| 摘要: | 細胞表面的滯留序列 (Cell Retention Sequence or CRS) 是一連串的鹼性胺基酸組成的。CRSBP-1 是一個存在細胞膜的受體蛋白。它的生理配體 (physiological ligands) 是若干重要的生長因子(如 PDGF-BB 和VEGF-A)。這些生長因子因帶有滯留序列 (CRS), 在細胞内合成分泌之後被CRSBP-1 結合而滯留在細胞表面上。我們利用基因克隆 (Cloning) 及其在細胞的表現，發現CRSBP-1 與另一種蛋白(LYVE-1)相同。LYVE-1 是已知的玻尿酸 (hyaluronic acid or HA)受體, CRSBP-1 能夠與玻尿酸及含有CRS 序列的生長因子結合, 因此CRSBP-1 具有雙重功能。因LYVE-1 特別表現在淋巴管的內皮細胞 (Endothelial Cells), 這表示CRSBP-1 可能在淋巴管的功能扮演一個重要的角色為了要研究 CRSBP-1 在體內所扮演的角色，我們最近培育出CRSBP-1 基因敲除小老鼠 (CRSBP-1-null mice)。這種小老鼠外觀正常也有正常的生育能力，但是我們發現這種小老鼠有不正常的淋巴管的構造及功能。它們的淋巴管呈現擴張的管口 (distended lumens)，它的組織間質到淋巴管的流通 (interstitial-lymphatic flow) 也增強。我們更進一步發現CRSBP-1 的生理的配體 (physiological ligands) 如PDGF-BB 及HA(玻尿酸)以及其特殊的配體 (specific ligands) 如PDGF-peptide 及 VEGF-peptide，也能使野生型(wild-type)老鼠的淋巴管管口 (lumen) 擴張以及流通增強，但在 CRSBP-1 基因敲除的小老鼠卻沒有這種功用。PDGF-peptide 及VEGF-peptide 帶有PDGF-BB 及 VEGFA 的CRS 序列，它們是小分子的多泩肽，它們只與CRSBP-1 作用而不與PDGF β-type receptor (PDGFR)及VEGF receptor 3 (VEGFR-3)作用。這些結果表示CRSBP-1 可以調控組織間質到淋巴管的流通。最近我們利用 SVEC4-10 細胞做為淋巴管內皮細胞的模型，來研究CRSBP-1 在淋巴管內皮細胞的功能及其調控組織間質到淋巴管流通的機制。最近有報告指出在SVEC4-10 細胞, VEGF-A 可以引起細胞表面上的VE-cadherin 内化而造成細胞與細胞的連接處張開。我們也發現CRSBP-1 的特殊配體如PDGF-peptide 及VEGF-peptide 也可以引起細胞表面上的VE-cadherin 内化。我們更發現在 SVEC4-10 細胞, PDGFR 與 CRSBP-1 行成complex, CRBBP-1 的特殊配體並且能促進PDGFR 的 tyrosine phosphorylation。基於這些的發現，我們提出一個假說: “CRBBP-1 是在調控細胞與細胞間接合處的功能”。這個提案的目標是要證實這個假說並研究 CRSBP-1 如何調控組織間質到淋巴管流通的機制。我們兩個方案： (1) 利用野生型老鼠及 CRSBP-1 基因敲除小老鼠來研究CRSBP-1 在控制組織間質與淋巴管的流通的功能。 (2) 利用 SVEC4-10 細胞，來研究CRSBP-1 的特殊配體 (PDGF-peptide 及VEGF-peptide) 如何使細胞之間連接處張開。這些研究將使我們瞭解 CRSBP-1 在調控組織間質到淋巴管流通的機制，也能供給關於如何調控組織間質到淋巴管流通新的知識，進而發展藥物來增強免疫反應與消減淋巴水腫及癌細胞經由淋巴管的轉移。 ; Cell-surface retention sequence (CRS) binding protein-1 (CRSBP-1) was first identified by its ability to bind oligopeptides PDGF-peptide and VEGF-peptide which contain the CRS motifs (a cluster of basic amino acid residues) of PDGF-BB and VEGF-A, respectively. CRSBP-1 was demonstrated to mediate cell-surface retention (after synthesis and secretion) of the v-sis gene product (PDGF-BB) in v-sis-transformed cells. cDNA cloning and expression of CRSBP-1 revealed that CRSBP-1 is a type I membrane glycoprotein and is identical to the independently discovered LYVE-1 (lymphatic vessel endothelial hyaluronic acid receptor-1). CRSBP-1 exhibits a dual ligand (CRS-containing proteins or peptides and hyaluronic acid) binding activity. The specific localization of LYVE-1/CRSBP-1 in intercellular junctions of lymphatic endothelial cells (LECs) implies its potential importance in the function of lymphatic capillary vessels. To determine the in vivo role of CRSBP-1, we recently generated CRSBP-1-null mice. These mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic capillary vessels marked by distended lumens and increased interstitial-lymphatic flow as determined by immunohistochemical analysis and FITC-dextran egress assay, respectively. In addition, CRSBP-1 ligands (PDGF-BB and hyaluronic acid) induce increased interstitial-lymphatic flow in wild-type mice but not in CRSBP- 1-null mice. A recent report suggests that VEGF-A, a ligand for both CRSBP-1 and the VEGF receptor 3 (VEGFR3), induces opening of intercellular junctions by causing disruption of VE-cadherin-mediated intercellular adhesion in SVEC4-10 cells, a LEC-like cell line. More recently, we found that, like PDGF-BB and VEGF-A, specific CRSBP-1 ligands (PDGF-peptide and VEGF-peptide), which do not interact with either PDGFR or VDGFR3, induce increased interstitial–lymphatic flow in wild-type mice but not CRSBP-1-null mice. We also found that CRSBP-1 and PDGFR form complexes in a ligand-independent manner in SVEC4-10 cells and that specific CRSBP-1 ligands stimulate tyrosine phosphorylation of PDGFR and induce internalization of cell-surface VE-cadherein in these cells. Based on these findings, we hypothesize that CRSBP-1 is involved in the maintenance of the functional integrity of intercellular junctions of LECs and that the CRSBP-1-null mutation and CRSBP-1 ligands cause disruption of VE-cadherin-mediated cellular adhesion between LECs by inducing internalization of cell-surface VE-cadherin, resulting in opening of intercellular junctions and increased interstitial-lymphatic flow. The goal of this proposal is to test this hypothesis and to determine the mechanism by which CRSBP-1 regulates interstitial-lymphatic flow. There are two specific aims: 1) To define the function of CRSBP-1 in regulating interstitial-lymphatic flow using wild-type and CRSBP-1-null mice and 2) To determine the mechanism by which specific CRSBP-1 ligands induce opening of intercellular junctions using SVEC4-10 cells. These studies should define the function of CRSBP-1 in regulating interstitial-lymphatic flow and provide new insights into the novel regulatory mechanism of interstitial-lymphatic flow. These studies may also lead to the development of novel agents for enhancing immune responses and for ameliorating lymphedema and carcinoma metastasis via lymphatic routes. ; 研究期間 9711 ~ 9807 |
