Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are “borderline” chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single‐cell RNA‐seq analysis revealed subpopulations of Col2a1‐creER‐marked neonatal chondrocytes and their cell type–specific markers. A tamoxifen pulse to Pthrp‐creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)‐GFP+ osteoblasts and Cxcl12‐GFPhigh reticular stromal “CAR” cells. Interestingly, these borderline chondrocyte‐derived marrow cells were short‐lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage‐tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/1/jbmr3719_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/2/jbmr3719-sup-0001-Suppl_Info_JBMR_021819.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/3/jbmr3719.pd

骨芽細胞誘導因子OBIFは、骨芽細胞形成に関連して骨形成に重要な役割を果たす

京都大学0048新制・課程博士博士(医学)甲第17777号医博第3803号新制||医||999(附属図書館)30584京都大学大学院医学研究科医学専攻(主査)教授 戸口田 淳也, 教授 開 祐司, 教授 妻木 範行学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

<p>Abstract</p> <p>Background</p> <p>While several cell types are known to contribute to bone formation, the major player is a common bone matrix-secreting cell type, the osteoblast. Chondrocytes, which plays critical roles at several stages of endochondral ossification, and osteoblasts are derived from common precursors, and both intrinsic cues and signals from extrinsic cues play critical roles in the lineage decision of these cell types. Several studies have shown that cell fate commitment within the osteoblast lineage requires sequential, stage-specific signaling to promote osteoblastic differentiation programs. In osteoblastic differentiation, the functional mechanisms of transcriptional regulators have been well elucidated, however the exact roles of extrinsic molecules in osteoblastic differentiation are less clear.</p> <p>Results</p> <p>We identify a novel gene, <it>obif </it>(<it>osteoblast induction factor</it>), encoding a transmembrane protein that is predominantly expressed in osteoblasts. During mouse development, <it>obif </it>is initially observed in the limb bud in a complementary pattern to <it>Sox9 </it>expression. Later in development, <it>obif </it>is highly expressed in osteoblasts at the stage of endochondral ossification. In cell line models, <it>obif </it>is up-regulated during osteoblastic differentiation. Exogenous <it>obif </it>expression stimulates osteoblastic differentiation and <it>obif </it>knockdown inhibits osteoblastic differentiation in preosteblastic MC3T3-E1 cells. In addition, the extracellular domain of obif protein exhibits functions similar to the full-length obif protein in induction of MC3T3-E1 differentiation.</p> <p>Conclusions</p> <p>Our results suggest that <it>obif </it>plays a role in osteoblastic differentiation by acting as a ligand.</p

Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice

<div><p>Background</p><p>Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that <i>Obif</i> (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that <i>Obif^−/−</i> mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in <i>Obif^−/−</i> mice.</p><p>Results</p><p>First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that <i>Obif^−/−</i> mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that <i>Obif^−/−</i> mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that <i>Obif</i> is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the <i>Obif^−/−</i> testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in <i>Obif^−/−</i> mice compared with wild-type mice, although this was not statistically significant.</p><p>Conclusions</p><p>Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that <i>Obif</i> is required for normal bone mineralization and late testicular differentiation <i>in vivo</i>. These findings suggest that <i>Obif</i> plays essential roles in the development of multiple tissues.</p></div

<i>Obif</i> is required for normal spermatogenesis.

<p><b>(A)</b> Gross appearance of testes in male wild-type and <i>Obif</i>^−/− mice at 12 wks. <b>(B)</b> Comparison of testicular weight in wild-type (white box), <i>Obif</i>^+/− (grey box), and <i>Obif</i>^−/− (black box) mice at 5 wks and 12 wks (n = 6). <b>(C)</b> Comparison of epididymis weight between wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(D)</b> Comparison of sperm number from cauda epididymis between wild-type, <i>Obif</i>^+/−, and <i>Obif</i>^−/− at 16 wks and 24 wks (n = 4). <b>(E)</b> The level of serum testosterone in wild-type and <i>Obif</i>^−/− mice at 12 wks (n = 6). <b>(F)</b> H&E staining of testis sections from wild-type and <i>Obif</i>^−/− mice at 12 wks. Scale bars represent 5 mm (<b>A</b>), and 50 μm <b>(F)</b>. Error bars show the SEM. *P < 0.05.</p

Obif protein is O-glycosylated at serine residue 36.

<p><b>(A)</b> Potential O-glycosylation sites of human and mouse Obif proteins. The predicted amino acid sequences of human OBIF (NP_859075.2) and mouse Obif (NP_666274.1) were aligned by the ClustalW program (<a href="http://clustalw.ddbj.nig.ac.jp/" target="_blank">http://clustalw.ddbj.nig.ac.jp/</a>). Asterisks indicate identical amino acids. Colons and periods indicate similar amino acids. Yellow boxes indicate potential O-glycosylation sites. Red bracket indicates extracellular domain. Blue underline indicates N-terminal signal peptides. Green box indicates single transmembrane domain. (<b>B-C</b>) Analysis of O-glycosylation sites in the mouse Obif protein. Constructs of pCAGGS expression vector (CAG), FLAG-tagged GFP (GFP), or FLAG-tagged mObif with or without mutation(s) (wild-type (WT), S36A, S43A, T54A, T60A, T67A, or S36A/S43A/T54A/T60A/T67A (ALL)) were transfected into HEK293T cells. The HEK293T cells were cultured for 24 h. The cell lysates were analyzed by Western blotting using an anti-FLAG M2 antibody <b>(B)</b>. FLAG-tagged constructs expressing GFP (GFP), human CD55 (hCD55), or wild-type mouse Obif (mObif-WT) were transfected into HEK293T cells cultured in standard medium. The HEK293T cells were cultured for 24 h, and subsequently cultured for 3 days in medium with or without benzyl-GalNAc. The cell lysates were analyzed by Western blot analysis using the anti-FLAG M2 antibody <b>(C)</b>. Arrowheads indicate the 60 kDa band of O-glycosylated mObif. Arrows indicate the 37 kDa band is a nascent form of mObif-WT. Benzyl-GalNAc, benzyl 2-acetamido-2-deoxy- α-D-galactopyranoside, an O-glycosylation inhibitor.</p

Histological analysis of distal femoral epiphysis of <i>Obif</i>^−/− mice.

<p><b>(A-B)</b> Villanueva bone staining of distal femur sections from wild-type and <i>Obif</i>^−/− mice. The thickness of distal femoral growth plates was unaltered between wild-type (white box) and <i>Obif</i>^−/− mice (black box) <b>(A)</b>. In wild-type and <i>Obif</i>^−/− mice, the osteoblasts (indicated by arrowheads) and osteoclasts (indicated by arrows) were unchanged in number and size. Scale bars represent 100 μm <b>(A)</b> and 20 μm <b>(B)</b>. Error bars show the SEM (n = 3).</p

Fertility parameters.

<p>Male mice at 12 wks were placed with ICR fertile females at 10 wks for 28 days. Results are shown as mean ± SEM. All data were analyzed with the <i>F</i>-test to determine normality, and the appropriate <i>t</i>-test was applied at the level of 5%.</p><p>Fertility parameters.</p