Aharoni D, Meiri I, Atzmon R, Vlodavsky I, Amsterdam A
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Curr Biol. 1997 Jan 1;7(1):43-51. doi: 10.1016/s0960-9822(06)00026-1.
Epithelial cells are closely associated with a basement membrane, but the intimate relationships that affect growth, differentiation and survival remain enigmatic. We have previously reported that granulosa cells adjacent to the basement membrane of the ovarian follicle have a higher degree of differentiation compared with cells located distal to the basement membrane. By contrast, granulosa cells distal to the basement membrane are the first to undergo apoptosis during follicular atresia. Moreover, growth of granulosa cells in vitro on a naturally produced basement-membrane-like extracellular matrix (ECM) enhances progesterone production and the cellular response to gonadotropic hormones by an undefined mechanism.
To investigate the effect of the ECM on granulosa cell differentiation and death, primary granulosa cells were cultured on ECMs that lacked or contained bFGF (basic fibroblast growth factor). These otherwise identical ECMs were deposited by HR9 mouse endodermal cells, which do not synthesize bFGF, or by HR9 cells transfected with the bFGF gene. Both ECMs provided protection against apoptosis in serum-free medium, but only the bFGF-containing ECM maintained expression of the steroidogenic P450scc enzyme system and the production of progesterone. Moreover, culturing the cells on this ECM enhanced the expression of the 30 kDa steroid acute regulatory protein which plays a key role in steroid hormone biosynthesis. Laminin, but not fibronectin, was able to replace the ECM in protecting the cells from apoptosis; but not in maintaining steroidogenesis, whereas bFGF was able to enhance steroidogenesis without protecting the cells against apoptosis. Cells cultured on both ECMs or laminin had a well-developed actin cytoskeleton compared with cells cultured on non-coated dishes, which underwent apoptosis.
Cellular responses to ECM are mediated by the combined action of macromolecular constituents and regulatory molecules, such as bFGF, that are sequestered and stored in the ECM. ECM or laminin protects against cell death by interacting with specific integrin receptors and maintaining a well-developed actin cytoskeleton. ECM-bound bFGF provides differentiation signals for granulosa cells, which are in intimate contact with the ECM. Thus, a clear distinction can be made between the survival activity and the differentiation stimulus exerted by the ECM on epithelial cells.
上皮细胞与基底膜紧密相连，但影响其生长、分化和存活的密切关系仍不清楚。我们之前报道过，与卵巢卵泡基底膜相邻的颗粒细胞与位于基底膜远端的细胞相比，具有更高程度的分化。相比之下，在卵泡闭锁过程中，基底膜远端的颗粒细胞是最先发生凋亡的。此外，在天然产生的类基底膜细胞外基质（ECM）上体外培养颗粒细胞，通过一种未知机制增强了孕酮的产生以及细胞对促性腺激素的反应。
为了研究ECM对颗粒细胞分化和死亡的影响，将原代颗粒细胞培养在缺乏或含有碱性成纤维细胞生长因子（bFGF）的ECM上。这些在其他方面相同的ECM是由不合成bFGF的HR9小鼠内胚层细胞或转染了bFGF基因的HR9细胞沉积而成。两种ECM在无血清培养基中都提供了抗凋亡保护，但只有含bFGF的ECM维持了类固醇生成的P450scc酶系统的表达和孕酮的产生。此外，将细胞培养在这种ECM上增强了在类固醇激素生物合成中起关键作用的30 kDa类固醇急性调节蛋白的表达。层粘连蛋白而非纤连蛋白能够替代ECM保护细胞免于凋亡，但不能维持类固醇生成，而bFGF能够增强类固醇生成但不能保护细胞免于凋亡。与在未包被培养皿上培养并发生凋亡的细胞相比，在两种ECM或层粘连蛋白上培养的细胞具有发育良好的肌动蛋白细胞骨架。
细胞对ECM的反应是由大分子成分和调节分子（如bFGF）的联合作用介导的，这些分子被隔离并储存在ECM中。ECM或层粘连蛋白通过与特定整合素受体相互作用并维持发育良好的肌动蛋白细胞骨架来保护细胞免于死亡。与ECM紧密接触的颗粒细胞，ECM结合的bFGF为其提供分化信号。因此，ECM对上皮细胞施加的存活活性和分化刺激之间可以明确区分。
