Krushel L A, Prieto A L, Edelman G M, Crossin K L
Department of Neurobiology, Scripps Research Institute, La Jolla, California 92037.
J Cell Physiol. 1994 Dec;161(3):508-18. doi: 10.1002/jcp.1041610314.
Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi.
细胞趋触蛋白/腱生蛋白是一种多结构域的细胞外基质蛋白，它既能抑制细胞铺展，又能抑制细胞内碱化。该蛋白有多个不同结构域，与表皮生长因子、纤连蛋白和纤维蛋白原中的区域同源。在先前的研究中，我们制备了与这些结构域对应的非重叠融合蛋白，并研究了它们对细胞黏附和铺展的影响。基于它们促进或抑制细胞黏附的能力，这些融合蛋白中有两种显示出黏附性，两种显示出抗黏附性。为了确定不同细胞趋触蛋白/腱生蛋白结构域的黏附性和抗黏附性活性如何改变细胞内pH（称为pHi），我们在存在细胞趋触蛋白/腱生蛋白融合蛋白、完整的细胞趋触蛋白/腱生蛋白以及纤连蛋白的情况下，测量了NIH3T3和U251MG细胞中的pHi。在完整的细胞趋触蛋白/腱生蛋白或抗黏附融合蛋白存在下孵育的细胞，其pHi低于对照细胞。相反，黏附性融合蛋白或纤连蛋白的存在导致细胞的pHi值高于对照细胞。当同时呈现两个片段时，其中一个单独作用时会升高pHi，另一个单独作用时会降低pHi，主要作用是降低pHi。用源自细胞趋触蛋白/腱生蛋白序列的含RGD肽孵育，可抑制由含第二至第六个III型纤连蛋白重复序列的黏附片段促进的碱化，该片段已知可与整合素结合。用肝素酶I或III孵育细胞，可抑制在存在另一种含纤维蛋白原结构域的黏附融合蛋白时接种的细胞的细胞内碱化，这表明硫酸乙酰肝素蛋白聚糖参与了这些pHi变化。蛋白激酶C的活性似乎对所有这些蛋白介导的pHi变化都很重要。蛋白激酶C抑制剂Calphostin C可阻断黏附融合蛋白和纤连蛋白引起的pHi升高。此外，通过添加佛波酯激活蛋白激酶C，可增加接种在细胞趋触蛋白/腱生蛋白或抗黏附融合蛋白上的细胞的pHi，并逆转它们的作用。本研究结果支持以下假说：细胞趋触蛋白/腱生蛋白可与多种细胞表面受体结合，从而引发不同的生理反应。pHi降低与抗黏附现象相关，而升高pHi的能力与通过至少两种不同类型的细胞表面受体进行的细胞黏附有关。这些数据增加了一种可能性，即细胞趋触蛋白/腱生蛋白的结合可能通过对pHi的差异调节来影响诸如迁移和增殖等主要细胞过程。
