3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.     

Copy number alteration and uniparental disomy analysis categorizes Japanese papillary thyroid carcinomas into distinct groups

The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAF(V600E), 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAF(V600E) and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA(+), UPD(+), and no chromosomal aberrations. BRAF(V600E) mutational status did not correlate with any parameters of chromosomal defects.     

Extremely slow rate of evolution in the HOX cluster revealed by comparison between Tanzanian and Indonesian coelacanths

Coelacanths are known as "living fossils" because their morphology has changed very little from that in the fossil record. To elucidate why coelacanths have evolved so slowly is thus of primary importance in evolutionary biology. In the present study, we determined the entire sequence of the HOX cluster of the Tanzanian coelacanth (Latimeria chalumnae) and compared it with that of the Indonesian coelacanth (L. menadoensis), which was available in the literature. The most intriguing result was the extremely small genetic divergence between the two coelacanths. The synonymous divergence of the HOX coding region between the two coelacanths was estimated to be 0.07%, which is ~11-fold smaller than that of human-chimp. When we applied the estimated divergence time of the two coelacanths of 6 million years ago (MYA) and 30 MYA, which were proposed in independent mitochondrial DNA analyses, the synonymous substitution rate of the coelacanth HOX cluster was estimated to be ~11-fold and 56-fold smaller than that of human-chimp, respectively. Thus, the present study implies that the reduction of the nucleotide substitution rate in coelacanth HOX genes may account for the conservation of coelacanth morphology during evolution.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.     

RAG-heptamer interaction in the synaptic complex is a crucial biochemical checkpoint for the 12/23 recombination rule

In V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, recombination-activating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule.     

Heparan sulfate regulates self-renewal and pluripotency of embryonic stem cells

Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.     

Heparin inhibits osteoclastic differentiation and function

We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF-kappaB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGIC(TM) kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.     

Chronological change and the potential of recovery on the photosynthetic efficiency of Pyropia yezoensis f. narawaensis (Bangiales) during the sporelings frozen storage treatment in the Japanese Nori cultivation

The chronological change of photosynthetic efficiency in a frozen storage treatment of the Japanese Nori cultivation industry was examined in the cultivated red alga, Pyropia yezoensis f. narawaensis (Saga‐#5 Strain, Bangiales) by using pulse‐amplitude fluorometry. During the desiccation process that was conducted after the nursery cultivation season in November, the maximum quantum yield (F _v/F _m) of the gametophytic sporelings growing on the Nori‐net decreased monotonically with decreasing absolute water content (AWC), and was around 0.1 at 20% AWC. During frozen storage of the Nori‐net, the F _v/F _m of the frozen gametophyte was low but stable, and ranged between 0.10 ± 0.02 SD and 0.14 ± 0.05 SD. The magnitude of F _v/F _m for the gametophyte of the freezing treatment, after 10 min and 3 h of immersion in seawater, recovered quickly. After 10 min and 3 h of immersion, these values were 0.29 ± 0.12 SD and 0.47 ± 0.05 SD during the 14 days of freezing treatment, and 0.15 ± 0.02 SD and 0.29 ± 0.04 SD after 71 days of freezing treatment, and suggest that the ability to recover gradually decreased as the storage duration increased. The response of F _v/F _m from general cultivation (i.e., directly cultivated from the nursery cultivation season) and those after 47 days of freezing were almost identical, suggesting that the current Nori ‐net frozen storage period (6 or 7 weeks) was not detrimental to the gametophyte.