The Role of TIMPs in Cell Growth and Differentiation: Tumor Angiogenesis

摘要：EGF 刺激 A549 人肺癌细胞系表明，细胞生长反应的抑制是通过激活蛋白酪氨酸磷酸酶活性介导的，并导致 EGF 受体磷酸化减少。使用抗整合素抗体的竞争结合实验将整合素 α3β1 鉴定为人微血管内皮细胞 (hMVEC) 上 TIMP-2 的假定细胞表面受体。 Ala TIMP-2还抑制VEGF-A或FGF-2刺激的体外有丝分裂和体内血管生成，从而证明TIMP-2的血管抑制活性与MMP抑制是分离的。这种效应的机制涉及生长因子受体信号传导的整联蛋白受体失活，称为异源受体失活。这是首次证明整合素可以负向调节受体酪氨酸激酶的激活。这项工作通过证明 TIMP 是多功能蛋白质，具有细胞表面受体，并且通过与这些受体相互作用，可以直接影响细胞行为，从而定义了 TIMP 生物学的新范例。使用体外和体内模型，我们当前和未来的工作重点是鉴定 TIMP-2 中的 α3β1 整合素结合域，并进一步了解 TIMP-2 与正常细胞和肿瘤细胞中的 α3β1 相互作用后的细胞效应，以及肿瘤微环境的后续变化。我们的目标是进一步表征 TIMP 在肿瘤微环境中的 MMP 独立和 MMP 依赖性作用及其对肿瘤抑制和/或进展的相对贡献。这些研究应该确定正常组织和肿瘤微环境中细胞外基质调节细胞行为的关键机制，并可能为癌症治疗带来新的治疗策略。这些发现表明，定义负责 TIMP-2 与 alpha3beta1 结合的结构域对于进一步剖析这种复杂分子的多种生物活性以及定义该活性对正常和恶性组织中微环境的功能贡献至关重要。该项目的重点是确定 Ala TIMP-2 的抗血管生成和抗肿瘤生成作用的机制。对人类微血管内皮细胞的初步研究已经证明了一种称为异源受体失活的机制。在这种作用中，TIMP-2 受体 α3β1 通过激活称为 Shp-1 的磷酸酪氨酸磷酸酶，减少受体酪氨酸激酶的磷酸化和激活，例如血管内皮生长因子受体 (VEGFR)-2、成纤维细胞生长因子受体 (FGFR)-1 和表皮生长因子受体 (EGFR)。然而，最近在肿瘤细胞和内皮细胞中进行的实验表明，Ala TIMP-2 的生长抑制活性更为复杂，似乎涉及细胞凋亡途径和上皮间质转化的基因表达变化，这对于肿瘤侵袭和转移至关重要。该项目的目的是识别和描绘这些途径，旨在将 Ala TIMP-2 开发为新型癌症治疗剂并确定潜在的新治疗靶点。金属蛋白酶组织抑制剂 2 (TIMP-2) 通过多种机制抑制血管生成，包括抑制 MMP 或直接与内皮细胞结合。本研究的主要目的是鉴定参与与先前鉴定的受体整合素α3β1结合的TIMP-2区域，并确定源自该区域的合成肽是否保留血管抑制和肿瘤抑制活性。我们证明 TIMP-2 (N-TIMP-2) 的 N 端结构域与 alpha3beta1 结合，并在体外抑制血管内皮生长因子刺激的内皮细胞生长，表明 TIMP-2 的 alpha3beta1 结合结构域和生长抑制活性均定位于 N 端结构域。使用肽阵列方法，我们鉴定了 TIMP-2 一级序列的 24 个氨基酸区域，由残基 Ile43-Ala66 组成，显示出 alpha3beta1 结合活性。随后我们证明，来自该区域的合成肽竞争 TIMP-2 与 alpha3beta1 的结合，并在体外抑制内皮生长。我们定义了一个最小的肽序列（肽8-9），它具有血管抑制作用，并且使用卡波西肉瘤的鼠异种移植模型，具有体内抗肿瘤发生活性。因此，α3β1 结合和血管抑制活性共同定位于 TIMP-2 一级序列中暴露于溶剂的柔性区域，与 TIMP 家族的其他成员相比，该区域的氨基酸序列是独特的。此外，该区域中 TIMP-2 和 TIMP-1 蛋白 3-D 结构的比较也发现了独特的结构差异。我们的研究结果表明，TIMP-2 的整合素结合、肿瘤生长抑制剂和体内血管抑制活性在独特的序列/结构环（B-C 环）内密切相关。

Summary: EGF-stimulation of the A549 human lung carcinoma cell line demonstrated that the suppression of cell growth response was mediated by the activation of protein tyrosine phosphatase activity and resulted in reduced EGF receptor phosphorylation. Competition binding experiments using anti-integrin antibodies identified integrin alpha3beta1 as a putative cell surface receptor for TIMP-2 on human microvascular endothelial cells (hMVECs). Ala+TIMP-2 also inhibited VEGF-A or FGF-2 stimulated mitogenesis in vitro and angiogenesis in vivo a, thus demonstrating that the angio-inhibitory activity of TIMP-2 is dissociable from MMP-inhibition. The mechanism of this effect involves an integrin receptor inactivation of growth factor receptor signaling, known as heterologous receptor inactivation. This was the first demonstration that integrins could negatively regulate activation of a receptor tyrosine kinase This work has defined a new paradigm for TIMP biology by demonstrating that TIMPs are multifunctional proteins, with cell surface receptors and through interaction with these receptors they can directly influence cellular behavior. Using both in vitro and in vivo models our current and future work is focused on identifying the alpha3beta1 integrin binding domain(s) in TIMP-2 and furthering our understanding of the cellular effects following TIMP-2 interaction with alpha3beta1 in both normal and neoplastic cells, as well as the subsequent alterations in the tumor microenvironment. It is our goal to further characterize the MMP-independent and MMP-dependent effects of TIMPs in the tumor microenvironment and their relative contribution to tumor suppression and/or progression. These studies should identify crucial mechanisms in the regulation of cell behavior by the extracellular matrix in normal tissues and the tumor microenvironment, and possibly lead to new therapeutic strategies for cancer treatment. These findings suggest that defining the domain(s) responsible for TIMP-2-binding to alpha3beta1 will be critical to further dissecting the multiple biological activities of this complex molecule, as well as defining the functional contributions of this activity to the microenvironment in both normal and malignant tissues. The focus of this project is to determine the mechanisms of the anti-angiogenic and anti-tumorigenic effects of Ala+TIMP-2. Preliminary work with human microvascular endothelial cells has demonstrated a mechanism known as heterologous receptor inactivation. In this effect the TIMP-2 receptor alpha3beta1 decreases phosphorylation and activation of receptor tyrosine kinases such as the vascular endothelial growth factor receptor (VEGFR)-2, fibroblast growth factor recetpor (FGFR)-1 and epidermal growth factor receptor (EGFR) by activation a phosphotyrosine phosphatase known as Shp-1. However, recent experiments in tumor cells and endothelial cells have revealed that the growth suppressor activity of Ala+TIMP-2 is more complex and appears to involve apoptotic pathways and changes in gene expression of the epithelial to mesenchymal transition that is essential to tumor invasion and metastasis. It is the purpose of this project to identify and delineate these pathways with the aim of developing Ala+TIMP-2 as a novel cancer therapeutic and identifying potential new therapeutic targets. Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits angiogenesis by several mechanisms involving either MMP inhibition or direct endothelial cell binding. The primary aim of this study was to identify the TIMP-2 region involved in binding to the previously identified receptor integrin alpha3beta1, and to determine whether synthetic peptides derived from this region retained angio-inhibitory and tumor suppressor activity. We demonstrated that the N-terminal domain of TIMP-2 (N-TIMP-2) binds to alpha3beta1 and inhibits vascular endothelial growth factor-stimulated endothelial cell growth in vitro, suggesting that both the alpha3beta1-binding domain and growth suppressor activity of TIMP-2 localize to the N-terminal domain. Using a peptide array approach we identify a 24 amino acid region of TIMP-2 primary sequence, consisting of residues Ile43-Ala66, which shows alpha3beta1-binding activity. Subsequently we demonstrate that synthetic peptides from this region compete for TIMP-2 binding to alpha3beta1 and suppress endothelial growth in vitro. We define a minimal peptide sequence (peptide 8-9) that posses both angio-inhibitory and, using a murine xenograft model of Kaposis sarcoma, anti-tumorigenic activity in vivo. Thus, both the alpha3beta1-binding and angio-inhibitory activities co-localize to a solvent exposed, flexible region in the TIMP-2 primary sequence that is unique in amino acid sequence compared with other members of the TIMP family. Furthermore, comparison of the TIMP-2 and TIMP-1 protein 3-D structures in this region also identified unique structural differences. Our findings demonstrate that the integrin binding, tumor growth suppressor and in vivo angio-inhibitory activities of TIMP-2 are intimately associated within a unique sequence/structural loop (B-C loop).