Berrou E, Breton M, Deudon E, Picard J
Laboratoire de Biochimie, Inserm U. 181, Faculté de Médecine Saint-Antoine, Paris, France.
J Cell Physiol. 1991 Dec;149(3):436-43. doi: 10.1002/jcp.1041490312.
We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.
我们之前已经表明（Berrou等人，《细胞生理学杂志》，137:430 - 438，1988年），猪内皮细胞条件培养基（ECCM）可刺激猪主动脉平滑肌细胞的蛋白聚糖合成。ECCM刺激需要蛋白质核心来起始糖胺聚糖链，并且伴随着分泌到培养基中的蛋白聚糖流体力学尺寸的增加。这项工作研究了ECCM效应所涉及的机制。1）用放线菌酮处理并未抑制对照和ECCM刺激的蛋白聚糖合成（通过20分钟的[35S] - 硫酸盐标记试验测量），这表明蛋白聚糖由预先存在的蛋白质核心组成，并且ECCM刺激这些蛋白质核心的糖基化。2）虽然ECCM刺激[35S] - 甲硫氨酸掺入分泌蛋白仅在孵育6小时后发生，但在1小时后观察到[35S]甲硫氨酸标记的蛋白聚糖增加，并且这种增加至少持续16小时保持稳定。3）通过SDS电泳分析，软骨素酶消化从[14C]丝氨酸标记的蛋白聚糖产生了7个高表观分子量（550 - 200 kDa）的蛋白质核心和1个47 kDa的蛋白质核心。两个最高表观分子量（550和460 kDa）的蛋白质核心，而非47 kDa的蛋白质核心，显示出响应ECCM时[14C] - 丝氨酸掺入增加（通过琼脂糖CL - 6B层析测量为51%）。4）最后，通过琼脂糖CL - 6B层析测定[35S] - 硫酸盐掺入软骨素酶产生的蛋白质核心上的糖胺聚糖连接残基：ECCM并未改变受刺激蛋白质核心中的[35S]/[14C]比率，表明ECCM不影响糖胺聚糖链的数量。这些研究结果表明：1）内皮细胞分泌因子，优先刺激最大的平滑肌细胞蛋白聚糖的合成且不进行结构修饰；2）这种刺激通过增强木糖基化的蛋白质核心的糖基化，随后增强蛋白质合成来进行。
