Effects of interleukin-6 and tumor necrosis factor-α on the expression of angiogenic and collagenolytic factors in premature and mature adipocytes.

OBJECTIVE: The remodeling of the vascular network and collagen in the extracellular matrix is closely associated with the expansion and dysfunction of adipose tissue. In the present study, we investigated the effects of interleukin (IL)-6 and tumor necrosis factor (TNF)-α on the expression of angiogenic factors, collagen, and collagenase and its endogenous inhibitor in premature and mature adipocytes. METHODS: Premature and mature adipocytes were differentiated from 3T3-L1 cells and stimulated with IL-6 or TNF-α to mimic the early and late phases of obesity development. The levels of expression of angiogenic factors, including vascular endothelial cell growth factor a (Vegfa), hepatocyte growth factor (Hgf), angiopoietin (Angpt)1, and Angpt2, as well as type I collagen, matrix metallopeptidase (Mmp) 13, and tissue inhibitor of Mmp (Timp) 1, were determined using real-time reverse transcription polymerase chain reaction or enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells were grown with the culture supernatant of adipocytes stimulated with/without IL-6 or TNF-α, and the formation of tube structures was evaluated. RESULTS: IL-6 and TNF-α induced the expression of Vegfa, Hgf, and Angpt2 and decreased the expression of Angpt1 in premature adipocytes, whereas, they decreased the expression of Vegfa and Hgf in mature adipocytes. The culture supernatant of IL-6- or TNF-α-stimulated premature adipocytes induced the formation of tube structures. IL-6 and TNF-α had no effects on type I collagen expression in both premature and mature adipocytes but suppressed the expression of Mmp13 and Timp1 in mature and premature adipocytes, respectively. CONCLUSION: The effects of IL-6 and TNF-α on the expression of angiogenic and collagenolytic factors differed between premature and mature adipocytes. This finding suggests that these inflammatory cytokines induce expansion and dysfunction of adipose tissue via angiogenesis and collagen turnover in premature and mature adipocytes.

Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution.

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Selective separation method of aggregates from IgG solution by aqueous two-phase system.

Aggregation of immunoglobulin G (IgG) is a serious concern that results in immunogenicity in pharmaceutical applications. Removal of the small and soluble aggregates in protein solutions through a simple method remains challenging. Here we show that an aqueous two-phase system (ATPS) can be used for the elimination of soluble aggregates from IgG solution. Polyethylene glycol (PEG) and dextran (DEX) were selected as components of the ATPS. As expected, IgG monomers were partitioned into the top or bottom phases of ATPS. Interestingly, almost all the small and soluble aggregates of IgG were extracted to the interface between top and bottom phases, rather than in the liquid phases. The partitioning of monomers and aggregates of IgG can be attributed to the solubility of these protein states in PEG and DEX. Thus, ATPS using PEG and DEX can be employed for the simple removal method of soluble aggregates from IgG solution.

Salt-containing aqueous two-phase system shows predictable partition of proteins with surface amino acids residues.

Aqueous two-phase system (ATPS) containing salts has been used for protein purification and enrichment. However, it is unclear how proteins are partitioned in the top or bottom phases of the system. In this study, we demonstrate that the partition of proteins in salt-containing ATPS (SATPS) depends only on the relation between the protein-surface amino acids and the type of salt using SATPS. The partition coefficients of four proteins changed depending on the kind of salt, according to the Hofmeister series. Interestingly, the partition coefficients of the proteins correlated to those of the combination of the amino acids in the surface of the protein with the correlation coefficients of >0.9. The results suggest that the interaction between the protein surface and aqueous ions plays an indispensable role for the partition of proteins in SATPS that can help in the design of protein partition in ATPS for purification and enrichment.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4.

AIMS: During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. MAIN METHODS: The cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF-which was generated by increasing the volume of culture medium in the wells of a 96-well plate-in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. KEY FINDINGS: In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4-another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. SIGNIFICANCE: Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.