Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA
Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA
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DOI：
10.1128/mcb.17.4.1938
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发表时间：
1997-04-01
影响因子：
5.3
通讯作者：
Yayon, A
中科院分区：
文献类型：
--
作者：
Aviezer, D;Iozzo, RV;Yayon, A
硫酸乙酰肝素蛋白聚糖（HSPG）在形成独特的成纤维细胞生长因子（FGF）-HS复合物中起关键作用，增强高亲和力结合和受体活化，串珠素（Perlecan）是增殖细胞中丰富的分泌型HSPG，能够在体外诱导FGF-受体相互作用和在体内诱导血管生成。通过表达对应于串珠素的N-末端和HS附着结构域的反义cDNA，已经实现了小鼠NIH 3 T3成纤维细胞和人转移性黑素瘤细胞中串珠素水平的稳定和特异性降低。通过使用对小鼠或人串珠素特异的DNA探针和抗体的北方印迹分析、免疫印迹和免疫组织化学所示的串珠素mRNA和核心蛋白水平的降低来证明串珠素的长期下调。与野生型或载体转染的细胞相比，表达反义串珠蛋白聚糖的细胞对增加浓度的碱性FGF（bFGF）的反应显著降低，如通过胸苷掺入和增殖速率所测量的。此外，用I-125-bFGF转染的反义串珠蛋白聚糖细胞的受体结合和亲和标记被显著抑制，表明消除串珠素表达导致高亲和力bFGF结合降低。反义串珠素表达克隆对bFGF的结合和促有丝分裂反应都可以被外源性肝素或串珠素所拯救。这些结果支持串珠素是小鼠成纤维细胞和人黑色素瘤中bFGF的主要辅助受体的观点，并指出串珠素反义构建体可能用作bFGF介导的反应的特异性调节剂。
Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation, Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation, Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with I-125-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan, These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.