It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.

Ameloblastoma is an odontogenic tumor located in the bone jaw with clinical characteristics of extensive bone resorption. It is a locally invasive tumor with a high recurrence rate despite adequate surgical removal. In bone disease, tumors and other cells including osteoblasts, osteoclasts, and osteocytes in the bone microenvironment contribute to the pathogenesis of tumor growth. However, the effect of osteoblasts on ameloblastoma cells is not well-understood, and there has been limited research on interactions between them. This study investigated interactions between ameloblastoma cells and osteoblasts using a human ameloblastoma cell line (AM-3 ameloblastoma cells) and a murine pre-osteoblast cell line (MC3T3-E1 cells). We treated each cell type with the conditioned medium by the other cell type. We analyzed the effect on cytokine production by MC3T3-E1 cells and the production of MMPs by AM-3 cells. Treatment with AM-3-conditioned medium induced inflammatory cytokine production of IL-6, MCP-1, and RANTES from MC3T3-E1 cells. The use of an IL-1 receptor antagonist suppressed the production of these inflammatory cytokines by MC3T3-E1 cells stimulated with AM-3-conditioned medium. The MC3T3-E1-conditioned medium triggered the expression of MMP-2 from AM-3 cells. Furthermore, we have shown that the proliferation and migration activity of AM-3 cells were accelerated by MC3T3-E1 conditioned media. In conclusion, these intercellular signalings between ameloblastoma cells and osteoblasts may play multiple roles in the pathogenesis of ameloblastoma.

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.

Osteoblasts (OBs) play an important role in bone fracture healing, yet the extreme adverse microenvironment in fracture sites has a negative impact on the survival of OBs. Therefore, it is important to study how OBs behave in the complex fracture microenvironment. Studies have shown that autophagy plays a pivotal role in maintaining cellular homeostasis and defending the cell against adverse microenvironments. In this study we found the induction of autophagy in OBs at femoral bone fracture sites, which may be a result of ischemia, oxidative stress and hypoxia within the local area. At fracture sites a low pH environment also developed. Until now it has been unclear whether the induction of autophagy in osteoblasts is triggered by the acidic pH environment. Therefore, we cultured OBs in vitro in media of different pH values, and found both autophagy and apoptosis increased in OBs in acidic conditions. However, when autophagy inhibitor chloroquine (CQ) was used, apoptosis increased significantly compared with that without CQ. Thus indicating that inhibition of autophagy may promote apoptosis in OBs in an acidic environment, which may provide a new therapeutic strategy to decrease cell apoptosis in OBs through the use of drugs that modulate the autophagic state.

The bone-adipose axis requires complex homeostasis in energy and global metabolism. The bioenergetics of bone establishes the necessary energy balance to coordinate endocrine functions that are affected by various factors and is not limited to matrix proteins only. UCP1 is an uncoupling protein of adipocytes, commonly known for its unique feature of promoting thermogenesis, mainly in brown fat; however, the effects of UCP1 in other cell types remain unreported.

Prostate cancer (PCa) metastasizes to bone, where the bone marrow microenvironment controls disease progression. However, the cellular interactions that result in active bone marrow metastases are poorly understood. A better understanding of these interactions is critical to success in the pursuit of effective treatments for this life ending disease. Anecdotally, we observe that after intracardiac injection of PCa cells, one of the greatest tools to investigate the mechanisms of bone-metastatic disease, animals frequently present with mandible metastasis before hind limb metastasis. Therefore, in this study, we investigated whether the bone cells derived from the mouse mandible influence PCa progression differently than those from the hind limb. Interestingly, we found that osteoblasts harvested from mouse mandibles grew faster, expressed more vascular endothelial growth factor (VEGF), increased vascularity and formed more bone, and stimulated faster growth of PCa cells when cultured together than osteoblasts harvested from mouse hind limbs. Additionally, these findings were confirmed in vivo when mouse mandible osteoblasts were co-implanted into mice with PCa cells. Importantly, the enhancement of PCa growth mediated by mandible osteoblasts was not shown to be due to their differentiation or proliferation activities, but may be partly due to increased vascularization and expression of VEGF.

FasL is a well-known actor in the apoptotic pathways but recent reports have pointed to its important novel roles beyond cell death, as observed also for bone cells. This is supported by non-apoptotic appearance of FasL during osteogenesis and by significant bone alterations unrelated to apoptosis in FasL deficient (gld) mice. The molecular mechanism behind this novel role has not yet been revealed. In this report, intramembranous bone, where osteoblasts differentiate directly from mesenchymal precursors without intermediary chondrogenic step, was investigated. Mouse mandibular bone surrounding the first lower molar was used as a model. The stage where a complex set of bone cells (osteoblasts, osteocytes, osteoclasts) is first present during development was selected for an initial examination. Immunohistochemical staining detected FasL in non-apoptotic cells at this stage. Further, FasL deficient vs. wild type samples subjected to osteogenic PCR Array analysis displayed a significantly decreased expression of Mmp2 in gld bone. To examine the possibility of this novel FasL-Mmp2 relationship, intramembranous bone-derived osteoblastic cells (MC3T3-E1) were treated with anti-FasL antibody or rmFasL. Indeed, the FasL neutralization caused a decreased expression of Mmp2 and rmFasL added to the cells resulted in the opposite effect. Since Mmp2 -/- mice display age-dependent alterations in the intramembranous bone, early stages of gld mandibular bone were examined and age-dependent phenotype was confirmed also in gld mice. Taken together, the present in vivo and in vitro findings point to a new non-apoptotic function of FasL in bone development associated with Mmp2 expression.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1Beetlejuice (Prickle1Bj). The Prickle1Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/β-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.

GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts. [BMB Reports 2016; 49(6): 343-348].

The functionality of living cells is inherently linked to subunits with dimensions ranging from several micrometers down to the nanometer scale. The cell surface plays a particularly important role. Electric signaling, including information processing, takes place at the membrane, as well as adhesion and contact. For osteoblasts, adhesion and spreading are crucial processes with regard to bone implants. Here we present a comprehensive characterization of the 3D nanomorphology of living, as well as fixed, osteoblastic cells using scanning ion conductance microscopy (SICM), which is a nanoprobing method that largely avoids mechanical perturbations. Dynamic ruffles are observed, manifesting themselves in characteristic membrane protrusions. They contribute to the overall surface corrugation, which we systematically study by introducing the relative 3D excess area as a function of the projected adhesion area. A clear anticorrelation between the two parameters is found upon analysis of ca. 40 different cells on glass and on amine-covered surfaces. At the rim of lamellipodia, characteristic edge heights between 100 and 300 nm are observed. Power spectral densities of membrane fluctuations show frequency-dependent decay exponents with absolute values greater than 2 on living osteoblasts. We discuss the capability of apical membrane features and fluctuation dynamics in aiding the assessment of adhesion and migration properties on a single-cell basis.

Estrogens, which bind to estrogen receptor alpha (ERα), are important for proper bone mineral density. When women go through menopause, estrogen levels decrease, and there is a decrease in bone quality, along with an increased risk for fractures. We previously identified an enhancer near FOXC1 as the most significantly enriched binding site for estrogen receptor alpha (ERα) in osteoblasts. FOXC1 is a transcription factor belonging to a large group of proteins known as forkhead box genes and is an important regulator of bone formation. Here, we demonstrate that 17β-estradiol (E2) increases the mRNA and protein levels of FOXC1 in primary mouse and human osteoblasts. GATA4 is a pioneer factor for ERα and it is also recruited to enhancers near Foxc1. Knockdown of Gata4 in mouse osteoblasts in vitro decreases Foxc1 expression as does knockout of Gata4 in vivo. Functionally, GATA4 and FOXC1 interact and regulate osteoblast proteins such as RUNX2, as demonstrated by ChIP-reChIP and luciferase assays. The most enriched motif in GATA4 binding sites from ChIP-seq is for FOXC1, supporting the notion that GATA4 and FOXC1 cooperate in regulating osteoblast differentiation. Together, these data demonstrate the interactions of the transcription factors ERα, GATA4, and FOXC1 to regulate each other's expression and other osteoblast differentiation genes.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-kappaB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

Dexamethasone (DEX) can exert a cytotoxic effect on cultured osteoblasts. The current study explored the potential osteoblast cytoprotective effect of fibroblast growth factor 23 (FGF23). In OB-6 human osteoblastic cells and primary murine osteoblasts, FGF23 induced phosphorylation of the receptor FGFR1 and activated the downstream Akt-S6K1 signaling. FGF23-induced FGFR1-Akt-S6K phosphorylation was largely inhibited by FGFR1 shRNA, but augmented with ectopic FGFR1 expression in OB-6 cells. FGF23 attenuated DEX-induced death and apoptosis in OB-6 cells and murine osteoblasts. Its cytoprotective effects were abolished by FGFR1 shRNA, Akt inhibition or Akt1 knockout. Conversely, forced activation of Akt inhibited DEX-induced cytotoxicity in OB-6 cells. Furthermore, FGF23 activated Akt downstream nuclear-factor-E2-related factor 2 (Nrf2) signaling to alleviate DEX-induced oxidative injury. On the contrary, Nrf2 shRNA or knockout almost reversed FGF23-induced osteoblast cytoprotection against DEX. Collectively, FGF23 activates FGFR1-Akt and Nrf2 signaling cascades to protect osteoblasts from DEX-induced oxidative injury and cell death.

Dexamethasone (Dex) exerts cytotoxic effects to cultured osteoblasts. The potential effect of MHY1485, a small-molecular mammalian target of rapamycin (mTOR) activator, against the process was studied here. In both osteoblastic MC3T3-E1 cells and primary murine osteoblasts, treatment with MHY1485 significantly ameliorated Dex-induced cell death and apoptosis. mTOR inhibition, through mTOR kinase inhibitor OSI-027 or mTOR shRNAs, abolished MHY1485-mediated osteoblast cytoprotection against Dex. Intriguingly, activation of mTOR complex (mTORC1), but not mTORC2, is required for MHY1485's anti-Dex activity. mTORC1 inhibitors (rapamycin and RAD001) or Raptor knockdown almost reversed MHY1485-induced osteoblast cytoprotection. mTORC2 inhibition, via shRNA knockdown of Rictor, failed to affect MHY1485's activity in MC3T3-E1 cells. Further studies showed that MHY1485 treatment in MC3T3-E1 cells and primary murine osteoblasts significantly inhibited Dex-induced mitochondrial death pathway activation, the latter was tested by mitochondrial depolarization, cyclophilin D-ANT-1 association and cytochrome C cytosol release. Together, these results suggest that MHY1485 activates mTORC1 signaling to protect osteoblasts from Dex.

Parathyroid hormone (PTH) and the sympathetic tone promote Rankl expression in osteoblasts and osteoclast differentiation by enhancing cyclic adenosine monophosphate production through an unidentified transcription factor for PTH and through ATF4 for the sympathetic tone. How two extracellular cues using the same second messenger in the same cell elicit different transcriptional events is unknown. In this paper, we show that PTH favors Rankl expression by triggering the ubiquitination of HDAC4, a class II histone deacetylase, via Smurf2. HDAC4 degradation releases MEF2c, which transactivates the Rankl promoter. Conversely, sympathetic signaling in osteoblasts favors the accumulation of HDAC4 in the nucleus and its association with ATF4. In this context, HDAC4 increases Rankl expression. Because of its ability to differentially connect two extracellular cues to the genome of osteoblasts, HDAC4 is a critical regulator of osteoclast differentiation.

Mechanical stimuli play crucial roles in bone remodeling and resorption. Osteopontin (OPN), a marker for osteoblasts, is important in cell communication and matrix mineralization, and is known to function during mechanotransduction. Hypergravity is a convenient approach to forge mechanical stimuli on cells. It has positive effects on certain markers of osteoblast maturation, making it a possible strategy for bone tissue engineering. We investigated the effects of hypergravity on OPN expression and cell signaling in osteoblasts. Hypergravity treatment at 20 g for 24 hours upregulated OPN expression in MC3T3-E1 cells at the protein as well as mRNA level. Hypergravity promoted OPN expression by facilitating focal adhesion assembly, strengthening actin bundles, and increasing Runx2 expression. In the hypergravity-triggered OPN expression pathway, focal adhesion assembly-associated FAK phosphorylation was upstream of actin bundle assembly.

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Communication between osteoblasts and endothelial cells (ECs) is essential for bone turnover, but the molecular mechanisms of such communication are not well defined. Here we identify Cxcl9 as an angiostatic factor secreted by osteoblasts in the bone marrow microenvironment. We show that Cxcl9 produced by osteoblasts interacts with vascular endothelial growth factor and prevents its binding to ECs and osteoblasts, thus abrogating angiogenesis and osteogenesis both in mouse bone and in vitro. The mechanistic target of rapamycin complex 1 activates Cxcl9 expression by transcriptional upregulation of STAT1 and increases binding of STAT1 to the Cxcl9 promoter in osteoblasts. These findings reveal the essential role of osteoblast-produced Cxcl9 in angiogenesis and osteogenesis in bone, and Cxcl9 can be targeted to elevate bone angiogenesis and prevent bone loss-related diseases.

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases.