細胞外基質（extracellular matrix）會經由影響生長因子的訊息傳遞而改變細胞的生理反應。以乳腺上皮細胞而言，若將細胞培養在基底膜上，胰島素的訊息傳遞則可順利進行；若將細胞培養在塑膠盤或膠原蛋白（collagen）上，雖然胰島素受體（insulin receptor，IR）的酪氨酸磷酸化不受影響，但胰島素受體受質-1（insulin receptor substrate，IRS-1）的酪氨酸磷酸化卻大幅降低。因此，缺乏基底膜時，胰島素的訊息傳遞會被阻斷在IR。在本實驗中，我們將細胞培養在塑膠盤或基底膜，以探討調節胰島素訊息傳遞的機轉，尤其是受體近端的訊息路徑。我們發現在乳腺上皮細胞中，胰島素的訊息傳遞應該不是發生在lipid rafts，因為破壞這些構造並未造成任何影響；然而細胞骨架的完整性對於胰島素的訊息傳遞卻是相當重要的。另外，aspirin和PGJ2也不會影響胰島素訊息傳遞，而phosphatidylinositol 3- kinase（PI3K）的抑制劑wortmannin卻可提高IRS-1接合到IR/IGF-IR的程度，以及IRS-1的酪氨酸磷酸化。此種效果又以培養在塑膠盤上的細胞較顯著。因此，培養在塑膠盤上的細胞之所以無法順利傳遞胰島素所誘發的訊號，可能是因為PI3K的作用。最後，我們檢測酪氨酸去磷酸（protein tyrosine phosphatase）LAR、PTP1B、SHP-2與RPTPα在細胞中的表現。不論是以基底膜或塑膠作為附著層，PTP1B、SHP-2與RPTP的表現量並無差異，而培養在基底膜的細胞其LAR的表現量則較高。所以目前為止，我們還無法確認出抑制胰島素訊息傳遞的去磷酸。
Extracellular matrix (ECM) affects a number of cellular responses through modulation of growth factor signaling. For mammary epithelia, insulin-stimulated signaling is propagated properly when cells are in contact with basement membrane, but it is hindered when cells are cultured on plastic or collagen I. Under the latter situation, tyrosine phosphorylation of insulin receptor (IR) is not affected but phosphorylation of insulin receptor substrate-1 (IRS-1) is substantially impaired. Therefore, a novel ECM-dependent control point in insulin signaling is at the level of tyrosine phosphorylation of IRS-1. In this study, we investigated into the regulatory mechanisms for insulin signaling in mammary epithelial cells, especially focusing on the membrane proximal events. We found that insulin signaling probably did not take place in lipid rafts since disruption of them exerted no effect on signaling; however, cytoskeleton integrity was critical for it. Incubation of aspirin and PGJ2 that have been shown to revert insulin resistance did not increase the extent of IRS-1 tyrosine phosphorylation. By contrast, inhibition of phosphatidylinositol 3 kinase (PI3K) by wortmannin led to greater degree of IRS-1 recruitment to the receptor, and tyrosine phosphorylation of IRS-1, especially for those cells cultured on plastic. This suggests that the inertness of the cells cultured on plastic to insulin signaling is probably mediated by PI3K. Lastly, we examined the expression of protein tyrosine phosphatases (PTP) in cells cultured on plastic and basement membrane as it plays an important role in controlling the homeostasis of tyrosine phosphorylation. We found that the levels of PTP1B, SHP-2 and RPTP were comparable in cells on both substrata, but the expression of LAR was more prominent in cells cultured on BM. Thus, we were still not clear which PTP might be involved in regulation of insulin signaling in cells cultured on plastic.
