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Background: The zinc finger transcription factor Egr-1 (Early growth response 1) is central to several growth factors and represents an important activator of target genes not only involved in physiological processes like embryogenesis and neonatal development, but also in a variety of pathophysiological processes, for example atherosclerosis or cancer. Current options to investigate its transcription and activation in vivo are end-point measurements that do not provide insights into dynamic changes in the living organism. Results: We developed a transgenic mouse (Egr-1-luc) in which the luciferase reporter gene is under the control of the murine Egr-1 promoter providing a versatile tool to study the time course of Egr-1 activation in vivo. In neonatal mice, bioluminescence imaging revealed a high Egr-1 promoter activity reaching basal levels three weeks after birth with activity at snout, ears and paws. Using a model of partial hepatectomy we could show that Egr-1 promoter activity and Egr-1 mRNA levels were increased in the regenerating liver. In a model of wound healing, we demonstrated that Egr-1 promoter activity was upregulated at the site of injury. Conclusion: Taken together, we have developed a transgenic mouse model that allows real time in vivo imaging of the Egr-1 promoter activity. The ability to monitor and quantify Egr-1 activity in the living organism may facilitate a better understanding of Egr-1 function in vivo. Additional File 1: BLI of adult Egr-1-luc mice with opened body cavity. Transgenic Egr-1-luc mice (one month old) received 6 mg luciferin in 100 μl PBS by intraperitoneal injection. Ten minutes thereafter the animal was killed by cervical dislocation, the body cavity opened immediately, skin from the ventral side partially removed and BLI measurement was carried out (10 min signal collection, setting 'high resolution'). A representative animal is shown with similar amplification setting as in Figure 2A.
Vascular occlusive diseases are one of the leading mortality causes in westernised countries. Occlusions of one of the major arteries can be overcome without devastating consequences provided a timely induction of compensating collateral arteries occurs. Perhaps the most outstanding feature of collateral vessel growth is the proliferation of smooth muscle cells (SMCs). Understanding the molecular mechanisms and identifying key molecular players of SMC proliferation would contribute significantly to the development of efficient therapies to intervene with all processes involving neointima formation, including collateral growth. mRNA and protein coding for co-transcription factor Egr1 were found to be up-regulated in growing collateral vessels 6, 12 or 24 hours following femoral artery ligation in mice. Since Egr1 is required for SMC proliferation in vitro and in vivo and likely to be implicated in the initiation of collateral artery growth, the key signalling mediators regulating Egr1 expression specifically in proliferating vascular SMCs were investigated. Northern blot and Western blot analysis revealed a strong up-regulation of Egr1 within 2 hours of stimulation with PDGF-AB and FGF-2. These two potent SMC mitogens involved in neointima formation were used to stimulate vascular SMCs not only to delineate the regulators of Egr1 expression but also to identify additional key mediators of SMC proliferation. FGF-2 but not PDGF-AB led to a drastic reduction of desmin amount in proliferating SMCs, correlating closely with the phenotypic modulation of SMCs in vivo. Both growth factors triggered a dramatic increase in DNA-synthesis rate with a concomitant loss of p27 exp Kip1. Stimulation with PDGF-AB and FGF-2 triggered a rapid and transient activation of PDGFRβ and FGFR1 respectively, thus providing the basis for activation of down-stream targets. Analysis of an array of signalling pathways demonstrated a strong activation of the Ras-Raf-MEK-ERK cascade in response to both factors as measured by the level of phosphorylation of prominent members MEK, ERK1/2 and c-Myc. SAPK/JNK and p38, which also belong to the superfamily of MAP kinases, did not become activated following stimulation with either PDGF-AB or FGF-2. The analysis of various PKC isoforms identified PKCδ and PKCθ to be the key mediators of PDGF-AB- and FGF-2-induced mitogenesis in proliferating SMCs. Whereas PDGF-AB potently stimulated PKB/Akt with concomitant GSK3β phosphorylation, FGF-2-induced inactivation of GSK3β was independent of PKB/Akt. Specific inhibition in order to evaluate the contribution of individual pathways to Egr1 expression and vascular SMC proliferation revealed that inhibition of the Raf-MEK-ERK module by UO126 completely abolished DNA-synthesis and Egr1 expression without a compensation by alternative pathways. Surprisingly, inhibition of PI3K led to a switch to the mitogenic RafMEK-ERK signalling cascade which resulted in an augmented Egr1 expression. In conclusion, in porcine vascular SMCs, activation of the Ras-Raf-MEK-ERK signalling module appears to be the main prerequisite for Egr1 expression and DNA synthesis induction in response to PDGF-AB and FGF-2 whereas related kinases SAPK/JNK and p38 play no significant role. Inhibition of the PI3K-Akt cascade represents an alternative way to activate ERK1/2 and induce Egr1 expression. Whereas MEK is the central regulator of mitogenic effects in proliferating vascular SMCs, the PI3K-Akt pathway most likely exerts survival function. Inactivation of MEK by its specific inhibitors identified hyperphosphorylation as ayet unknown mechanism of kinase inhibition.