Subject(s)
Acetylglucosaminidase/metabolism , Chondroitin Lyases/metabolism , Glycosaminoglycans/metabolism , Polysaccharide-Lyases/metabolism , Reagent Kits, Diagnostic , Skin Aging , Skin/radiation effects , Sunlight/adverse effects , Adult , Aged , Biomarkers/metabolism , Chondroitin ABC Lyase/metabolism , Humans , Predictive Value of Tests , Skin/metabolism , Young AdultSubject(s)
ABO Blood-Group System/biosynthesis , Dermatologic Agents/administration & dosage , Extracellular Matrix/metabolism , Keratinocytes/drug effects , Monosaccharides/administration & dosage , Plant Extracts/administration & dosage , Skin Aging/drug effects , Skin/drug effects , Administration, Cutaneous , Adult , Aged , Cell Line , Dermatologic Agents/isolation & purification , Double-Blind Method , Female , Humans , Keratinocytes/metabolism , Middle Aged , Phytotherapy , Plant Extracts/isolation & purification , Plants, Medicinal , Skin/cytology , Skin/metabolism , Time Factors , Up-RegulationABSTRACT
BACKGROUND: Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin. OBJECTIVE: To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts. METHODS: UV irradiation-induced or xylosyltransferase (XYLT) 1 siRNA-mediated smaller-sized protein bands detected by Western blot using BGN antibodies were identified as monoglycosylated forms of BGN, using BGN siRNA-mediated knockdown and chondroitinase ABC (ChABC). Differential activity of XYLT1 and 2 on BGN core protein was investigated by size shift of S42A- and S47A-BGN mutants to core protein size caused by XYLT1 siRNA transfection or UV irradiation. RESULTS: After UV irradiation, intact form of BGN protein (I-BGN) and core protein form were reduced in cultured fibroblasts, but other smaller-sized bands were observed to be increased. These smaller-sized ones were reduced by transfection of BGN siRNA, and shifted to the core protein size by treatment with ChABC, suggesting that they are defectively-glycosylated forms of BGN (D-BGN) protein. UV irradiation also decreased mRNA expression levels of XYLT1 and 2, which are responsible for initiation of GAG chain synthesis. UV-mediated reduction of XYLT1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of D-BGN and the decrease of I-BGN, while down-regulation of XYLT2 resulted in no change of D-BGN and I-BGN, suggesting that the XYLT1 may react with both GAG-attaching serine sites of BGN; however, XYLT2 may prefer to react one of them. Another dermatan sulfate (DS) proteoglycan, decorin, showed no or a little change of its molecular weight by UV irradiation or XYLT1 siRNA transfection, suggesting that DS synthesis may not be a critical factor in formation of D-BGN. Co-transfection with XYLT1, 2 siRNAs and wild-type or mutant forms of BGN overexpression vectors revealed that S42A-BGN showed size reduction to core protein size by XYLT1 downregulation, but S47A-BGN did not, suggesting that XYLT2 can react only with S42 on BGN core protein. With UV irradiation, both S42A-BGN and S47A-BGN showed size reduction, which is probably because UV-caused downregulation of both XYLTs and overexpression condition resulted in incomplete glycosylation and secretion. CONCLUSIONS: UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity, which acts only at S42 on BGN core protein, caused by UV-mediated stronger reduction of XYLT1.
Subject(s)
Biglycan/biosynthesis , Biglycan/genetics , Glycosaminoglycans/biosynthesis , Pentosyltransferases/metabolism , Ultraviolet Rays , Cells, Cultured , Decorin/metabolism , Down-Regulation/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Silencing , Glycosaminoglycans/radiation effects , Glycosylation/radiation effects , Humans , Molecular Weight , Pentosyltransferases/genetics , Pentosyltransferases/radiation effects , Protein Biosynthesis/radiation effects , RNA, Messenger/metabolism , Skin Physiological Phenomena/radiation effects , Xylose/metabolism , UDP Xylose-Protein XylosyltransferaseABSTRACT
Most studies of cancer metastasis focus on cancer cell invasion utilizing adhesion assays that are performed independently, and are thus limited in their ability to mimic complex cancer metastasis on a chip. Here we report the development of an integrated cell-based microfluidic chip for intra- and extravasation that combines two assays on one chip for the study of the complex cascade of cancer metastasis. This device consists of two parts; one is an intravasation chamber for the three-dimensional (3-D) culture of cancer cells using a Matrigel matrix, and the other is an extravasation chamber for the detection of metastasized cancer cells by adhesion molecules expressed by epithelial cells. In this novel system, the intravasation and extravasation processes of cancer metastasis can be studied simultaneously using four screw valves. Metastatic LOVO and non-metastatic SW480 cells were used in this study, and the invasion of LOVOs was found to be higher compared to SW480. In contrast, invasion of cells treated with metalloproteinase (MMP) inhibitors decreased within the intravasation chamber. Degraded cancer cells from the intravasation chamber were detected within the extravasation chamber under physiological conditions of shear stress, and differences in binding efficiency were also detected when CA19-9 antibody, an inhibitor of cancer cell adhesion, was used to treat degraded cancer cells. Our results support the potential usefulness of this new 3D cell-based microfluidic system as a drug screening tool to select targets for the development of new drugs and to verify their effectiveness.