Hirotaka Yoshioka

The current paradigm of osteoblast fate is that the majority undergo apoptosis, while some further differentiate into osteocytes and others flatten and cover bone surfaces as bone lining cells. Osteoblasts have been described to exhibit heterogeneous expression of a variety of osteoblast markers at both transcriptional and protein levels. To explore further this heterogeneity and its biological significance, Venus‐positive (Venus⁺) cells expressing the fluorescent protein Venus under the control of the 2.3‐kb Col1a1 promoter were isolated from newborn mouse calvariae and subjected to single‐cell RNA sequencing. Functional annotation of the genes expressed in 272 Venus⁺ single cells indicated that Venus⁺ cells are osteoblasts that can be categorized into four clusters. Of these, three clusters (clusters 1 to 3) exhibited similarities in their expression of osteoblast markers, while one (cluster 4) was distinctly different. We identified a total of 1920 cluster‐specific genes and pseudotime ordering analyses based on established concepts and known markers showed that clusters 1 to 3 captured osteoblasts at different maturational stages. Analysis of gene co‐expression networks showed that genes involved in protein synthesis and protein trafficking between endoplasmic reticulum (ER) and Golgi are active in these clusters. However, the cells in these clusters were also defined by extensive heterogeneity of gene expression, independently of maturational stage. Cells of cluster 4 expressed Cd34 and Cxcl12 with relatively lower levels of osteoblast markers, suggesting that this cell type differs from actively bone‐forming osteoblasts and retain or reacquire progenitor properties. Based on expression and machine learning analyses of the transcriptomes of individual osteoblasts, we also identified genes that may be useful as new markers of osteoblast maturational stages. Taken together, our data show much more extensive heterogeneity of osteoblasts than previously documented, with gene profiles supporting diversity of osteoblast functional activities and developmental fates. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Communication between osteoblasts and osteoclasts plays a key role in bone metabolism. We describe here an unexpected role for matrix vesicles (MVs), which bud from bone-forming osteoblasts and have a well-established role in initiation of bone mineralization, in osteoclastogenesis. We show that the MV cargo miR-125b accumulates in the bone matrix, with increased accumulation in transgenic (Tg) mice overexpressing miR-125b in osteoblasts. Bone formation and osteoblasts in Tg mice are normal, but the number of bone-resorbing osteoclasts is reduced, leading to higher trabecular bone mass. miR-125b in the bone matrix targets and degrades Prdm1, a transcriptional repressor of anti-osteoclastogenic factors, in osteoclast precursors. Overexpressing miR-125b in osteoblasts abrogates bone loss in different mouse models. Our results show that the MV cargo miR-125b is a regulatory element of osteoblast-osteoclast communication, and that bone matrix provides extracellular storage of miR-125b that is functionally active in bone resorption.

The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient (kl/kl) mice and cell and organ cultures. sKL induced FGF23 signaling in bone and exacerbated the hypomineralization without exacerbating the hyperphosphatemia, hypercalcemia and hypervitaminosis D in kl/kl mice. The same effects were seen in rodent bone models in vitro, in which we also detected formation of a sKL complex with FGF23-FGFR and decreased Phex (gene responsible for X-linked hypophosphatemic rickets (XLH)/osteomalacia) expression. Further, sKL-FGF23-dependent hypomineralization in vitro was rescued by soluble PHEX. These data suggest that exogenously added sKL directly participates in FGF23 signaling in bone and that PHEX is a downstream effector of the sKL-FGF23-FGFR axis in bone.

Osteoblasts and adipocytes share a common mesenchymal progenitor in the bone marrow. This implies that a reciprocal relationship exists between osteogenic and adipogenic differentiation. Further, cells of osteoblast lineage transdifferentiate into adipocytes under some circumstances. Dysregulation of osteo-adipogenic fate-determination leads to bone diseases such as osteoporosis, accompanied by an increase in bone marrow adipose tissue. Thus, the fine-tuning of osteo-adipogenesis is necessary for bone homeostasis. Osteo-adipogenic progression is governed by a complex crosstalk of extrinsic signals, transcription factors, and epigenetic factors. Long non-protein-coding RNAs (lncRNAs) act in part as epigenetic regulators in a broad range of biological activities, such as chromatin organization, transcriptional regulation, post-translational modifications, and histone modification. In this review, we highlight the roles of epigenetic regulators, particularly lncRNAs, in the osteo-adipogenic lineage commitment of bone marrow mesenchymal stem cells and the adipogenic transdifferentiation of osteoblasts.

Purpose: Mutation of the klotho gene in mice elicits a syndrome resembling accelerated human aging. However, there is limited evidence for the role of Klotho in the kidney. We conducted a comparative proteome analysis of wild‐type (WT) and klotho‐knockout (kl^−/−) mouse kidneys to identify proteins involved in Klotho deficiency.Experimental design: MALDI imaging MS (MALDI‐IMS) of frozen kidney sections from 7‐wk‐old male WT and kl^−/− mice was used to determine genotype‐specific differences in the MS distribution. Proteins uniquely distributed in kl^−/− kidneys were identified by subsequent analysis of adjacent trypsinized sections by MALDI‐IMS in combination with LC‐MS/MS. Immunohistochemistry and western blotting were adopted in qualitative and quantitation analysis.Results: Ninety‐seven and 69 proteins identified by LC‐MS/MS were matched to the MALDI‐IMS spectra in WT and kl^−/− mouse kidneys, respectively. Among protein types matched, nucleic acid binding proteins were most abundant, followed by enzymes. We identified secretogranin‐1 (SCG1), which was predominately distributed in the glomeruli and renal tubules of kl^−/− mouse kidneys. Immunohistochemistry for SCG1 mirrored images of MALDI‐IMS.Conclusions: SCG1 may be a candidate protein involved in Klotho deficiency. Although further research is needed to investigate the role of SCG1 in the kidney, we show the usefulness of MALDI‐IMS combined with LC‐MS/MS.

Matrix vesicles (MVs) play a key role in bone mineralization. These nano-sized vesicles bud from apical microvilli of osteoblasts and accumulate in osteoid, and initial hydroxyapatite crystals form within these vesicles. MVs appear to share a common feature with exosomes. Recent studies have demonstrated that exosomes include microRNAs (miRNAs) as mediators of intercellular communication. We purified MVs from MC3T3-E1 cell cultures by ultracentrifugation and found approximately 200 miRNAs. MVs suppressed RANKL-dependent osteoclast formation and bone resorption in mouse bone marrow macrophage (BMM) and/or RAW-D cell cultures with decreased levels of osteoclast marker genes such as Acp5, Ctsk and Dcstamp. There were no obvious effects of MVs on rat and human osteoblast and MC3T3-E1 cell cultures. We then focused on miRNAs with predicted target genes involved in osteoclast formation. In both human and rodent models, miR-125b was highly abundant in MVs, compared to osteoblasts and BMMs. Moreover, we found that miR-125b was complexed with Ago2 and stored in the extracellular matrix of MC3T3-E1 cell cultures and mouse bone. Besides being a suppression of osteoclast formation, miR-125b decreased the expression of osteoclast marker genes. The transcriptional repressor Prdm1 was downregulated by miR-125b, resulting in increases in the expression of the anti-osteoclastogenic genes, Irf8 and Mafb. To determine whether miR-125b affects bone in vivo, we generated transgenic mice overexpressing miR-125b under the control of the human osteocalcin promoter (Tg). Overexpressing miR-125b in osteoblasts in Tg mice led to high bone mass by decreasing the number of osteoclasts without any changes in osteoblasts. miR-125b suppressed osteoclastic osteolysis in the lipopolysaccharide -induced mouse osteolysis model. These findings suggest an additional role of MVs and that miR-125b embedded in bone regulates osteoclastogenesis, which may serve as a novel mechanism for osteoblast-osteoclast communication.

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear. Here, we have uncovered the spatial distributions of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) to determine epigenetic events in the mandibular incisors of mice. Immunohistochemistry and dot blotting showed that 5-hmC in ameloblasts increased from the secretory stage to the later maturation stage. We also demonstrated the distribution of 5-mC-positive ameloblasts with punctate nuclear labeling from sometime after the initiation of the secretory stage to the later maturation stage; however, dot blotting failed to detect this change. No obvious alteration of 5-mC/5-hmC staining in odontoblasts and dental pulp cells was observed. Concomitant with quantitative expression data, immunohistochemistry showed that maintenance DNA methyltransferase DNMT1 was highly expressed in immature dental epithelial cells and subsequently decreased at later stages of development. Meanwhile, de novo DNA methyltransferase Dnmt3a and Dnmt3b and DNA demethylase Tet family genes were universally expressed, except Tet1 that was highly expressed in immature dental epithelial cells. Thus, DNMT1 may sustain the undifferentiated status of dental epithelial cells through the maintenance of DNA methylation, while the hydroxylation of 5-mC may occur through the whole differentiation process by TET activity. Taken together, these data indicate that the dynamic changes of 5-mC and 5-hmC may be critical for the regulation of amelogenesis.