We previously demonstrated that cyclic strain, like shear stress, upregulates PDGF-B gene transcription in bovine aortic EC. We have recently observed that membrane type 1 matrix metalloproteinase 1 (MT 1-MMP) transcription is differentially regulated by oscillatory shear and cyclic strain. Exposure of rat microvessel EC to cyclic strain leads to increase in MT-1 MMP gene and protein; exposure to oscillatory shear decreases MT1-MMP transcription. Both the PDGF-B and MT 1-MMP gene are regulated by the sp 1 and early growth response factor 1 (egr-1) transcription factors and increases in egr-1 can displace the ubiquitously present sp 1 that is bound to the promoter sites. However, our preliminary findings indicate that nuclear egr-1 levels are increased in both cyclic strain and oscillatory shear and yet there is differential regulation of MT-1 MMP. Oscillatory shear forces are complex and likely composed of both steady and impulse flow. We hypothesize that both cyclic strain and pulsatile shear up regulate egr1 mRNA and protein through a similar pathway that involves phosphatidylinositol 3-phosphate (PI3K) and ERK 1/2 activation. Sp1 is ubiquitously expressed in EC and occupy binding on G + C rich regions of the promoter regions of MT 1-MMP and PDGF-B. Under cyclic strain, egr1 can displace sp1 and transactivate the MT-1MMP and PDGF-B genes. Pulsatile shear, however, induces Sp1 phosphorylation in EC through an atypical PKC-z pathway and this increases sp1 DNA binding affinity. Increased levels of Egr-1 cannot displace the phosphorylated Sp1 and this inhibits MT 1-MMP transcription. PDGF-B is increased with shear stress despite sp1 phosphorylation because of the interaction of other transcription factors.
The specific aims are: 1. Define how egr-1 is regulated by mechanical forces, a) Assess egrl transcription by different forces, b) Assess egr1 post-translational modification by different forces. 2. Define how sp1 and sp3 are regulated by mechanical forces a) Assess sp1 and sp3 transcription by different forces b) Assess sp1 posttranslational modification by different forces 3. Assess MT1-MMP and PDGF-B response to different physical forces. Assess relevance of egr-1 and sp-1 in MT 1-MMP and PDGF-B transcriptional activation. 4. Assess egr-1 and sp1 binding in MT1-MMP and PDGF-B promoters under mechanical stress 5.Assess the functional relevance of egr-1 in a low flow carotid model using an egr-1 knock-out mouse.
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