A novel nuclear complex of DRR1, F-actin and COMMD1 involved in NF-κB degradation and cell growth suppression in neuroblastoma.

Downregulated in renal cell carcinoma 1 (DRR1) has important roles in tumor cell growth, neuron survival and spine formation, and was recently shown to bind actin. However, the roles of nuclear DRR1 remain largely unexplored. Here, we identified an interaction between filamentous actin (F-actin) and DRR1 in the nucleus, and demonstrated that copper metabolism MURR1 domain-containing 1 (COMMD1) is another binding partner of DRR1. Accordingly, DRR1, F-actin and COMMD1 were shown to form a complex in the nucleus, and the stability of COMMD1 was enhanced in this complex. Increased nuclear COMMD1 in turn promoted the degradation of NF-κB. In addition, DRR1 and COMMD1 suppressed the cyclin D1 expression, G1/S transition and cell proliferation of neuroblastoma cells. The binding between DRR1 and F-actin in the nucleus was required for these events. Consistent with these facts, low expressions of DRR1 were associated with tumorigenesis of human neuroblastoma and its mouse model. This study has thus revealed a novel nuclear complex of F-actin, DRR1 and COMMD1 that is involved in NF-κB degradation and cell cycle suppression in neuroblastoma cells.

Involvement of midkine in neuroblastoma tumourigenesis.

UNLABELLED: Midkine is highly expressed in various cancers, including neuroblastoma, one of the most malignant paediatric solid tumours known. Also, it has been shown to be useful as a tumour marker, a prognosis factor and a target of molecular therapy. Several molecular tools (e.g. siRNA, antibodies and RNA aptamer) have been used to establish a midkine-targeted therapy. The involvement of midkine in tumourigenesis has been demonstrated in vivo in a mouse neuroblastoma model, where targeting it with an RNA aptamer was shown to be an effective treatment for xenografted tumours. Chemoresistance is one of the notable phenotypes regulated by midkine in various cancer cell types. In pancreatic tumours and glioma cells, midkine is expressed in chemoresistant cells and is involved in the survival of these cells in the presence of anticancer drugs. In contrast to these tumours, midkine was found to be expressed in every neuroblastoma cell line tested and the knockdown of midkine alone was sufficient to suppress their growth. These results indicate that neuroblastoma cells are highly dependent on midkine and that a midkine-targeted therapy could exert a significant effect in these cells. However, to achieve a midkine-targeted therapy for high-risk neuroblastoma patients, the further refinement of the RNA aptamer or antibody as tools and the elucidation of midkine signalling are immediate issues that need to be resolved. Regarding the latter, although it has been shown that Notch2 functions as a receptor in neuroblastoma cells, it is likely that other receptors (e.g. anaplastic lymphoma kinase) are also involved in midkine signalling. LINKED ARTICLES: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Recovery of the blood flow around the femoral head during early corticosteroid therapy: dynamic magnetic resonance imaging in systemic lupus erythematosus patients.

Disturbance of blood supply to the femoral head is a risk factor for corticosteroid-associated osteonecrosis. The aim was to measure blood supply of the proximal femur during corticosteroid therapy in systemic lupus erythematosus (SLE) patients. We repeatedly performed 78 dynamic MRIs of 19 hip joints in 19 SLE patients after initiation of corticosteroid administration for one year. Blood supply of the femoral head (epiphysis, growth plate, and metaphysis), the femoral neck, and the medial circumflex femoral artery were measured in terms of peak percent enhancement. At the first month, blood supply of the growth plate was significantly higher in the pediatric group (<15 years old) than in the adolescent and adult group (>15 years old). At the fourth month, blood supply in every part of the femoral head (epiphysis, growth plate, and metaphysis) was significantly higher in the pediatric group than in the adolescent and adult group. Multiple regression analysis revealed that blood supply to the femoral head depended on the number of days after initiation of corticosteroid administration and the age at the time of dynamic MRI. Blood supply to the femoral head is abundant in pediatric patients and is a function of the number of days after initiation of corticosteroid administration.

Proton magnetic resonance spectroscopy of the thalamus in patients with osteoarthritis of the hip.

OBJECTIVES: The purpose of this study was to assess N-acetyl aspartate changes in the thalamus in patients with osteoarthritis of the hip using proton magnetic resonance spectroscopy. METHODS: Nine patients with osteoarthritis of the hip (symptomatic group, nine women; mean age 61.4 years (48 to 78)) and nine healthy volunteers (control group, six men, three women; mean age 30.0 years (26 to 38)) underwent proton magnetic resonance spectroscopy to assess the changes of N-acetyl aspartate in the thalamus. RESULTS: The ratio of N-acetyl aspartate to creatine plus phosphocreatine in the thalamus contralateral to the symptomatic hip in patients with osteoarthritis of the hip was significantly lower than the ratio of N-acetyl aspartate to creatine plus phosphocreatine in the thalamus in the control group (1.611 (range; 1.194-1.882) vs 1.355 (range; 1.043-1.502), p < 0.001). And, a strong negative correlation was detected between the ratio of N-acetyl aspartate to creatine plus phosphocreatine in the thalamus contralateral to the symptomatic hip in patients with osteoarthritis of the hip and pain duration (r = -0.83, p = 0.018). CONCLUSIONS: We evaluated the ratio of N-acetyl aspartate to creatine plus phosphocreatine in the thalamus of patients with osteoarthritis of the hip by using proton magnetic resonance spectroscopy. We concluded that the ratio of N-acetyl aspartate to creatine plus phosphocreatine in the thalamus contralateral to the symptomatic hip in patients with osteoarthritis of the hip were significantly lower than those in the thalamus of the control group, and that pain duration was strongly related to the decrease of the ratio of N-acetyl aspartate to creatine plus phosphocreatine.

Spontaneous repair of asymptomatic osteonecrosis associated with corticosteroid therapy in systemic lupus erythematosus: 10-year minimum follow-up with MRI.

Systemic lupus erythematosus (SLE) patients are at high risk of developing osteonecrosis. This study utilized MRI to document the long-term natural history of asymptomatic osteonecrosis associated with corticosteroid therapy in SLE patients. Two hundred and one SLE patients treated with high-dose corticosteroids were prospectively observed from 1986 to 1997. The inclusion criterion was that patients had received periodic MRI examinations of all their hip and knee joints for ≥10 years. Joints that were already collapsed and symptomatic at the first examination were excluded. Five hundred and thirty-seven joints (251 hips and 286 knees) were identified in 144 patients, with a mean follow-up period of 13.6 years (range, 10-20 years) and a follow-up rate of 73%. Mean age of SLE onset was 26 years, and the mean highest oral corticosteroid dosage was 57 mg/day. Osteonecrosis developed in 238 (44%) of 537 joints. At final follow-up, 117 (49%) of these 238 joints demonstrated spontaneous repair in the necrotic area. Osteonecrosis completely disappeared in 21 joints. Enlargement of osteonecrosis was noted in 35 joints (15%) following increased steroid dosage because of SLE recurrence. Finally, 52 joints (22%) were collapsed. Spontaneous repair of asymptomatic osteonecrosis was observed, whereas enlargement occurred only after corticosteroid dosage increases.

Laparoscopic gastrectomy with regional lymph node dissection for upper gastric cancer.

BACKGROUND: The technique and results of laparoscopic gastrectomy in 110 patients with gastric cancer located in the upper third of the stomach are presented. METHODS: Proximal gastrectomy was performed for lesions in the upper third of the stomach, and total gastrectomy for those that spread over both the upper and middle third. D1 and D2 lymph node dissection was undertaken in patients with T1 or T2 lesions. Anastomosis of the oesophagus was performed intracorporeally using a conventional circular stapling device or a laparoscopic linear stapler. RESULTS: Median operating time was 247 min for proximal gastrectomy and 285 min for total gastrectomy; median blood loss was 207 and 334 ml respectively. A median of 23 lymph nodes was harvested from patients in the proximal gastrectomy group and 34 from those having a total gastrectomy. There was minimal morbidity and fast recovery after surgery. Postoperative recurrence occurred in only one patient, giving a recurrence rate of 0.9 per cent. CONCLUSION: Laparoscopic gastrectomy for upper gastric cancer appears to be a safe and curative procedure.

Laparoscopic distal gastrectomy with regional lymph node dissection for gastric cancer.

BACKGROUND: Recent advances in surgical techniques have led to widespread acceptance of laparoscopic gastrectomy for gastric cancer. We performed distal gastrectomy with regional lymph node dissection in 235 patients with gastric cancer located in the middle and lower third of the stomach. METHODS: In 171 cases, reconstruction was done using the Billroth I method intracorporeally and the aid of laparoscopic linear stapling devices. The Billroth II and Roux-en-Y methods were used in the remaining 56 and eight patients, respectively, RESULTS: Patients who underwent laparoscopic distal gastrectomy had a more rapid postoperative recovery than those treated via the open approach. Postoperative complications with this technique were within a permissible range. In terms of the survival curve, there was no statistical difference between the laparoscopic group diagnosed as clinical T2N0 (c T2N0) Preoperatively and the open group. CONCLUSION: The laparoscopic technique is not only less invasive, but is also similarly safe and curative compared to open gastrectomy.

A novel laparoscopic technique for stapled colon and rectal anastomosis.

BACKGROUND: We present new techniques of stapling anastomosis at laparoscopic colorectal surgery with retrospective review of data. METHODS: A triangulating stapling technique (T method) was performed in 101 laparoscopic colectomies. Adouble stapling technique (DST) with rectal division by a conventional linear stapler (Abd method) was used in 5 cases of upper/middle rectal cancers and subsequent eversion of the distal rectum from the anus (Ev method) was used for 4 low rectal cancers. Four hundred ninety-six colectomies and 280 rectal surgeries were reviewed. RESULTS: Leakage was lower in the T group (0.5%, n=196) than in the hand-sewn group (3.0%, n=233). Leakage of the DST using a laparoscopic linear stapler (12.1%, n=91) was significantly higher than with conventional DST (2.1%, n=189). There was no leakage with either Abd method or Ev method. The T-method is acceptable after laparoscopic colectomy. CONCLUSION: New methods of rectal division using conventional devices are expected to yield reliable anastomosis at laparoscopic rectal surgery.

Facial myokymia associated with an isolated lesion of the facial nucleus.

We report on a patient with transient facial myokymia. He had an isolated lesion of the right facial nucleus in the pontine tegmentum. Facial myokymia is a rare symptom and its pathogenesis is not known. Our case had a very localized lesion and we attempted to determine the case of the facial myokymia.

Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation.

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Synergistic activation of the Wnt signaling pathway by Dvl and casein kinase Iepsilon.

Although casein kinase Iepsilon (CKIepsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIepsilon activates the Wnt signaling pathway in co-operation with Dvl. CKIepsilon and Axin associated with different sites of Dvl, and CKIepsilon and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIepsilon alone did not accumulate beta-catenin, but their co-expression accumulated greatly. Dvl and CKIepsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIepsilon activated Tcf, it did not synergize with CKIepsilon. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIepsilon. Furthermore, Dvl and CKIepsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dvl and CKIepsilon synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIepsilon to Axin is necessary for their synergistic action.

Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway.

Interleukin-1 (IL-1) is a proinflammatory cytokine that recognizes a surface receptor complex and generates multiple cellular responses. IL-1 stimulation activates the mitogen-activated protein kinase kinase kinase TAK1, which in turn mediates activation of c-Jun N-terminal kinase and NF-kappaB. TAB2 has previously been shown to interact with both TAK1 and TRAF6 and promote their association, thereby triggering subsequent IL-1 signaling events. The serine/threonine kinase IL-1 receptor-associated kinase (IRAK) also plays a role in IL-1 signaling, being recruited to the IL-1 receptor complex early in the signal cascade. In this report, we investigate the role of IRAK in the activation of TAK1. Genetic analysis reveals that IRAK is required for IL-1-induced activation of TAK1. We show that IL-1 stimulation induces the rapid but transient association of IRAK, TRAF6, TAB2, and TAK1. TAB2 is recruited to this complex following translocation from the membrane to the cytosol upon IL-1 stimulation. In IRAK-deficient cells, TAB2 translocation and its association with TRAF6 are abolished. These results suggest that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of a TRAF6-TAB2-TAK1 complex. Formation of this complex is an essential step in the activation of TAK1 in the IL-1 signaling pathway.

Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein.

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

Effects of rat Axin domains on axis formation in Xenopus embryos.

Wnt signaling plays an important role in axis formation in early vertebrate development. Axin is one Wnt signaling regulator that inhibits this pathway. The effects of the injection of mRNA of several rat Axin (rAxin) mutants on axis formation in Xenopus embryos were examined. It was found that rAxin mutants containing only a regulation of G-protein signaling (RGS) domain fragment or with deletion of the RGS domain induced axis formation. Because the RGS domain is a major adenomatous polyposis coli gene product (APC)-binding domain, APC association with glycogen synthase kinase 3beta (GSK3beta) on the Axin molecule may be important in inhibition of axis formation. The ventralizing activities of wild-type rAxin and a mutant in which the Dishevelled and Axin (DIX) domain was deleted (deltaDIX mutant) were examined. Histological examination and gene expression revealed that the ventralizing activity of the deltaDIX mutant was weaker than that of wild-type rAxin. This finding suggests that the C-terminus of rAxin contributes to the inhibition of Wnt signaling in Xenopus embryos. Furthermore, an rAxin mutant that contained both the RGS and GSK3beta-binding domains affected both the dorsal and ventral sides of blastomeres, mediated ectodermal fate and induced expansion of notochord and/or endoderm, but did not induce axis formation.

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation.

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.

Inhibition of Wnt signaling pathway by a novel axin-binding protein.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.

Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin.

Adenomatous polyposis coli gene product (APC) functions as a tumor suppressor and its mutations in familial adenomatous polyposis and colorectal cancers lead to the accumulation of cytoplasmic beta-catenin. The molecular mechanism by which APC regulates the stability of beta-catenin was investigated. The central region of APC, APC-(1211-2075), has the beta-catenin- and Axin-binding sites and down-regulates beta-catenin. Glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated beta-catenin slightly in the presence of either APC-(1211-2075) or Axin(delta)(beta)(-catenin), in which the beta-catenin-binding site is deleted, and greatly in the presence of both proteins. The enhancement of the GSK-3 beta-dependent phosphorylation of beta-catenin was eliminated by the APC-binding site of Axin. Axin down-regulated beta-catenin in SW480 cells, but not Axin(delta)(beta)(-catenin). In L cells where APC is intact, Axin(delta)(beta)(-catenin) inhibited Wnt-dependent accumulation of beta-catenin but not Axin-(298-832)(delta)(beta)(-catenin) in which the APC- and beta-catenin-binding sites are deleted. These results indicate that the complex formation of APC and Axin enhances the phosphorylation of beta-catenin by GSK-3 beta, leading to the down-regulation of beta-catenin.

TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway.

The TAK1 MAPKKK mediates activation of JNK and NF-KB in the IL-1-activated signaling pathway. Here we report the identification of TAB2, a novel intermediate in the IL-1 pathway that functionally links TAK1 to TRAF6. Expression of TAB2 induces JNK and NF-kappaB activation, whereas a dominant-negative mutant TAB2 impairs their activation by IL-1. IL-1 stimulates translocation of TAB2 from the membrane to the cytosol where it mediates the IL-1-dependent association of TAK1 with TRAF6. These results define TAB2 as an adaptor linking TAK1 and TRAF6 and as a mediator of TAK1 activation in the IL-1 signaling pathway.