Goro Sugiyama

Inhibition of BMP2-Induced Bone Formation by the p65 Subunit of NF-κB via an Interaction With Smad4

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.

Regulated interaction of protein phosphatase 1 and protein phosphatase 2A with phospholipase C-related but catalytically inactive protein.

Protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) are major members of the protein serine/threonine phosphatase families. We have identified PP1 and PP2A as interacting partners of PRIP (phospholipase C-related but catalytically inactive protein), a protein isolated in our laboratory. We first investigated the interaction of PRIP with two phosphatases, using purified recombinant proteins. PRIP immobilized on beads pulled down the catalytic subunits of both PP1 and PP2A, indicating that the interactions were in a direct manner, and the binding of PP1 and the binding of PP2A to PRIP were mutually exclusive. Site-directed mutagenesis experiments revealed that the binding sites for PP1 and PP2A on PRIP were not identical, but similar. Phosphorylation of PRIP by protein kinase A (PKA) resulted in the weakened binding of PP1, but not PP2A. Rather, the dissociation of PP1 from PRIP by phosphorylation accompanied the strengthened binding of PP2A in in vitro experiments. This regulation of binding of PP1 and PP2A to PRIP by PKA-dependent phosphorylation was also observed in living cells treated with forskolin or isoproterenol. These results suggested that PRIP directly interacts with the catalytic subunits of two distinct phosphatases in a mutually exclusive manner and the interactions are regulated by phosphorylation, thus functioning as a scaffold to regulate the activities and subcellular localizations of both PP1 and PP2A in phospho-dependent cellular signaling.

Metastatic adenocarcinoma of the mandibular condyle from uterine cervix: Report of a case

Abstract Metastasis to the mandibular condyle is rare, and such lesions should be identified by both clinical and pathological examinations. We experienced a case of adenocarcinoma occurring in the right mandibular condyle. A 65-year-old female with uterine cervical cancer showed condylar dysfunction. Imaging examinations revealed a tumor with bone destruction and a rapidly progressive course, while pathological examinations suggested metastasis originating from another site. Based on the clinical and pathological findings, the patient was diagnosed with condylar metastasis derived from the uterine cervix, in addition to a recurrence of uterine cervical cancer.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF‐κB) suppresses BMP‐induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF‐κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4‐binding domain (SBD), an amino‐terminal region of TA2 that associates with the MH1 domain of Smad4. Cell‐permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2‐induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF‐κB signaling. SBD peptide enhanced the binding of the BMP2‐inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id‐1) compared with control peptide. Although SBD peptide did not affect BMP2‐induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2‐induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2‐induced bone regeneration without inhibiting NF‐κB activity.

High Le Fort I osteotomy for correction of mid-face deformity in Crouzon syndrome

An 18‐year‐old woman with mild Crouzon syndrome was referred with malocclusion and mandibular protrusion. Examination revealed Class III canine and molar relationships, hypoplastic maxilla, 1‐mm overbite, and −2‐mm overjet. Analysis showed 69° sella‐nasion‐A, 73.6° sella‐nasion‐B, and −4.6° A point‐nasion‐B point angles. Polysomnography revealed respiratory disturbance and 6.3% oxygen desaturation indices of 5.4/h and 9.0/h. We performed double‐jaw surgery using high Le Fort I osteotomy and bilateral sagittal split ramus osteotomy for midfacial deformity correction. Twelve months post‐surgery, her measures were 70.8°, 72°, −1.2°, 3.0/h, and 6.1/h, respectively. Esthetics were satisfactory. High Le Fort I osteotomy is effective for midfacial deformity correction in patients with Crouzon syndrome.

The Distinct Distributions of Immunocompetent Cells in Rat Dentin Pulp After Pulpotomy

Pulpotomy involves the removal of the coronal portion of pulp, including the diseased tissue, with the intent of maintaining the vitality of the remaining pulpal tissue via a therapeutic dressing. Once odontoblasts suffer injuries, the differentiation of mesenchymal cells is induced from the precursor cell population in the dental pulp, and these cells are recruited to the injured site to differentiate into odontoblasts. However, the involvement of immunocompetent cells during pulpal regeneration remains unclear. Thus, the purpose of this study was to investigate the properties of macrophages that infiltrated wound healing sites in rats between 1 and 28 days after pulpotomy (dap). During the inflammatory phase, ED1+ (CD68+) macrophages significantly increased throughout root pulp, especially apical to the demarcation zone, and this population persisted until 3 dap before decreasing gradually until 28 dap. OX6+ macrophages expressing class II MHC also increased in the apical pulp at 1 dap and declined thereafter. However, OX6+ cells appeared prior to dentin bridge formation at 3 dap and appeared again apical to the dentin bridge during the healing stage at 14 dap. The shift from ED1+ cells in the inflammation phase to OX6+ cells during dentin bridge formation might contribute to wound healing. Anat Rec, 298:741–749, 2015.

Beckwith-Wiedemann syndrome with asymmetrical mosaic of paternal disomy causing hemihyperplasia

Beckwith-Wiedemann syndrome (BWS) is a congenital disorder with 3 main features-overgrowth in infancy, macroglossia, and abdominal wall defects. Here, we report on a 5-month old girl with hemihyperplasia and macroglossia caused by paternal uniparental disomy (pUPD) asymmetric mosaic on chromosome 11p15.5. She could not retract her tongue into her mouth and the midline of the tongue was shifted to the left. Glossectomy was performed at age 1 year. A specimen of the tongue showed normal skeletal muscle, but the muscle fibers were closely spaced, and there were fewer stroma components in the tissue from the right side of the tongue than that from the left side. With respect to pUPD of chromosome 11p15.5, microsatellite marker analysis of the tongue tissue specimen revealed a higher mosaic rate in the tissue from the right side of the tongue (average 48.3%) than that from the left side (average 16.9%). Methylation analysis of Kv differentially methylated region (DMR) 1 (KvDMR1) and H19DMR revealed hypomethylation of KvDMR1 and hypermethylation of H19DMR in the tissue on the right side of the tongue (hyperplastic side). In this case, the difference in mosaic rate of pUPD in the 11p15.5 region was hypothesized to influence the expression level of insulin-like growth factor 2. This result may be helpful to clinicians, especially surgeons, when planning plastic surgery for hemihyperplasia.

Efficient regulation of branching morphogenesis via fibroblast growth factor receptor 2c in early-stage embryonic mouse salivary glands.

Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.

Involvement of the T-box transcription factor Brachyury in early-stage embryonic mouse salivary gland

The mouse submandibular gland (SMG) is important organ for embryonic development, and branching morphogenesis is regulated by many molecules containing transcription factors. Real-time reverse transcriptase polymerase chain reaction revealed that the expression of Brachyury increased in the SMG and peaked between E12.5-E13.5, concomitant with the early stage of branching morphogenesis. The expression of Brachyury in SMG rudiments between E12.5-E13.5 was confirmed by western blotting. In addition, fibronectin and Btbd7 (regulated by fibronectin), which are both essential for cleft formation, were expressed strongly during the same period. The Sox2 and Wnt3a, which regulate cell growth, were also expressed strongly during E12.5-E13.5. On the other hand, cleft formation and branching morphogenesis was suppressed by knockdown of Brachyury gene, suggesting that Brachyury plays a central role in regulating cell growth and cleft formation in early-stage embryonic mouse salivary gland development.