Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)-a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor ß superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Carboxymethyl-chitin promotes chondrogenesis by inducing the production of growth factors from immune cells.

Many techniques have been tested for their ability to restore cartilage defects, but several problems still remain in the complete healing of injured cartilage. In our previous study, we found that a carboxymethyl-chitin/beta-tricalcium phosphate (CM-chitin/beta-TCP) composite induced cartilage regeneration in the osteochondral defects of rabbits in vivo. We also found that CM-chitin stimulated peritoneal exudate cells (PEC) in mice and induced several kinds of inflammatory cytokines and transforming growth factor beta-1 (TGF-beta1). In this study, we examined whether CM-chitin is responsible for the induction of chondrogenesis via the production of TGF-beta1 in vitro. The murine pluripotent cell line C3H10T1/2 was maintained as a micromass culture in conditioned medium prepared from PEC stimulated with and without CM-chitin. CM-chitin-conditioned medium induced RNA expression of the chondrogenic-factor Sox9 and the matrix proteins aggrecan, Col2a1, and Comp. Their expression levels were decreased in the presence of anti-TGF-beta1 antibody. The micromass tissues cultured in CM-chitin conditioned medium at day 21 were clearly stained by Toluidine blue or Alcian blue (histological staining) and collagen II antibody (immunohistological staining), showing the expression of acidic glycosaminoglycan and type II collagen. Similar results were observed in micromass tissue stimulated with TGF-beta1 as a positive control. However, no chondrogenesis occurred when CM-chitin was added directly to a C3H10T1/2 cell culture. These results indicated that CM-chitin is a potent inducer of chondrogenesis via the induction of TGF-beta1 in immune cells.

Biological roles of carboxymethyl-chitin associated for the growth factor production.

Many techniques to restore cartilage defection have been tried. However, the development is still under way because of problems, including loosening of artificial joint, degenerative change of compensated tissue, risk of viral transmission via allograft/autograft, and cost of therapeutic materials for repair. In the previous research, we found that complementing cartilage defective part with carboxymethyl-chitin (CM-chitin)/beta-tricalcium phosphate composite induced regeneration of cartilage in rabbits in vivo, and it is presumable that CM-chitin plays a key role in chondrogenesis causing the regeneration of cartilage. However, the induction mechanism of chondrogenesis with CM-chitin is still unclear. In this study, we investigated the cell responses to CM-chitin by using peritoneal exudate cell (PEC) in mice and found that CM-chitin induced the expression of inflammatory cytokines and growth factors, both of which are both considered to correlate with chondrogenesis. After intraperitoneal injection CM-chitin showed enhanced expressions of mRNA of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), keratinocyte-derived chemokine, tumor necrosis factor-alpha, and transforming growth factor-beta1 (TGF-beta1) in PEC as observed by reverse transcriptase polymerase chain reaction. Productions of TGF-beta1 protein were confirmed by enzyme linked immunosorbant assay. It was also shown that mononuclear cells in PEC were responsible for the TGF-beta1 production. These results suggest that CM-chitin is an inductor of inflammatory cytokines and growth factors and may contribute to regeneration of cartilage.

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus.

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.

Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components.

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of Toll-like receptor 2 (TLR2). In this study, we demonstrated that lipoproteins obtained from S. aureus are potent activators of TLR2. A fraction obtained by Triton X-114 phase partitioning activated cells through TLR2. The fraction contained proteins and LTA. The activity was detected in compounds in a mass range of 12-40 kDa. Proteinase K digested the active compounds into lower molecular weight active materials <10 kDa. In contrast, hydrofluoric acid treatment, which decomposes LTA, did not alter the molecular mass of the active compounds. Further, most of the activity was abrogated by lipoprotein lipase digestion. These results suggested that lipoproteins are predominant TLR2 ligands in S. aureus cell wall components.