探討乳腺細胞泌乳激素與第一型類胰島素生長因子訊息傳遞之調控機轉

Regulatory mechanisms involved in prolactin and IGF-I signaling in breast cells

杜軍毅(Jyun-Yi Du)

指導教授 ： 李宜儒

中山醫學大學/醫學院/微生物免疫研究所/博士(2014年)

摘要

第一部分:
對乳腺上皮細胞而言，泌乳激素所誘發的訊息傳遞與基因表現是需要細胞藉由插入蛋白黏附於基底膜上。細胞在缺乏對基底膜的黏附時，像是將細胞培養在塗有第一型膠原蛋白的塑膠盤上時，泌乳激素的訊息傳遞會大幅減弱。本篇研究證實，對於培養在第一型膠原蛋白上的乳腺細胞，其泌乳激素訊息傳遞受阻是RhoA-Rok-myosin II路徑所造成的。在這些細胞，RhoA路徑被活化，因而抑制了泌乳激素接受器的表現並降低泌乳激素的訊息傳遞。迫使培養在基底膜上細胞提高RhoA活性則會抑制泌乳激素接受器的表現量並阻礙泌乳激素所誘發的Stat5酪胺酸磷酸化以及乙型酪蛋白的表現。反之，在培養於第一型膠原蛋白上的細胞中表現dominant negative RhoA或是抑制Rok活性時，則能提高泌乳激素接受器的表現與改善泌乳激素的訊息傳遞。以blebbistatin抑制myosin II ATPase活性也得到類似的效果，顯示細胞因應膠原蛋白基質所產生之張力，連同RhoA-Rok-myosin II路徑的活化造成泌乳激素訊息傳遞的失效。除此之外，將細胞培養在塗有基膜素（laminin）的塑膠盤上時，其細胞形態、RhoA活性和泌乳激素訊息傳遞的能力皆與培養在第一型膠原蛋白上的細胞相似，證明了基質軟硬度對於訊息傳遞的重要性。因此， RhoA在細胞與基質之間的交互作用中扮演了影響細胞命運的重要角色。

第二部分:
第一型類胰島素生長因子（IGF-I）與雌激素在乳腺發育中皆扮演重要的角色。然而，當這些生長因子與荷爾蒙不正常的增加時，將會促進乳癌生成以及造成乳癌病人預後不良的情形發生。已有許多文獻顯示IGF-I與雌激素所誘導的訊息傳遞路徑對於促進細胞增生具有協同作用，而目前僅知可能的原因為雌激素可以促進類胰島素生長因子受體（IGF-IR）與胰島素受體受質（IRS-1）的表現，但其他的機制仍不清楚。因此，我們想要更進一步探討IRS-1的表現量是否受到ERα的調控。我們意外地發現提高ERα表現量反而抑制初代小鼠乳腺上皮細胞IRS-1蛋白的表現量。此外，IGF-I所誘導的IRS-1酪胺酸磷酸化也同樣受到抑制。我們使用初代人類乳腺上皮細胞和正常小鼠乳腺上皮細胞株NMUMG也得到相似的結果。接下來，我們使用ER陰性乳癌細胞株Hs578T與MDA-MB-231做為實驗材料。結果顯示提高ERα表現量抑制MDA-MB-231的IRS-1蛋白表現；相反的，卻是增加Hs578T的IRS-1蛋白表現量，但此二種乳癌細胞株的IGF-I訊息傳遞都受到ERα的抑制。這個結果可能是因為類胰島素生長因子結合蛋白（IGFBPs）表現量上升所致。雖然正常細胞與癌細胞有著相似的結果，但是正常乳腺細胞對於ERα調控的敏感性是比較高的。藉由以上結果我們推測正常細胞本身可能對於不適當的外界刺激具有其防衛機制，以避免其造成過度的不正常反應。然而，此機制的效果在轉變成癌細胞之後或許消失或受到一定程度的影響。

關鍵字

乳腺 ； 泌乳激素 ； 第一型類胰島素生長因子

並列摘要

part1
In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions.
part 2
Insulin-like growth factor-I (IGF-I) and estrogens play important roles in the development of mammary glands. However, the abnormal increase of these hormones has been implicated in the development, progression and adverse outcome of breast cancer. Accumulating evidence reveals a crosstalk between IGF-I and estrogens-triggered signaling pathways, resulting in a synergistic effect on cell proliferation. The underlying mechanism is not completely elucidated, but upregulation of IGF-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) by estrogens might have a part in it. Here we found that overexpression of estrogen receptor a (ERa)in primary mouse mammary epithelial cells did not increase IRS-1 levels; instead, it decreased them in a dose-dependent manner. In addition, IGF-I-induced IRS-1 tyrosine phosphorylation was hampered. Similar results were obtained in primary human mammary epithelial cells and a normal mouse mammary cell line NMuMG. In ERa-negative human breast cancer cell lines, overexpression of ERa increased IRS-1 levels in Hs578T but decreased them in MDA-MB-231. Surprisingly, IRS-1 tyrosine phosphorylation was inhibited in both cell lines. This might be due to an increase in levels of IGF-binding proteins (IGFBPs). Although enhanced ERa expression in primary cells and cancer cells lead to the same result, primary cells are far more sensitive than cancer cells. We speculate that normal mammary cells possess safe-guard mechanisms to prevent over-stimulation when they are exposed to improper insults, such as overexpression of ERa. This protective mechanism might be still retained in certain cancer cells but the sensitivity is decreased.

並列關鍵字

mammary gland ； prolactin ； IGF-I

參考文獻

1. Visvader, J.E., Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis. Genes Dev, 2009. 23(22): p. 2563-77.

2. Sternlicht, M.D., Key stages in mammary gland development: the cues that regulate ductal branching morphogenesis. Breast Cancer Res, 2006. 8(1): p. 201.

3. Sternlicht, M.D., et al., Hormonal and local control of mammary branching morphogenesis. Differentiation, 2006. 74(7): p. 365-81.

5. Aoki, N., Regulation and functional relevance of milk fat globules and their components in the mammary gland. Biosci Biotechnol Biochem, 2006. 70(9): p. 2019-27.

7. Watson, C.J., Involution: apoptosis and tissue remodelling that convert the mammary gland from milk factory to a quiescent organ. Breast Cancer Res, 2006. 8(2): p. 203.