Regulation of type IV collagenase expression

描述（由申请人提供）：92 kDa IV型胶原酶（MMP-9）有助于口腔癌的扩散，了解其表达如何调节可能最终产生抑制其表达并减少肿瘤侵袭的新药物。虽然我们先前在670个碱基对的启动子中鉴定了多个调节元件（AP-1、NF-κ B、PEA 3、Sp1），但是这些结合位点的有限数目使得MMP-9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，新兴研究表明染色质环境（由包裹在组蛋白核心周围的DNA构成）在基因表达的调节中发挥着作用。我们在此表明，Mtal，促进组蛋白去乙酰化，抑制MMP-9的表达。此外，在产生MMP-9的口腔癌中检测不到Mtal表达。在特异性目的1中，使用基因组足迹和染色质免疫沉淀测定（Chip），我们将鉴定转录因子结合的顺式元件和乙酰化组蛋白，其位于Mtal靶向的670个碱基对MMP-9启动子中的这些位点，以实现MMP-9抑制。MMP-9表达的生物抑制因子也可以提供用于鉴定染色质化启动子中的调节元件的工具。我们在此表明，转移抑制基因KiSS-1减弱MMP-9的转录部分通过减少NF-κ B结合染色质化的启动子。然而，NF-κ B结合减少的程度只能部分解释转录的减少。因此，在特异性目标2中，我们将鉴定位于MMP-9启动子中这些位点的反式激活的顺式元件和乙酰化组蛋白，它们介导MMP-9的KiSS-1依赖性抑制。类似地，MEK 1抑制剂PD 098059抑制MMP-9表达，在特异性目标3中，我们将确定该抑制剂的转录靶点是否与MMP-9相同或不同，Mtal和KiSS-1。如果我们确定转录靶点不同，我们将确定联合PD 098059和KiSS-1或Mtal是否证明在降低MMP-9表达和口腔癌侵袭性方面上级单独的方式。由于在我们以前的MMP-9转录研究中使用了染色体外报告基因，因此依赖于染色质环境的调控元件可能无法检测到。因此，为了鉴定特异性目的4中的这种新的顺式元件，我们将采用DNA酶超敏性、基因组足迹法（genomic footprinting）和DNA印迹11文库筛选来鉴定MMP-9表达的另外的反式激活因子/阻遏因子。最终，这些研究的目标是确定MMP-9启动子中的新转录靶点，从而允许治疗干预来抑制这种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.