Multivalent proteoglycan modulation of FGF mitogenic responses in perivascular cells.
Multivalent proteoglycan modulation of FGF mitogenic responses in perivascular cells.
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DOI：
10.1007/s10456-012-9316-7
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发表时间：
2013-04
期刊：
影响因子：
9.8
通讯作者：
Perris, Roberto
中科院分区：
文献类型：
--
作者：
Cattaruzza, Sabrina;Ozerdem, Ugur;Denzel, Martin;Ranscht, Barbara;Bulian, Pietro;Cavallaro, Ugo;Zanocco, Daniela;Colombatti, Alfonso;Stallcup, William B.;Perris, Roberto
血管新生血管周围细胞的萌发被认为高度依赖于自分泌和旁分泌生长因子的刺激。因此，我们报道了在NG2/CSPG4蛋白多糖(PG)缺失的小鼠中，异位移植的成纤维细胞生长因子诱导的角膜血管生成受到了严重的损害，这些小鼠已知存在周细胞增殖/动员缺陷。相反，当使用VEGF作为血管分泌因子时，野生型和敲除型角膜之间没有显著差异。NG2缺陷型小鼠的周细胞对多种FGFs刺激的旁分泌和自分泌的干扰反应在NG2缺陷型小鼠细胞中得到证实，而其他生长因子诱导的细胞增殖在野生型和基因敲除型细胞中相同。在siRNA介导的NG2基因敲除后，获得了相同的结果，在这些缺失NG2的细胞中检测到的成纤维细胞生长因子受体磷酸化水平的降低也证明了这一点。在活细胞上进行的重组蛋白结合分析和分子相互作用分析表明，成纤维细胞生长因子-2以糖胺糖非依赖性的核心蛋白介导的方式与NG2结合，并且PG单独能够将成纤维细胞生长因子-2保留在细胞膜上以供随后的受体呈递。利用显性-负性突变细胞，通过联合转导NG2缺失结构和下调内源PG的siRNA，使我们能够确定NG2的成纤维细胞生长因子联合受体活性完全由其胞外部分介导。事实上，强制过度表达NG2胞外区可增加其由成纤维细胞生长因子-2诱导的有丝分裂，并补偿低水平的成纤维细胞生长因子受体表面表达，其方式等同于过表达全长NG2所产生的结果。与成纤维细胞生长因子结合后，NG2的胞质结构域被磷酸化，但没有证据表明这一事件引发的信号转导可以绕过FGFR介导的信号转导。下拉实验、蛋白质结合分析和流式细胞仪FRET一致地揭示了NG2与FGFR1和FGFR3的选择性配体无关的关联。NG2与这些受体的合作在功能上也得到了成纤维细胞生长因子-2处理细胞的结果的证实，这些细胞被设计成表达不同的NG2/FGFR组合。综上所述，研究结果提示，血管周围NG2可能是成纤维细胞生长因子在细胞膜上的可获得性/可及性的双重调节器，以及由此产生的FGFR转导活性。
Sprouting of angiogenic perivascular cells is thought to be highly dependent upon autocrine and paracrine growth factor stimulation. Accordingly, we report that corneal angiogenesis induced by ectopic FGF implantation is strongly impaired in NG2/CSPG4 proteoglycan (PG) null mice known to harbour a putative deficit in pericyte proliferation/mobilization. Conversely, no significant differences were seen between wild type and knockout corneas when VEGF was used as an angiocrine factor. Perturbed responsiveness of NG2-deficient pericytes to paracrine and autocrine stimulation by several FGFs could be confirmed in cells isolated from NG2 null mice, while proliferation induced by other growth factors was equivalent in wild type and knockout cells. Identical results were obtained after siRNA-mediated knock-down of NG2 in human smooth muscle-like cell lines, as also demonstrated by the decreased levels of FGF receptor phosphorylation detected in these NG2 deprived cells. Binding assays with recombinant proteins and molecular interactions examined on live cells asserted that FGF-2 bound to NG2 in a glycosaminoglycan-independent, core protein-mediated manner and that the PG was alone capable of retaining FGF-2 on the cell membrane for subsequent receptor presentation. The use of dominant-negative mutant cells, engineered by combined transduction of NG2 deletion constructs and siRNA knock-down of the endogenous PG, allowed us to establish that the FGF co-receptor activity of NG2 is entirely mediated by its extracellular portion. In fact, forced overexpression of the NG2 ectodomain in human smooth muscle-like cells increased their FGF-2-induced mitosis and compensated for low levels of FGF receptor surface expression, in a manner equivalent to that produced by overexpression of the full-length NG2. Upon FGF binding, the cytoplasmic domain of NG2 is phosphorylated, but there is no evidence that this event elicits signal transductions that could bypass the FGFR-mediated ones. Pull-down experiments, protein-protein binding assays and flow cytometry FRET coherently revealed an elective ligand-independent association of NG2 with FGFR1 and FGFR3. The NG2 cooperation with these receptors was also corroborated functionally by the outcome of FGF-2 treatments of cells engineered to express diverse NG2/FGFR combinations. Comprehensively, the findings suggest that perivascular NG2 may serve as a dual modulator of the availability/accessibility of FGF at the cell membrane, as well as the resulting FGFR transducing activity.
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影响因子：
4.8
作者：
Goretzki, L;Burg, MA;Stallcup, WB
通讯作者：
Stallcup, WB
影响因子：
4.8
作者：
Loo, BM;Kreuger, J;Salmivirta, M
通讯作者：
Salmivirta, M
DOI：
10.1083/jcb.200106075
发表时间：
2001-11-26
期刊：
The Journal of cell biology
影响因子：
--
作者：
Allen BL;Filla MS;Rapraeger AC
通讯作者：
Rapraeger AC
影响因子：
5.3
作者：
Gotoh, N;Manova, K;Schlessinger, J
通讯作者：
Schlessinger, J
影响因子：
64.8
作者：
Armulik, Annika;Genove, Guillem;Betsholtz, Christer
通讯作者：
Betsholtz, Christer