Absence of the transcriptional regulator Zfp521 results in decreased bone formation by osteoblasts and increased osteoclast differentiation, largely via Zfp521’s regulation of the transcription factor Ebf1.
Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521+/− mice was rescued in Zfp521+/−:Ebf1+/− mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis.
Although the clinicopathologic features and prognosis of Borrmann type advanced gastric cancer
has been well characterized, those of advanced gastric cancer simulating early gastric cancer
(AGC simulating EGC) still remains unclear.
We reviewed 1985 gastric cancer patients who had undergone gastrectomy at our hospital to
determine the clinicopathologic characteristics, susceptible sites for lymph node metastasis, and
prognosis of AGC simulating EGC in comparison with Borrmann type advanced gastric cancer.
Among 102 patients with AGC simulating EGC, 100 patients (98%) had tumors with depressed type
appearance. The frequencies of serosal invasion, lymph node metastasis, lymphatic vessel invasion,
blood vessel invasion, and liver metastasis were significantly lower in AGC simulating EGC than in
Borrmann type tumors. The prognosis of AGC simulating EGC was significantly better than that of the
Borrmann type tumors. Multivariate analysis indicated that the gross appearance was an independent
prognostic factor. In patients with AGC simulating EGC which invaded to the the muscularis propria
(MP), most lymph node metastasis was restricted with the perigastric lymph nodes (1st-titer lymph
nodes) and lymph node metastasis to 2nd-titer lymph nodes was only observed at station 8a.
AGC simulating EGC is less advanced in comparison with Borrmann type advanced gastric cancer.
Based on the results of susceptible sites for lymph node metastasis in the current study, limited
lymph node dissection could be indicated for AGC simulating EGC whose depth of invasion is
gastric cancer; gross appearance; lymph node dissection; prognosis
Suitable chemotherapy is needed to prolong the survival of patients with unresectable
advanced or recurrent colorectal cancer. We scored the periodical changes of several
prognostic markers during chemotherapy in patients with this type of cancer to discern
the effectiveness of chemotherapy.
Twenty consecutive patients with unresectable advanced or recurrent colorectal cancer were
enrolled. All patients underwent combination chemotherapy with oxaliplatin or irinotecan plus
5-fluorouracil/leucovorin. Neutrophil/lymphocyte ratio (NLR), serum C-reactive protein (CRP),
serum carcinoembryonic antigen (CEA) and serum albumin (ALB) were compared between the
two periods (before chemotherapy and 3 months after it was started) in each patient.
The scoring system was as follows: points are added when a patient shows a decrease of NLR,
CRP and CEA and an increase of ALB at 3 months after the start of chemotherapy with
a possible final score of +4. On the other hand, points are reduced if a patient shows an
elevation of NLR, CRP and CEA and a decrease of ALB at 3 months after the start of
chemotherapy with a possible final score of −4.
At 3 months after the start of first line chemotherapy, 13 patients showed positive scores
but 7 patients showed zero or minus scores. According to our scoring system, we found
the mean survival time (MST) of the 13 patients with plus scores was 34 months and
this was significantly better than that of the 7 patients who showed zero or minus
scores (P = 0.0008).
Our new scoring system is useful but when we find that first line chemotherapy is ineffective,
we need to change it to second line chemotherapy as soon as possible. That may be the best
treatment for patients with unresectable advanced or recurrent colorectal cancer.
chemotherapy; colorectal cancer; inflammation; immunity; nutrition
Because of their mechanical strength, chemical stability, and low molecular weight, carbon nanotubes (CNTs) are attractive biological implant materials. Biomaterials are typically implanted into subcutaneous tissue or bone; however, the long-term biopersistence of CNTs in these tissues is unknown. Here, tangled oxidized multi-walled CNTs (t-ox-MWCNTs) were implanted into rat subcutaneous tissues and structural changes in the t-ox-MWCNTs located inside and outside of macrophages were studied for 2 years post-implantation. The majority of the large agglomerates were present in the intercellular space, maintained a layered structure, and did not undergo degradation. By contrast, small agglomerates were found inside macrophages, where they were gradually degraded in lysosomes. None of the rats displayed symptoms of cancer or severe inflammatory reactions such as necrosis. These results indicate that t-ox-MWCNTs have high biopersistence and do not evoke adverse events in rat subcutaneous tissue in vivo, demonstrating their potential utility as implantable biomaterials.
The regulation of bone and fat homeostasis and its relationship to energy expenditure has recently been the focus of increased attention due to its potential relevance to osteoporosis, obesity and diabetes. Although central effectors within the hypothalamus have been shown to contribute to the regulation of both energy balance and bone homeostasis, little is known of the underlying mechanisms, including the possible involvement of transcriptional factors within the hypothalamus. Transgenic mice overexpressing ΔFosB, a splice variant of the AP1 transcription factor FosB with mixed agonist-antagonistic properties, have increased energy expenditure and bone mass. Since these mice express ΔFosB in bone, fat and hypothalamus, we sought to determine 1) whether overexpression of ΔFosB within the hypothalamus was sufficient to regulate energy expenditure and whether it would also regulate bone mass, and 2) whether these effects were due to antagonism to AP1. Our results show that stereotactic injection of an adeno-associated virus vector to restrict overexpression of ΔFosB to the ventral hypothalamus of wildtype mice induced a profound increase in both energy expenditure and bone formation and bone mass. This effect was phenocopied, at an even stronger level, by overexpressiong of a dominant-negative DNJunD, a pure AP1 antagonist. Taken together these results suggest that downregulation of AP1 activity in the hypothalamus profoundly increases energy expenditure and bone formation, leading to both a decrease in adipose mass and an increase in bone mass. These findings may have physiological implications since ΔFosB is expressed and regulated in the hypothalamus.
AP-1; hypothalamus; energy expenditure; bone formation
Proximal gastrectomy (PG) has been introduced for patients who are preoperatively diagnosed with early gastric cancer located in the upper third of the stomach. In the present study, we compared the prognosis of patients who underwent PG with that of patients who underwent total gastrectomy (TG). Between 1997 and 2006, 51 patients were diagnosed with early gastric cancer located in the upper third of the stomach and underwent PG. In the same period, 35 patients were diagnosed with early gastric cancer and underwent TG. Of these, in 24 patients, the cancer was localized in the middle to upper part of the stomach, and 11 patients had multiple cancers. We compared the clinicopathologic differences and prognoses between the two groups. Significantly fewer lymph nodes were dissected in the PG group (mean, 18.2) than in the TG group (mean, 36.6;P < 0.001). Complications were detected in 17.6% of patients in the PG group and in 14.3% of patients in the TG group, which was not significant (P = 0.678). The overall and disease-specific 5-year survival rates in the 51 patients who underwent PG (88.7% and 97.1%, respectively) were not different from those in the 35 patients who underwent TG (87.6% and 93.4%; P = 0.971 and P = 0.553; respectively). These findings indicate that PG can be performed safely and may have various advantages compared with TG in terms of patients' daily lives.
Early gastric cancer; Proximal gastrectomy; Prognosis; Total gastrectomy
To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.
To determine the clinicopathologic characteristics and prognosis of gastric cancer in
young patients, a total of 1985 gastric cancer patients who had undergone gastrectomy at
our hospital were reviewed. The male-to-female ratio was significantly lower in the young
patients than in either the middle-aged (P < 0.0001) or elderly
patients (P < 0.0001). Undifferentiated carcinoma was observed more
frequently in the young patients compared with either the middle-aged (P
< 0.0001) or elderly patients (P < 0.0001). Furthermore, peritoneal
metastasis was observed more frequently in the young patients than in either the
middle-aged (P < 0.005) or elderly patients (P <
0.005). Five-year survival rates were 61.0, 73.6 and 68.1% in the young, middle-aged and
elderly patients, respectively. The prognosis of the middle-aged patients was
significantly better than that of either the young or the elderly patients
(P < 0.05). Multivariate analysis indicated that age was an
independent prognostic factor. Peritoneal recurrence was more frequently observed in the
young patients than either the middle-aged or the elderly patients (P
< 0.05). Gastric cancer in young patients has unique characteristics, namely, a
predominance of female patients and a high frequency of undifferentiated cancer and
peritoneal metastasis and recurrence.
age; gastric cancer; prognosis
Heterotrophic activities on sinking particulate matter (SPM) play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP) in seawater (HBPSW) and SPM (HBPSPM) as well as potential extracellular enzyme activity (EEA) in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N), the Kuroshio Extension area (35°N), and the subtropical gyre (20°N). Depth-integrated HBPSW from the surface to 500 m was comparable between the locations, whereas HBPSPM at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC) export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase) activity, β-glucosidase (BGase) activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production.
sinking particulate matter; sediment trap; heterotrophic bacterial activity; extracellular enzyme activity; western North Pacific
Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23−/− and Klotho−/− (Kl−/−) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23−/− mice ameliorated the phenotype in Fgf23−/−/PTH−/− mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23−/− mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl−/− (Kl−/−/PTH−/− or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl−/−/PTH−/− mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23−/−/PTH−/− mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl−/−/PTH−/− mice. Moreover, continuous PTH infusion of Kl−/− mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl−/−, but not of Fgf23−/− mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho.
Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. PTH, Klotho, and FGF23 are the key regulators of blood mineral ion homeostasis. Klotho is a type-I membrane protein and has been identified as cofactor required for FGF23 to bind and activate its receptor. Loss of either Klotho or Fgf23 activity results in a similar abnormal phenotype, including severe defects in skeletal mineralization and alterations in mineral ion balance. Here we describe a new mouse model in which we eliminated PTH from Kl−/− mice, and we can show that the skeletal mineralization defect was completely rescued in Kl−/−/PTH−/− mice and that this phenomenon was accompanied by a reduction in the high levels of osteopontin in bone and serum. We also present additional data showing that continuous infusion of Kl−/− mice with PTH results in an elevation in Opn levels and subsequently increased osteoid volume. Interestingly, this result differs from our previous report in which we describe that the osteomalacia and the high Opn levels in Fgf23−/−/PTH−/− mice persisted. Our finding suggests that PTH, possibly by regulating osteopontin, is responsible for the skeletal mineralization defect in Kl−/− mice, but not in Fgf23−/− mice.
Surgery has been reported to suppress cell-mediated immunity; however, the detailed
mechanisms responsible for this remain unclear. We obtained information on expressions
of T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and programmed cell
death 1 (PD-1) on both CD4+ and CD8+ T cells postoperatively from colorectal cancer
patients and evaluated by multicolor flow cytometry. The functions of CD4+ and CD8+ T
cells based on TIM-3 and PD-1 expressions were determined by quantitating the
concentration of interferon-γ (IFN-γ) by enzyme-linked immunosorbent
assay. The study demonstrated a rapid and significant decrease in number of total
lymphocytes, CD4+ T cells and CD8+ T cells. These immune cells reached the minimum
on day 1 and then increased, but still significantly decreased on days 3 and 7.
The frequency of PD-1+CD4+ T cells and TIM-3+CD4+ T cells significantly increased after
colorectal surgery. IFN-γ secretion by PD-1+TIM-3+CD4+ T cells was
significantly decreased compared to secretion by either PD-1+TIM3-CD4+ T cells or
PD-1–TIM-3–CD4+ T cells. Furthermore, IFN-γ secretion
by PD-1+TIM-3+CD8+ T cells was significantly decreased compared to secretion by either
PD-1–TIM-3+CD8+ T cells or PD-1–TIM-3–CD8+ T cells. The expression of
PD-1 and TIM-3 was closely related to impaired cell-mediated immunity that was observed after
surgery for colorectal cancer. New treatment targeting TIM-3 and PD-1 on CD4+ and
CD8+ T cells during the perioperative period may provide a breakthrough in the treatment
of colorectal cancer patients.
colorectal cancer; programmed cell death 1; surgical trauma; T-cell immunoglobulin domain and mucin domain 3
Laparosocpy-assisted pylorus-preserving gastrectomy (LAPPG) is a widely accepted surgical
procedure for the treatment of early gastric cancer in the middle third of the stomach. We
have been performing this operation since 2007. Compared with traditional distal
gastrectomy, LAPPG has postoperative nutritional benefits for patients. However, this
procedure preserves only the pyloric branch of the vagus nerve and not the celiac branch.
We found that patients retain a large amount of residual food in the gastric remnant,
which interferes with the detection of secondary cancer on endoscopic follow-up. To
improve the pyloric function and postoperative gastrointestinal motility, we changed our
procedure during 2009 to preserve both the pyloric and celiac branches of the vagus nerve,
and we named this new procedure laparoscopy-assisted vagus nerve and pylorus-preserving
gastrectomy (LAVNPPG). From 2009 to 2011, 11 patients underwent LAVNPPG at our hospital.
Retrospective comparison of the safety of operation, postoperative complications, and
condition of the gastric remnant between LAPPG (n = 13) and LAVNPPG
(n = 11) found that the occurrence of postprandial stasis and food
residue in the gastric remnant tended to be lower following LAVNPPG, though the
differences were not significant. These findings indicate that LAVNPPG may be an operative
procedure that could replace LAPPG.
gastrectomy; gastric cancer; laparoscopy-assisted; pylorus; vagus nerve
The aim of this study was to evaluate the nutritional advantages of pylorus-preserving gastrectomy (PPG) in comparison with distal gastrectomy with Billroth I anastomosis (DG) in early gastric cancer (EGC). Between 2005 and 2007, 24 patients underwent PPG and 30 underwent DG. Subjective global assessment, objective data assessment, and endoscopic findings of the remnant stomach were compared between the two groups. Two years after surgery, the patients’ body weights recovered to 97% in PPG, but they continued to decrease in DG. Postoperative blood lymphocyte counts remained low in DG, but recovered to preoperative levels 6 months after surgery in PPG. Food residue in the gastric remnant was frequently observed in PPG (71.4%) than in DG (15.8%, P = 0.001). In nutritional aspect, PPG may be a more ideal operation than DG. However, food residue in the gastric remnant should be considered in PPG.
Electronic supplementary material
The online version of this article (doi:10.1007/s12262-010-0167-4) contains supplementary material, which is available to authorized users.
Gastrectomy; Gastric cancer; Pylorus; Surgical procedures; Treatment outcome
We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.
In the growth plate, the interplay between Parathyroid Hormone-Related Peptide (PTHrP) and Indian Hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional co-regulator, in pre-hypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP-/- and chondrocyte-specific PTHR1-/- mice, with decreased chondrocyte proliferation, early hypertrophic transition and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased cyclin D1 and Bcl-2 expression while increasing caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to up-regulate cyclin D1 and to antagonize Runx2, Ihh and Collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.
Zfp521 regulates osteoblast development during lineage commitment and osteoblast maturation by suppressing Runx2 transcriptional activity.
Runx2 is indispensable for osteoblast lineage commitment and early differentiation but also blocks osteoblast maturation, thereby causing bone loss in Runx2 transgenic mice. Zinc finger protein 521 (Zfp521) antagonizes Runx2 in vivo. Eliminating one Zfp521 allele mitigates the cleidocranial dysplasia–like phenotype of newborn Runx2+/− mice, whereas overexpressing Zfp521 exacerbates it. Overexpressing Zfp521 also reverses the severe osteopenia of adult Runx2 transgenic mice. Zfp521 binds to both Runx2 and histone deacetylase 3 (HDAC3), promotes their association, and antagonizes Runx2 transcriptional activity in an HDAC3-dependent manner. Mutating the Zfp521 zinc finger domains 6 and 26 reduces the binding of Zfp521 to Runx2 and inhibition of Runx2 activity. These data provide evidence that Zfp521 antagonizes Runx2 in vivo and thereby regulates two stages of osteoblast development, early during mesenchymal cell lineage commitment and later during osteoblast maturation. Thus, the balance and molecular interplay between Zfp521 and Runx2 contribute to the control of osteoblast differentiation, skeletal development, and bone homeostasis.
Expression of the costimulatory molecule B7-H4, a member of the inhibitory B7 family, has
been reported to be upregulated on tumor-associated macrophages, and this overexpression
may be involved in immune evasion in cancer patients. The present study was designed to
investigate B7-H4 expression on monocytes/macrophages and its relationship with immune
evasion in gastric cancer patients. B7-H4 expression on circulating monocytes and
tumor-associated macrophages was evaluated by multicolor flow cytometry.
Carboxyfluorescein succinimidyl ester proliferation assays and quantitative
interferon-gamma enzyme-linked immunosorbent assays were carried out to determine the
inhibitory effect of B7-H4+ monocytes on CD4+ T cells. B7-H4 expression on circulating
monocytes was upregulated and these B7-H4+ monocytes showed immunosuppressive properties.
B7-H4 expression was closely related to the depth of invasion, as well as the presence of
lymphatic and venous invasion. There were significant correlations between B7-H4
expression and B7-H1 or HLA-DR expression on monocytes/macrophages in gastric cancer
patients. B7-H4 expression was remarkably upregulated in gastric cancer tissues compared
with peripheral blood samples. Complete surgical removal of the tumor decreased B7-H4
expression on circulating monocytes from gastric cancer patients. Cocultures of gastric
cancer cell lines and monocytes led to upregulation of B7-H4 expression on monocytes.
Increased B7-H4 expression on circulating monocytes and tumor-associated macrophages may
be one of the key mechanisms responsible for immune evasion by tumors in gastric
B7-H1; B7-H4; gastric cancer; macrophage; monocyte
Parathyroid hormone (PTH) suppresses Dickkopf 1 (Dkk1) expression in osteoblasts. To determine whether this suppression is essential for PTH-mediated Wnt signaling and bone formation, we examined mice that overexpress Dkk1 in osteoblasts (Dkk1 mice). Dkk1 mice were osteopenic due to abnormal osteoblast and osteoclast activity. When fed a low calcium diet, and in two other models of hyperparathyroidism, these mice failed to develop the peritrabecular stromal cell response (“osteitis fibrosis”) and new bone formation seen in wild type mice. Despite these effects of Dkk1 overexpression, PTH still activated Wnt signaling in Dkk1 mice and in osteoblastic cells cultured from these mice. In cultured MC3T3E1 preosteoblastic cells, PTH dramatically suppressed Dkk1 expression, induced PKA-mediated phosphorylation of β-catenin and significantly enhanced Lef1 expression. Our findings indicate that the full actions of PTH require intact Wnt signaling but that PTH can activate the Wnt pathway despite overexpression of Dkk1.
PTH; PTH/PTHrP receptor; Dkk1; Wnt signaling
Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.
Kremen1 and Kremen2 (Krm1 and Krm2) are transmembrane coreceptors for Dickkopf1 (Dkk1), an antagonist of Wnt/β-catenin signaling. The physiological relevance of Kremen proteins in mammals as Wnt modulators is unresolved. We generated and characterized Krm mutant mice and found that double mutants show enhanced Wnt signaling accompanied by ectopic postaxial forelimb digits and expanded apical ectodermal ridges. Triple mutant Krm1−/− Krm2−/− Dkk1+/− mice show enhanced growth of ectopic digits, indicating that Dkk1 and Krm genes genetically interact during limb development. Wnt/β-catenin signaling also plays a critical role in bone formation. Single Krm mutants show normal bone formation and bone mass, while double mutants show increased bone volume and bone formation parameters. Our study provides the first genetic evidence for a functional interaction of Kremen proteins with Dkk1 as negative regulators of Wnt/β-catenin signaling and reveals that Kremen proteins are not universally required for Dkk1 function.
The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity.
In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8+ T cells in 2-week and/or 3-week cultures. CD8+ T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens.
We found that DCs loaded with killed breast cancer cells can prime naïve CD8+ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201- breast cancer cells can kill HLA-A*0201+ breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin.
This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.
Activating receptor activator of NF-κB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-α–induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF–induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-α promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.
The lipid compositions of barophilic bacterial strains which contained docosahexaenoic acid (DHA [22:6n-3]) were examined, and the adaptive changes of these compositions were analyzed in response to growth pressure. In the facultatively barophilic strain 16C1, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were major components which had the same fatty acid chains. However, in PE, monounsaturated fatty acids such as hexadecenoic acid were major components, and DHA accounted for only 3.7% of the total fatty acids, while in PG, DHA accounted for 29.6% of the total fatty acids. In response to an increase in growth pressure in strain 16C1, the amounts of saturated fatty acids in PE were reduced, and these decreases were mainly balanced by an increase in unsaturated fatty acids, including DHA. In PG, the decrease in saturated fatty acids was mainly balanced by an increase in DHA. Similar adaptive changes in fatty acid composition were observed in response to growth pressure in obligately barophilic strain 2D2. Furthermore, these adaptive changes in response were also observed in response to low temperature in strain 16C1. These results confirm that the general shift from saturated to unsaturated fatty acids including DHA is one of the adaptive changes in response to increases in pressure and suggest that DHA may play a role in maintaining the proper fluidity of membrane lipids under high pressure.