Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: ( A) Serum proteins downregulated in four OPLL patients vs . four healthy controls and ( B ) quantitative analysis of one of these proteins. In proteomics analysis, a specific peak of mass 10300 Da was decreased in OPLL patients compared to healthy subjects. ( C ) SDS PAGE and silver staining of a purified sample of the peak of interest with/without dithiothreitol. ( D ) Serum chemokine (C-X-C motif) ligand 7 (CXCL7) concentration determined by ELISA (normal, n = 7; OPLL, n = 13). ( E ) CXCL7 levels measured by ELISA in OPLL subtypes (continuous, n = 17; mixed, n = 15; segmental, n = 7; circumscribed, n = 2). ( F ) Real-time PCR analysis of CXCL7 gene expression in total RNA extracted from human (normal, n = 7; OPLL, n = 13). Human OPLL tissues were also examined by scanning electron microscopy as follows: ( G ) Segmental-type OPLL tissue (bracket) and ( H) continuous-type OPLL tissue. The presence of OPLL fibers was confirmed, as indicated by the arrows below the fibers. Scanning electron microcopy of (I ) human PCL tissue and ( J ) human ACL tissue. ( K ) H&E staining of human OPLL tissue (scale bar; 100 μm). ( L ) Immunohistochemical staining and in situ hybridization of human OPLL tissue (axial direction) confirmed the expression of Has-miR-340 aatcaG(L)t5(L)aT(L)tG(L)cT(L)ttataa_N(6)_Y with Alexa Fluor 488 fluorescence and BMP-2 (Alexa Fluor 555 labeled). Nuclei were stained with DAPI.

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques: SDS Page, Silver Staining, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Electron Microscopy, Staining, Immunohistochemical staining, In Situ Hybridization, Fluorescence, Labeling

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: Body weight, volume ratios of subcutaneous and visceral fat, and biochemical data for wild-type and CXCL7 -null mice.

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques:

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: ( A ) CXCL7 -null mouse and wild-type mouse at 8 months of age. ( B ) CXCL7- null mouse transverse (arrow; OPLL) and ( C ) vertical image of L1–L3 (arrow; OPLL). ( D ) H&E and EVG staining of the lumbar OPLL tissue in a CXCL7 -null mouse and EVG staining of lumbar tissue in a wild-type mouse. Histological analysis of the posterior longitudinal ligament (PLL) of the wild-type mouse showed symmetrically arranged elastic fibers intercalated with collagen fibers. Posterior longitudinal ligament ossified tissue in the CXCL7 -null mouse adheres to the dura, and the fibrous tissue of the ligament tissue is unidentified (arrows). ( E ) Villanueva bone (VB) staining of the first lumbar vertebrae (L1-OPLL) in a CXCL7 -null mouse at 8 months of age. ( F ) Close-up of ligament ossification (arrow). ( G ) VB staining of L1-level ligament ossification in a CXCL7 -null mouse at 8 months of age. ( H and I ) Fluorescence labeling in Fig 2G. Osteoblasts in the periosteum on the spinal side in a CXCL7 -null mouse (indicated by arrows). ( J ) Immunohistochemical staining and in situ hybridization of OPLL tissue in a 10-month-old CXCL7 -null mouse confirms the expression of Hsa-miR-340 (Alexa Fluor 488 fluorescence) and BMP-2. Nuclei were stained with DAPI. L1 vertebrae-OPLL ( K ) CT image of a CXCL7 -null mouse with dysbasia at 18 months of age. The arrows indicate OPLL in L2-L5. Longitudinal ( L ) and cross-sectional ( M) images. ( N ) Subcutaneous fat (orange) and visceral fat (yellow) analysis of a spinal cross-section at L3 by 3D-CT. ( O ) Immunohistochemical staining for glucagon (Alexa Fluor 488; green) in the pancreatic islet of Langerhans from a CXCL7 -null mouse. Nuclei were counterstained with DAPI. ( P) H&E-stained islets of Langerhans in the pancreatic tissue of a CXCL7 -null T12-OPLL mouse. ( Q ) C3-level ligament ossification in a female CXCL7 -null mouse showing a bulging eye. ( R) PAS-stained optic lenses in wild-type and CXCL7 -null mice. ( S ) H&E-stained retinal tissue from wild-type and CXCL7 -null mice. GCL; ganglion cell layer, IPL; inner plexiform layer, OPL; outer plexiform layer, RODS and CONES; rod and cone layers. ( T) L12-OPLL edematous kidney tissue in a CXCL7 -null mouse. ( U and V ) periodic acid-Schiff (PAS)-stained and periodic acid methenamine silver-stained renal glomerulus of a CXCL7 -null mouse. (W) After intravenous injection of human recombinant CXCL7 into a CXCL7 -null mouse, the glomerular tissue of the kidney was examined by PAS staining after 3 months.

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques: Staining, Fluorescence, Labeling, Immunohistochemical staining, In Situ Hybridization, Expressing, Injection, Recombinant

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: Bone histomorphometric analysis of wild-type and CXCL7 -null mice. The parameters measured in cortical bone were determined at the midpoint of the tibia. Data are expressed as the mean ± SEM of eight bones/group. Abbreviations related to osteocyte parameters in Table 2 are described in S1 Table in the Supplement (Histomorphometric parameters are described according to the American Society for Bone and Mineral Research (ASBMR) nomenclature committee).

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques:

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: Proteomics revealed phosphorylation of (A) MAPK kinase kinase (MAP3K)15 at threonine 1076 and serine 1078, (B) E3 ubiquitin ligase at serine 419, and (C) protein kinase C alpha at serine 235 in OPLL ligament primary cells derived from CXCL7 -null mice. Function of the ubiquitin proteasome system in CXCL7 -null mice. ( D ) Ubiquitin levels in primary cells derived from CXCL7 -null mouse spinal ligaments cultured in the presence of 5 μM HLI 373 for 24 h and 36 h. ( E ) Wild-type spinal ligament primary cells, CXCL7 -null OPLL ligament primary cells, or CXCL7 -null OPLL ligament primary cells from CXCL7 animals treated with CXCL7 (i.v.) for 3 months were treated with the ubiquitin E3 ligase inhibitor HLI 373 (5 μM) or recombinant Hdm2 (1 μM). Cell lysates were separated by SDS-PAGE and stained with Coomassie brilliant blue (CBB) or western blotted for CXCL7. ( F ) Serum UCHL1 levels, as determined by ELISA in normal subjects, (n = 7), wild-type mice (n = 7), OPLL patients (n = 13), and CXCL7 -null mice (n = 7). ( G ) Serum UCHL1 concentrations as determined by ELISA in different human OPLL subtypes (continuous, n = 17; mixed, n = 15; segmental, n = 7; circumscribed, n = 2). ( H ) Western blotting analysis of UCHL1, PSMA2, UBE3C, and CaMK II in the sera of patients with OPLL and normal human subjects. Lanes 1 and 2, normal subjects; lanes 3 and 4, segmental-type OPLL; lanes 5 and 6, mixed-type OPLL; lanes 7 and 8, continuous-type OPLL. The bottom panel shows the SYPRO ruby stain used to normalize protein loading. (I) Immunohistochemical staining of OPLL tissues from human OPLL patients, CXCL7 -null mice and its primary cells, examining the expression of CaMK II (Alexa Fluor 488, left-hand side) and dual staining for UBE3C (Alexa Fluor 488; green), UCHL1 (Alexa Fluor 555; red), and ubiquitin (linkage-specific K48) (Alexa Fluor 555; red). The nuclei were stained with DAPI (blue) as indicated by the arrows. These images were obtained from the CXCL7 -null L-OPLL tissue section shown in . ( J ) White blood cells were extracted from patients with OPLL and K48 ubiquitination analysis to examine CXCL7 ubiquitination was performed, because CXCL7 protein was present at low levels or was defective. The standard band of CXCL7 at 33 kDa and the band of CXCL7 in the higher macromolecular region indicates the K48 ubiquitin modification. The ubiquitination band of CXCL7 was not seen in healthy subjects, but was present in OPLL patients of the circumscribed, segmental, mixed, and continuous types. Agarose beads (UM400, Life Sensors, Inc.; Cat. # UM400) were used as a control without conjugation of TUBE. ( K ) Immunohistochemical staining of white blood cells of continuous-type patients with OPLL was performed. The expression of K48 (Alexa Fluor 555: red) is shown; nuclear staining was performed with DAPI.

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques: Derivative Assay, Cell Culture, Recombinant, SDS Page, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Expressing, Modification, Conjugation Assay

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: Kyoto encyclopedia of genes and genomes pathway analysis. Data from renal tissue genomic arrays of wild-type and CXCL7 -null mice were analyzed using DAVID software.

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques: Software

Journal: PLoS ONE

Article Title: Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

doi: 10.1371/journal.pone.0196204

Figure Lengend Snippet: ( A) Upper panel. Real-time PCR analysis of osteogenic genes expressed in the osteoblastic cell line MC3T3-E1, the MC3T3-G2/PA6 preadipocyte cell line, and a co-culture of the two. MC3T3-E1 osteoblasts or MC3T3-G2/PA6 preadipocytes were transfected with shRNA-LacZ or shRNA-CXCL7 after which the shRNA-CXCL7-transfected cells were treated with the MEK inhibitor U0126 (10 μM for 15 min), Hdm2 (1 μM for 30 min), or HLI 373 (5 μM for 8 h). Lower panel. Staining of calcified nodules. Cells from the treatments shown in the upper panel were stained to detect calcification, as shown in red. Calcification was confirmed in MC3T3-E1 osteoblasts expressing sh- CXCL7 and following Hdm2 administration. ( B ) BMP-2 levels in cultures of MC3T3-E1 osteoblasts, MC3T3-G2/PA6 preadipocytes, or co-cultures of both, treated or not with recombinant Hdm2 (1 μM for 30 min) or HL1 373 (5 μM for 8 h). Similarly, even following western blotting, BMP-2 expression was enhanced following sh-RNA-CXCL7 transfection in osteoblastic MC3T3-E1 cells, and in addition BMP-2 levels were also increased following administration of Hdm2. Sex differences in OPLL. (C ) Ovarian and testicular expression of ring finger protein (Rnf)216 in wild-type and CXCL7 -null mice (each, n = 4).

Article Snippet: We administered human recombinant CXCL7 (Wako, Osaka, Japan) (1 mg/kg/week) to CXCL7 -null mice by intravenous injection and prepared primary cell cultures from spinal ligament tissue ( ).

Techniques: Real-time Polymerase Chain Reaction, Co-Culture Assay, Transfection, shRNA, Staining, Expressing, Recombinant, Western Blot