SIGNAL TRANSDUCTION BY INTEGRIN-MATRIX INTERACTION

研究了细胞-基质相互作用的分子机制，特别是整合素介导的细胞外基质信号转导，特别强调了间素α3β1与基底膜蛋白层粘连蛋白（LN）的特异性相互作用。获得的主要发现如下。(1) LN-8的纯化与鉴定：通过RT-PCR筛选了5种不同LN α链在35多种人细胞系中的表达。T98G胶质瘤细胞在5条LN α链中只表达α4链。我们对T98G细胞进行了大规模培养，并收获了用硫酸铵沉淀浓缩的培养基（约4升），然后使用抗ln β1单克隆抗体4F5进行凝胶过滤和免疫亲和层析。在非还原条件下，所得蛋白在SDS-PAGE上具有明显的同质性，由α4/β1/γi链组成，鉴定为LN-8。纯化后的LN-8对T98G细胞的粘附作用较弱，比LN-5和LN-10/11更强，其细胞粘附活性与LN-1相当。细胞粘附在LN-8上是通过两种主要的ln结合整合素α6β1和α3β1介导的。(2)细胞骨架对LN-10/11的调节：观察粘附在LN-10/11上是否能诱导应力纤维的形成和局灶性粘附。尽管LN-10/11具有很强的细胞粘附活性，但在粘附LN-10/11的细胞中，它不能诱导应力纤维或局灶性粘附。我们还发现LN-10/11没有检测到rho的激活，这与它在诱导应力纤维和局灶粘连方面的失败是一致的。(3)鉴定与整合素α3β1紧密结合的30 kDa蛋白CD151：制备了针对整合素α3亚基细胞质结构域的多克隆抗体，并从人胎盘中纯化了整合素α3β1。纯化后的整联素α3β1在SDS-PAGE上显示出3个主要的条带，即α3和β1链对应的150 kda/120 kda条带和一个出乎意料的30 kda条带。利用爱媛大学医学院Hitoshi Hasegawa博士提供的抗CD151抗体，通过免疫沉淀鉴定出30 kDa的条带为CD151，是跨膜4超家族蛋白之一。我们还制备了两种识别30kda蛋白的单克隆抗体，根据抗体与转染CD151 cDNA的NIH3T3细胞的反应性，证实抗体识别CD151。这些单克隆抗体将在CD151的生理功能研究中发挥重要作用。(4) LN α3链敲除小鼠的制备：我们成功制备了LN α3链基因缺失小鼠。这些小鼠在出生时就死于严重的上皮异常。整合素α6β4不能诱导突变小鼠基底膜的稳定粘附，与结缔组织出现水疱和半脂质体异常一致。我们还在缺乏LN-5的表皮基底膜中发现了一个新的整合素α3β1配体。我们还在缺乏LN-5的表皮基底膜中发现了一个新的整合素α3β1配体。我们还发现突变上皮细胞的存活缺陷可以通过外源性LN-5、胶原或α6 - β4抗体修复，这表明通过α6或β4整合素的信号传导足以维持存活。少

Molecular mechanisms of cell-matrix interaction, particularly integrin-mediated signal transduction from extracellular matrices, were investigated with special emphasis on the specific interaction of interin α3β1 with the basement membrane protein laminin (LN). Major findings obtained are as follows.(1) Purification and Characterization of LN-8 : We screened the expression of five different LN αchains in more than 35 human cell lines by RT-PCR. The T98G glioma cells were found to express only α4 chain among the five LN α chains. We grew T98G cells in a large scale and harvested the spent medium (approximately 4 liter) which was concentrated by ammonium sulfate precipitation, followed by gel filtration and immunoaffinity chromatography using the anti-LN β1 monoclonal antibody 4F5. The resulting protein was apparently homogeneous on SDS-PAGE under nonreducing conditions and identified to be LN-8 since it consisted of α4/β1/γi chains. The purified LN-8 was less potent in mediating adhesio … More n of T98G cells than LN-5 and LN-10/11 and comparable to LN-1 in its cell-adhesive activity. Cell adhesion onto LN-8 was mediated through integrin α6β1 and α3β1, two major LN-binding integrins.(2) Cytoskeletal Modulation on LN-10/11 : We examined whether adhesion onto LN-10/11 could induce formation of stress fibers and focal adhesions. Despite its very potent cell-adhesive activity, LN-10/11 failed to induce stress fibers or focal adhesions in cells adhered to LN-10/11. We also found that activation of rho was not detected on LN-10/11, consistent with its failure in inducing stress fibers and focal adhesions.(3) Identification of the 30 kDa Protein That Tightly Associates with Integrin α3β1 As CD151 : We produced polyclonal antibodies against the cytoplasmic domain of the integrin α3 subunit and purified integrin α3β1 from human placenta. Integrin α3β1 thus purified gave three major bands on SDS-PAGE, i. e. 150 kda/120 kDa bands corresponding to the α3 and β1 chains and an unexpected 30 kDa band. The 30 kDa band was identified as CD151, one of the transmembrane 4 superfamily proteins, by immunoprecipitation using the anti-CD151 antibody provided by Dr. Hitoshi Hasegawa (Ehime University Medical School). We also produced two monoclonal antibodies both recognizing the 30 kDa protein and the antibodies were confirmed to recognize CD151 based on their reactivity with the NIH3T3 cells transfected with the CD151 cDNA. These monoclonal antibodies should prove to be useful in the study of physiological functions of CD151.(4) Production of LN α3 Chain Knockout MIce : We succeeded in producing mice lacking the LN α3 chain gene. The mice died neonatally with profound epithelial abnormalities. The basement membrane of the mutant mice could not induce stable adheison by integrin α6β4, consistent with the presence of junctional blisters and abnornmal hemidesmosomes. We also detected a new ligand for integrin α3β1 in the epidermal basement membrane that worked in the absence of LN-5. We also detected a new ligand for integrin α3β1 in the epidermal basement membrane that worked in the absence of LN-5.We also identified a survival defect in mutant epithelial cells that could be rescued by exogenous LN-5, collagen, or antibody against α6β4, suggesting that signaling through α6 or β4 integrins is sufficient for survival. Less