A Study On The Mechanism Of BIGLYCAN In Photoaging Process

Background:Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two chondroitin sulfate (CS)/dermatan sulfate (DS) glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin. The synthesis of BGN-CS/DS chain starts in the ER while the core protein is being synthesized, and continues in the Golgi apparatus. Many steps mediated by various GTs are involved in the process of CS/DS-GAG synthesis. Although it is abundant in many tissues, its biological functions remain poorly understood. BGN is considered to have an organizing role in the assembly of the ECM. Targeted disruption of the BGN gene results in abnormal collagen fibril morphology and an osteoporosis-like phenotype. BGN interacts via its core protein or GAG chains with numerous components of the extracellular matrix (ECM), and has critical roles in type Ⅰ, Ⅱ, Ⅲ, and Ⅵ collagen and elastic fibril formation. Recently, the BGN has emerged as a key factor associated with inflammation. The Bgn-null mice have a considerable survival benefit compared with WT animals, in LPS-induced sepsis mice model, BGN modulates inflammation by regulating Toll-like receptors and P2X7 receptor signaling in macrophages, leading to secretion of TNF-a and mature IL-1b,promotes inflammatory reactions. BGN plays an important role as a reservoir for balancing the pathogen recognition (TLRs, resistance genes in plants, CD14, nucleotide-binding oligomerization domain proteins) to control inflammation. However, functions of BGN during photoaging and their regulatory mechanisms have not been well established. This study is focus on the role of BGN in photoaging process.PART ONEObjectives: To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts.Materials and 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).Results:After UV irradiation, intact form of BGN protein (1-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 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 XYLT 1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of defectively-glycosylated forms of BGN and the decrease of 1-BGN, while down-regulation of XYLT2 resulted in no increase of defectively-glycosylated forms of BGN and no decrease of I-BGN. By co-transfection with XYLT1,2 siRNAs and wild-type or mutant forms of BGN overexpression vectors, it was observed 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.Conclusions:UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity caused by UV-mediated stronger reduction of XYLT1.PART TWOObjectives:To investigate the role of BGN in UV-induced inflammatory response.Materials and methods:To make clear the correlation between BGN and TLR, fibroblasts were transfected with BGN siRNA or TLR4 siRNA, and then irradiated with UV. Thereafter, pro-inflammatory cytokines were observed by using RT-PCR. Next, fibroblasts were transfected with BGN siRNA and stimulated with TLR4 specific ligands LPS. Here could demonstrate the extracellular matrix BGN regulation of inflammation by interacting with TLR 4 receptors.Results:UV irradiation on dermal fibroblasts increased proinflammatory cytokines, and knockdown of BGN and TLR4 (BGN-siRNA and TLR4-siRNA) significantly decreased UV-induced inflammation. Importantly, LPS-induced proinflammatory factors were significantly reduced in BGN-siRNAtransfected fibroblasts. Our results indicate that extracellular matrix component BGN is signal transduction factor, plays important role in TLR4 mediated inflammation.Conclusions:The extracellular matrix component BGN promotes UV-induced inflammatory response via TLR4 signaling pathway.