Sung V, Stubbs J T, Fisher L, Aaron A D, Thompson E W
Department of Cell Biology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC, USA.
J Cell Physiol. 1998 Sep;176(3):482-94. doi: 10.1002/(SICI)1097-4652(199809)176:3<482::AID-JCP5>3.0.CO;2-K.
Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSP-RGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the alpha(v)beta3 vitronectin receptor, whereas adhesion and proliferation responses were alpha(v)beta5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for alpha(v)beta3 or alpha(v)beta5 respectively. Although some expression of the alternate alpha(v) integrin was still retained, the alpha(v)beta5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the alpha(v)beta3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs alpha(v)beta3 and alpha(v)beta5 integrin receptors.
骨唾液蛋白（BSP）是一种存在于骨基质中的分泌型糖蛋白，与人类乳腺癌（HBC）的乳腺微钙化形成及亲骨性转移有关。BSP具有一个整合素结合RGD（精氨酸-甘氨酸-天冬氨酸）结构域，该结构域可能促进HBC细胞与骨细胞外基质之间的相互作用。纯化的BSP、重组人BSP片段和BSP衍生的RGD肽在MDA-MB-231 HBC细胞系中可引发迁移、黏附和增殖反应。重组BSP片段分析将这些活性的一个重要成分定位到该蛋白的RGD结构域，带有BSP侧翼序列的合成RGD肽（BSP-RGD）也能引发这些反应。纤连蛋白衍生的RGD对应物GRGDSP（甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸）不能支持这些细胞反应，这突出了BSP构型的特异性。虽然大多数增殖和黏附反应可归因于RGD相互作用，但这些相互作用仅部分负责迁移反应。整合素阻断抗体实验表明，BSP-RGD诱导的迁移利用α(v)β3玻连蛋白受体，而黏附和增殖反应由α(v)β5介导。通过荧光激活细胞分选，我们分别选择了两个富含α(v)β3或α(v)β5的MDA-MB-231细胞亚群。虽然仍保留了一些交替α(v)整合素的表达，但富含α(v)β5的MDA-MB-231细胞显示出增强的增殖和黏附反应，而富含α(v)β3的亚群增殖和黏附受到抑制，但对BSP-RGD的迁移反应增强。此外，对另外两种HBC细胞系的类似分析显示，在对BSP片段的黏附和增殖方面，RGD依赖性趋势不太明显但相似。总体而言，这些数据证明了BSP对HBC细胞增殖、迁移和黏附功能的影响，以及RGD介导的成分对α(v)β3和α(v)β5整合素受体的不同利用。
