Regulation of type IV collagenase expression

描述(申请人提供)：92 kDa IV型胶原酶(MMP-9)有助于口腔癌的扩散，并了解其表达受调控如何最终产生新的药物来抑制其表达和降低肿瘤侵袭性。尽管我们先前在670碱基对启动子中发现了多个调控元件(AP-1、NF-kappaB、PEA3、Sp1)，但这些结合位点的有限数量使得基质金属蛋白酶-9的表达不太可能仅仅是通过这些基序反式激活的结果。事实上，新出现的研究表明，染色质环境(由包裹在组蛋白核心上的DNA构成)在基因表达调控中发挥了作用。我们在此表明，促进组蛋白去乙酰化的MTAL抑制了基质金属蛋白酶-9的表达。此外，在产生基质金属蛋白酶-9的口腔癌中检测不到Mtal的表达。在特定的目标1中，利用基因组足迹和染色质免疫沉淀分析(Chip)，我们将鉴定转录因子结合的顺式元件和乙酰化组蛋白，它们位于Mtal靶向的670个碱基对的MMP-9启动子上，以实现对基质金属蛋白酶-9的抑制。基质金属蛋白酶-9表达的生物抑制物也可以提供一种工具来鉴定显色化启动子中的调控元件。我们在此表明，转移抑制基因KiSS-1部分通过减少核因子-kappaB与显色化启动子的结合来抑制基质金属蛋白酶-9的转录。然而，核因子-kappaB结合减少的程度只能部分解释转录减少的原因。因此，在特定目标2中，我们将确定位于MMP-9启动子这些位置的反式激活的顺式元件和乙酰化组蛋白，它们介导KISS-1依赖的对MMP9的抑制。类似地，MEK1抑制剂PD098059抑制MMP9的表达，在特定目标3中，我们将确定该抑制因子的转录靶点是否与MTAL和KISS-1相同或不同。如果我们确定转录靶点不同，我们将确定PD098059与KISS-1或MTAL联合使用在减少MMP-9表达和口腔癌侵袭性方面是否优于单独使用。由于在我们之前对基质金属蛋白酶-9转录的研究中使用了染色体外的记者，依赖于染色质环境的调节元件可能逃脱了检测。因此，为了在特定的目的4中识别这些新的顺式元件，我们将使用DNA酶超敏反应、基因组足迹和lambdagt11文库筛选来识别其他的基质金属蛋白酶-9表达的反式激活/抑制因子。最终，这些研究的目标是确定基质金属蛋白酶-9启动子中的新转录靶点，以允许进行治疗干预以抑制胶原酶的表达和口腔癌的侵袭性。

DESCRIPTION (provided by applicant): The 92 kDa type IV collagenase (MMP-9) contributes to the spread of oral cancer and understanding how its expressions regulated could ultimately yield new agents to repress its expression and diminish tumor invasiveness. Although we previously identified multiple regulatory elements (AP-1, NF-kappaB, PEA3, Sp1) in the 670 base pair promoter, the limited number of these binding sites makes it unlikely that MMP-9 expression is solely the consequence of transactivation through these motifs. Indeed, emerging studies indicate a role for the chromatin environment (constituted by DNA wrapped around a histone core), in the regulation of gene expression. We show herein that Mtal, which promotes histone deacetylation, represses MMP-9 expression. Further, Mtal expression is undetectable in MMP-9-producing oral cancer. In Specific Aim 1 using genomic foot printing and chromatin immunoprecipitation assays (Chip) we will identify transcription factor-bound cis elements and acetylated histones localized at these sites in the 670 base pair MMP-9 promoter targeted by Mtal to achieve MMP-9 repression. Biological suppressors of MMP-9 expression may also provide a tool for identifying regulatory elements in the chromatinized promoter. We show herein that the metastasis suppressor gene KiSS-1 attenuates MMP-9 transcription partly by reducing NF-kappaB binding to the chromatinized promoter. Nevertheless, the degree to which NF-kappaB binding is reduced can only partly account for the diminished transcription. Therefore, in Specific Aim 2, we will identify transactivated cis elements and acetylated histones localized at these sites in the MMP-9 promoter that mediate KiSS-1-dependent repression of MMP-9.Similarly, the MEK1 inhibitor PD098059 represses MMP-9 expression and in Specific Aim 3 we will determine if the transcriptional targets of this repressor are identical to, or distinct from, those of Mtal and KiSS-1. If we determine that the transcriptional targets differ, we will determine whether combining PD098059 and KiSS-1 or Mtal proves superior to individual modalities in reducing MMP-9 expression and oral cancer invasiveness. Since extra-chromosomal reporters were used in our previous studies of MMP-9 transcription, regulatory elements, that depend on the chromatin environment, may have escaped detection. Thus, to identify such novel cis elements in Specific Aim 4, we will employ DNase hypersensitivity, genomic foot printing and lambdagt11 library screening to identify additional transactivators/repressors of MMP-9 expression. Ultimately, the goal of these studies is to identify new transcriptional targets in the MMP-9 promoter that allow for therapeutic intervention to repress expression of this collagenase and oral cancer invasiveness.