Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro
Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro
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DOI：
10.3109/08977199809002117
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发表时间：
1998-01-01
期刊：
影响因子：
1.8
通讯作者：
Brigstock, DR
中科院分区：
文献类型：
--
作者：
Steffen, CL;Ball-Mirth, DK;Brigstock, DR
结缔组织生长因子 (CTGF) 是培养的成纤维细胞的一种促有丝分裂和趋化因子，与伤口愈合、纤维化疾病和子宫功能有关。尽管最近已经描述了预计会分泌的小鼠、人和猪 CTGF（mCTGF、hCTGF、pCTGF）基因的主要翻译产物以及大约 M-r 38,000、10 kDa 生物活性形式的 pCTGF。在本报告中，我们表明人包皮成纤维细胞 (HFF) 和小鼠结缔组织成纤维细胞含有 2.4kb CTGF 转录物，用抗 CTGF[81-94] 肽抗血清呈阳性染色，并产生 38 kDa 的蛋白质，该蛋白质由抗 CTGF[247-260] 肽抗血清免疫沉淀。虽然 38 kDa CTGF 在细胞裂解物中很容易检测到，但在条件培养基中无法或几乎检测不到。 38 kDa CTGF 在合成后至少 5 天仍与细胞相关，并且不能通过用胰蛋白酶、肝素、1 M NaCl 或低 pH 处理细胞来释放。从人或小鼠成纤维细胞条件培养基中纯化 CTGF，可分离出 10-11 kDa CTGF 蛋白，这些蛋白具有肝素结合性、生物活性，并在蛋白质印迹上与抗 CTGF[247-260] 发生反应。尽管 10 kDa CTGF 刺激 3T3 细胞中 DNA 合成的程度与血小板衍生生长因子 (PDGF)-AA、-AB 或 -BB 相同，但它不会与 I-125-PDGF-BB 竞争与 α α、α β 或 β β PDGF 受体 (PDGF-R) 的结合，不会刺激 PDGF-α-R 或 -β-R 的酪氨酸磷酸化，并且不会被中和因子拮抗。 PDGF-R-α 抗血清。这些数据表明，在培养的成纤维细胞中，38 kDa CTGF 主要与细胞相关，而低质量形式的 CTGF 是可溶的且具有生物活性。他们进一步证明，与之前提出的 38 kDa CTGF 特性相反，10 kDa CTGF 不与 PDGF-R 结合，并通过不依赖于 PDGF-R 的机制刺激 Balb/c 3T3 细胞有丝分裂。
Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes an predicted to be secreted and of approximate M-r 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that a as immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non-or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-11 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with I-125-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum.These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.