Osteoblasts In Bone Tissue Research Articles

Acyl-protein thioesterase1 alleviates senile osteoporosis by promoting osteoblast differentiation via depalmitoylation of BMPR1a

ObjectiveSenile osteoporosis (SOP) is an aging-related disease. The depalmitoylating enzyme Acyl-protein thiesterase1 (APT1) is involved in disease regulation. This study explored the mechanism of APT1 in SOP. MethodsEight-week-old SAMP6 mice were selected as SOP models and SAMR1 mice were controls, while osteoblasts were isolated from the femoral surface-soft tissues of SOP and control mice as in vitro models. Mouse femur morphological, bone mineral density (BMD), femur maximum elastic stress and maximum load, and APT1 expression were detected by HE staining, X-ray bone densitometer, material testing machine, and RT-qPCR and Western blot (WB). Osteoprotegrin (OPG)-labeled osteoblasts and APT1 localization in bone tissues were detected by immunohistochemical staining. APT1 expression was promoted in SOP mice by tail vein injection of APT1 lentivirus or promoted/silenced in osteoblasts by transfection of pcDNA3.1-APT1 overexpression or si-APT1 plasmids. SOP mouse osteoblast differentiation (OD), OD-related protein levels, osteoblast proliferation, BMPR1a palmitoylation level, and BMP/Smad pathway were detected by alizarin red staining, ALP activity detection, WB, CCK-8, and IP-ABE method. The effects of the pathway inhibitor LDN-193189 on OD were detected. ResultsAPT1 was under-expressed in osteoblasts of bone tissue in SOP mice and mainly localized in osteoblasts. SOP mice manifested increased bone marrow cavity and bone trabecular space, thinned trabecular bone, decreased BMD, maximum elastic stress, maximum load, and reduced OPG-positive osteoblasts in bone tissues, which were averted by APT1 overexpression, thus alleviating SOP. APT1 overexpression increased osteoblast calcium nodules, ALP activity, OD-related protein levels, and cell proliferation. In mechanism, APT1 overexpression inhibited BMPR1a palmitoylation in SOP mouse osteoblasts and activated the BMP/Smad pathway, thus promoting OD. ConclusionAPT1 activated the BMP/Smad pathway and promoted OD by regulating BMPR1a depalmitoylation, thus alleviating mouse SOP.

PTH1-34 promotes osteoblast formation through Beclin1-dependent autophagic activation.

PTH1-34 can stimulate osteoblast formation, which contributes to the improvement of bone loss. PTH1-34 can activate autophagy, and autophagy plays a key role in osteoblast formation. This study aimed to explore the role of autophagy in PTH1-34-regulated osteoblastogenesis. In this study, the mice treated with ovariectomy (OVX mice) were used to observe the effect of PTH1-34 on the formation and autophagy of osteoblasts in trabecular bone in vivo. Osteoblast precursor cell line MC3T3-E1 was treated with PTH1-34, and then the autophagic parameters of osteoblast precursors (including autophagic proteins and autophagosome formation) were detected using Western Blotting and Transmission Electron Microscopy. Next, after using autophagic pharmacological inhibitor (3-MA) and silencing vectors of autophagic molecule Beclin1 to downregulate autophagic activity, the parameters related to osteogenesis (including ALP staining intensity, ALP activity, cell proliferation and osteoblastic protein expression) were evaluated using corresponding assays. In vivo results showed that PTH1-34 not only improved bone loss caused by OVX but also restored Beclin1 expression and autophagic activity of immature osteoblasts in bone tissues. In vitro assays also showed that treatment of PTH1-34 enhanced the autophagy in osteoblast precursors. Moreover, under PTH1-34 intervention, the upregulated osteogenic parameters were reversed by autophagic inhibition with 3-MA. Of note, Beclin1 silencing can recover the osteogenic activity enhanced by PTH1-34. PTH1-34 can enhance the autophagic activity of osteoblast precursors, which is involved in PTH1-34-regulated osteoblast formation. Furthermore, Beclin1, as a key autophagic regulator, plays a pivotal role in PTH1-34-regulated osteoblast precursor autophagy and osteoblastogenesis.

Triiodothyronine promotes the osteoblast formation by activating autophagy

Based on the osteogenic effect, triiodothyronine (T3) plays an important role in bone growth and development. Autophagy contributes to osteoblast formation and subsequent osteogenesis. Our study aims to explore the relationship among T3, autophagy and osteogenesis. In this study, cranial primary osteoblasts were obtained from 2 to 3 weeks-old Sprague Dawley (SD) rat fetuses. Osteoblasts were treated with T3, and then the autophagic parameters of Osteoblasts (including autophagic proteins, LC3 conversion rate and autophagosome formation) were observed through Western Blotting and Transmission Electron Microscopy. Next, after using autophagic pharmacological inhibitors (3-MA and chloroquine) and silencing vectors of autophagic genes (BECN1, Atg5 and Atg7) to downregulate autophagic activity, osteoblast proliferation and osteoblastic gene expression were detected using cell counting kit-8 (CCK-8) and quantitative real-time PCR (qRT-PCR) assays, respectively. Ultimately, the mice treated with partial thyroidectomy (PTx mice) were used to further observe the effect of T3 on the formation and autophagy of osteoblasts in trabecular bone in vivo. Our results show that T3 enhances osteoblast autophagy. Autophagy suppression with 3-MA, chloroquine or autophagy-genes knockdown reverses T3-promoted osteoblast formation. In vivo assays showed that the formation and autophagy of osteoblasts in bone tissue were reduced in T3-deficient young mice. Overall, T3 can promote osteoblast formation by activation of autophagy.

Osteoblasts are inherently programmed to repel sensory innervation

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.

Homeostasis and Disorder of Musculoskeletal System.Cellular dynamics in musculoskeletal system visualized by intravital imaging techniques.

Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts and osteoblasts, the fundamental process and dynamics have remained elusive. We originally established an advanced imaging system to visualize living bone tissues using intravital two-photon microscopy. By means of this system, we revealed the in vivo behavior of bone-resorbing osteoclasts and bone-forming osteoblasts in bone tissues. This approach facilitates investigation of cellular dynamics in the pathogenesis of musculoskeletal disorders, and would thus be useful for evaluating the efficacy of novel therapeutic agents.

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-β3, a unique component of Lm-332, indicated specific expression of laminin-β3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, β3, and γ2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

Hypoxia-conditioned media allows species-specific attraction of bone marrow stromal cells without need for recombinant proteins

BackgroundIn vivo tissue regeneration depends on migration of stem cells into injured areas, their differentiation into specific cell types, and their interaction with other cells that are necessary to generate new tissue. Human mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs), can migrate and differentiate into osteoblasts in bone tissue. This can be facilitated by recombinant growth factors and cytokines. In many animal species, the availability of genomic sequences, recombinant proteins, and/or antibodies is limited so that new approaches are needed to generate resources that facilitate migration of stem cells into tissue defect areas. Here we used bone marrow stromal cells of human, ovine, equine, and canine origin to generate hypoxia-conditioned media (HCM) in order to attract BMSCs of the respective species in migration assays.ResultsWe show that HCM contain attractors even more potent than vascular endothelial growth factor and can therefore be used in many animal species without the need for purified proteins.ConclusionGeneration of HCM is easy and cheap compared to preparation and purification of protein fractions and/or recombinant proteins. Hence, HCM could be applied in large animals (e.g. sheep, horse, dogs) for attraction of BMSCs into tissue defects caused by tumor resection or trauma.

Prostaglandin E Receptor EP4 Antagonist Suppresses Lipopolysaccharide-Induced Osteoclast Formation and Inflammatory Bone Loss

Prostaglandin E2 (PGE2) is mainly produced by osteoblasts in bone tissue, and acts as a potent stimulator of bone resorption. In osteoblasts, PGE2 production was greatly stimulated by lipopolysaccharide (LPS) following the expression of cyclooxygenase (COX)-2 and membrane-bound PGE synthase (mPGES)-1 mRNA. In the coculture of mouse bone marrow cells and osteoblasts, LPS induced PGE production and osteoclast formation, and EP4 antagonist completely suppressed osteoclast formation, indicating that the PGE2-mediated EP4 signal is essential for LPS-induced osteoclast formation. Inflammatory bone diseases including periodontitis are known to be accompanied by bone loss with increased osteoclastogenesis. To examine the role of EP4-mediated PGE2 action in periodontitis, we examined the effects of EP4 antagonist on LPS-induced bone resorption using mouse alveolar bone. In an organ culture of alveolar bone, LPS-induced bone resorbing activity and EP4 antagonist suppressed this LPS-induced bone resorption. In an experimental model of periodontitis, LPS was injected into the lower gingiva, and the bone mineral density of alveolar bone was measured. LPS-induced the loss of alveolar bone, which was recovered by the treatment with EP4 antagonist in vivo. Therefore, EP4 antagonist is a possible candidate for the therapy of inflammatory bone disease including periodontitis.

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.