探討人類纖維母細胞中PKC δ在bFGF誘導urokinase-type plasminogen activator 生成中之重要角色

Protein kinase C δ plays a critical role in bFGF-induced urokinase-type plasminogen activator production in human fibroblasts

廖志裕(Chih-Yu Liao)

指導教授 ： 鄧哲明

國立臺灣大學/醫學院/藥理學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01947

摘要

由最近的研究發現，癌細胞可以釋出多種生長因子，進而促使周邊細胞(如纖維母細胞)分泌 uPA，而幫助癌細胞轉移的發生。由本實驗結果發現，使用basic fibroblast growth factor (bFGF) 刺激纖維母細胞 MRC-5，確實可以明顯地增加 uPA 的產生。進一步給予不同的抑制劑來探討其調控的訊息傳遞路徑，發現PD98059 (an ERK 1/2 inhibitor)、Ro318220 和 staurosporine (two PKC inhibitors)、SP600125 (a JNK inhibitor) 及 PDTC (a NF-κB inhibitor) 皆會有效抑制 bFGF 誘發 uPA 產生。尤其可觀察到 PKC inhibitor 具有很強的抑制作用。由於 protein kinase C (PKC) 家族含有 12 種 isoforms，為了進ㄧ步釐清何種 isoform 為主要的調控者，於是給予專一性的 PKC inhibitors來探討。我們發現 rottlerin (a PKC δ inhibitor) 會明顯減少 uPA 的產生。接著以西方點墨法觀察到 rottlerin 可以抑制ERK 1/2磷酸化，但 PD98059 卻不會影響 PKC δ 的活化。由於 ERK 1/2 的活化會影響到 P70S6K 的磷酸化而促進細胞進入轉譯 (translation) 作用，產生大量的蛋白質。在我們的實驗結果中發現給予 PD98059 及 rottlerin 都會抑制 P70S6K 在 Thr421/Ser424 及 Thr389 位置的磷酸化。综合上述的實驗我們可以推測在 MRC-5 細胞中給予 bFGF 刺激 uPA 產生的過程中，藉由 PKCδ 調控 ERK 1/2 的活化，進一步促進 uPA 產生；同時也可能會促進 P70S6K 的磷酸化而使細胞進入轉譯 (translation)，增加蛋白質的生成。另一方面，由於 SP600125 及 PDTC 可以有效抑制 uPA 產生，知道 bFGF 也可能經由 JNK 及 NF-κB 的訊息傳遞路徑來促進 MRC-5 細胞產生 uPA。藉由釐清癌細胞和纖維母細胞之間的交互作用，我們發現癌細胞的侵入及轉移中，PKC δ扮演一個重要的角色，因此PKC δ可作為未來研發抗癌藥物的新標的。

關鍵字

PKC δ ； urokinase-type plasminogen activator

並列摘要

From current studies, cancer cells generate many growth factors to induce adjacent cells, for instance fibroblasts, to secrete uPA then render the occurrence of cancer metastasis. In this study, we found that basic fibroblast growth factor (bFGF) stimulated fibroblast (MRC-5) to produce high level of uPA. Next, by using different protein kinase inhibitors to elucidate the involved signaling pathways, we observed that PD98059 (an ERK 1/2 inhibitor)、Ro318220 and staurosporine (two PKC inhibitors)、SP600125 (a JNK inhibitor) and PDTC (a NF-κB inhibitor), all reduced bFGF-induced uPA. Especially PKC inhibitors had stronger inhibition. Because protein kinase C (PKC) family comprises 12 isoforms, we utilized different specific PKC inhibitors to ascertain which isoform is the main factor. We noted that rottlerin, a PKCδ inhibitor, effectively decreased uPA production. Then, we exploited Western blotting analysis to investigate the interaction of different signaling transducers. The data showed that rottlerin could inhibit ERK 1/2 phosphorylation, but PD98059 did not affect PKCδ activation. According to previous studies, ERK 1/2 activation would cause P70S6K phosphorylation that makes cells translate mRNA to synthesize lots of proteins. We found that both PD98059 and rottlerin down-regulated the phosphorylation on Thr421/Ser424 and Thr389 of P70S6K. In this study, we could consider in the process of bFGF-induced uPA in MRC-5 cells, PKCδ caused ERK 1/2 activation and stimulated uPA production; at the same time it might influence P70S6K phosphorylation to let cells go into translation to increase protein synthesis. On the other hand, SP600125 and PDTC lowered uPA level, so in MRC-5 cells, bFGF might apply JNK and NF-κB pathways to induce uPA production. By clarifying the interaction between tumor cells and fibroblasts, we could design new drugs according to each potential target. Our data demonstrate that PKC δ plays a critical role, so we supposed PKCδ could be one of the potential targets for anticancer drug development.

並列關鍵字

PKC δ ； urokinase-type plasminogen activator

參考文獻

Alonso, D. F., Farias, E. F., Ladeda, V., Davel, L., Puricelli, L., and Bal de Kier, J. E. (1996). Effects of synthetic urokinase inhibitors on local invasion and metastasis in a murine mammary tumor model. Breast Cancer Res. Treat. 40, 209-23.

Andreasen, P. A., Kjoller, L., Christensen, L., and Duffy, M. J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1-22.

Andreasen, P. A., Sottrup-Jensen, L., Kjoller, L., Nykjaer, A., Moestrup, S. K., Petersen, C. M., and Gliemann, J. (1994). Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes. FEBS Lett. 338, 239-45.

Bajou, K., Lewalle, J. M., Martinez, C. R., Soria, C., Lu, H., Noel, A., and Foidart, J. M. (2002). Human breast adenocarcinoma cell lines promote angiogenesis by providing cells with uPA-PAI-1 and by enhancing their expression. Int. J. Cancer 100, 501-6.

Bajpai, A., and Baker, J. B. (1985). Cryptic urokinase binding sites on human foreskin fibroblasts. Biochem. Biophys. Res. Commun. 133, 475-82.