Bailey Caleb M, Khalkhali-Ellis Zhila, Kondo Shinji, Margaryan Naira V, Seftor Richard E B, Wheaton William W, Amir Sumaira, Pins Michael R, Schutte Brian C, Hendrix Mary J C
Department of Anatomy and Cell Biology and Pediatrics, Roy A. and Lucille J. Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, USA.
J Biol Chem. 2005 Oct 7;280(40):34210-7. doi: 10.1074/jbc.M503523200. Epub 2005 Jul 26.
Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.
自1994年报道发现maspin（乳腺丝氨酸蛋白酶抑制剂）以来，它因其促进细胞凋亡和抑制细胞侵袭的能力而被归类为II类肿瘤抑制因子。Maspin在正常乳腺上皮细胞中高度表达，但在侵袭性乳腺癌中表达降低或缺失。然而，尽管人们努力阐明maspin作为肿瘤抑制因子发挥作用的机制，但其分子特征仍有些难以捉摸。因此，为了鉴定与maspin相互作用的蛋白质，从而深入了解maspin的功能途径，我们采用了以maspin为诱饵的酵母双杂交系统，随后鉴定出干扰素调节因子6（IRF6）为maspin结合蛋白。IRF6属于转录因子的IRF家族，该家族最出名的是在病原体刺激后对干扰素和干扰素诱导基因的调控。尽管许多IRF家族成员已得到充分表征，但IRF6仍了解甚少。我们报告称，IRF6在正常乳腺上皮细胞中表达，并且在酵母双杂交系统和体外它都直接与maspin结合。这种相互作用通过保守的IRF蛋白结合结构域发生，并受IRF6磷酸化的调节。我们已经表明，与maspin类似，IRF6的表达与乳腺癌侵袭性呈负相关。我们进一步证明，在乳腺癌细胞中瞬时重新表达IRF6会导致N-钙黏蛋白增加和波形蛋白重新分布，这与细胞形态的变化相一致，提示发生了上皮-间质转化事件。同时，我们表明maspin作为这一过程的负调节因子。这些发现有助于阐明maspin的分子机制，并提示maspin和IRF6在调节细胞表型方面存在相互作用，细胞表型的丧失可导致肿瘤转化。
