technologies). Transfection was performed at 37 °C for 4 h in accordance with the manufacturer’s protocol.

4.5. IL-6 Neutralization

In some experiments, neutralizing antibody anti-mIL-6 (AF-406-SP, R&D Systems, Minneapolis, MN, USA) was used at 1 $[eqn]$ g/mL.

4.6. Osteoclastogenesis

RAW 264.6 murine macrophages were cultured in α-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO_2_ at 37 °C. Cells were seeded at a density of 4000 cells/cm^2^ in a standard 24-well plate and treated with recombinant mouse RANKL protein (R&D Systems) at a concentration of 30 ng/mL for 7 days, adding 25 mM D-glucose or its vehicle (25 mM L-glucose) and the osteocyte secretome, with medium, mRANKL and CM renewal every 2–3 days. We evaluated osteoclast formation in culture by morphology, quantifying large multinucleated cells, defined as cells larger than macrophages and containing three or more nuclei, an accepted criterion for osteoclast identification [45].

4.7. Ex Vivo Bone Culture

All animal procedures were approved by the Institutional Animal Care and Use Committee of San Pablo CEU University, with animal care conducted according to institutional guidelines (Animal Research Ethics Board of CEU San Pablo University approval Code: MGI24JAA 401.998, approval Date: 8 April 2025). Male C57BL/6J mice aged 12 weeks were housed in groups of five per cage with free movement, provided water ad libitum, and maintained under a 12 h light–dark cycle at 20 $[eqn]$ 0.5 °C and 55 $[eqn]$ 0.5% relative humidity. All experiments were performed using male mice exclusively in order to minimize potential variability related to fluctuations in estrogen levels in female mice. This approach was chosen based on the well-established effects of estrogens on the activity and behavior of several bone cell types, particularly osteocytes. At 4 months of age, the mice were euthanized, and their tibias and femurs were collected. To create a hyperglycemic environment ex vivo, the bones were cultured in $[eqn]$ -MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin and 100 $[eqn]$ g/mL streptomycin for 48 h. At the onset of culture, bones were treated with 25 mM D-glucose or 25 mM L-glucose (vehicle) and 1 $[eqn]$ g/mL of a neutralizing anti-IL-6 antibody. Finally, tibias and femurs were homogenized for RNA extraction using TRIzol (Ambion, Life Technologies). All animal procedures were carried out in compliance with institutional regulations and in accordance with the ARRIVE guidelines for animal research.

4.8. Analysis of mRNA Expression

Total RNA was extracted using the guanidinium thiocyanate–phenol–chloroform separation method. The RNA concentration was then measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and 2 $[eqn]$ g of RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems, Grand Island, NY, USA) in an Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany), following the manufacturer’s instructions. Subsequently, quantitative PCR (qPCR) was performed on a QuantStudio™ 5 384-Well Block (Thermo Fisher Scientific) using SYBR Premix Ex Taq (RR420W, Takara, Otsu, Japan) and specific primers (Table 1) for MLOY4 and the ex vivo model, with 18S rRNA used as the endogenous control gene; PTH1R was evaluated using a specific TaqMan Probe, (Thermo Fisher Scientific) [Mm_00441046_m1], as well as 18s [4319413E] (for MLO-Y4 and ex vivo studies) or GAPDH [4326317E] (OCY-454) TaqMan probes as endogenous control genes.

4.9. Statistical Analysis

Experimental data were analyzed using GraphPad Prims v10.6.0 (GraphPad Software, San Diego, CA, USA). The results are expressed as mean ± Standard Deviation (SD). Datasets including three or more groups were evaluated by the non-parametric Kruskal–Wallis test, followed by the multiple comparison Dunn’s test. The Mann–Whitney U test was used to compare two datasets. In all statistical tests, a p-value < 0.05 was considered statistically significant.