| Cardiac hypertrophy is one of adaptive responses of the heart to a variety of pathological stimuli, including hypertension, myocardial infarction, congenital heart disease, heart valvula disease, and perturbations in sarcomeric function due to altered expression or mutations of contractile proteins. Increase in wall tension and hormonal stimuli are thought to directly activate various membrane-bound receptors and signal transduction cascade within cardiomyocytes resulting in the activation of immediate early genes and responsive genes such as c-jun, c-fos, c-myc, egr-1, ANF, BNP. Then the expression of construction proteins such as MLC-1, skeletal α-actin was unregulated. Receiving signals from any kinds of cascades, a number of transcription factors have been implicated as direct mediators of hypertrophic gene expression including SP1, Elk-1, SRF, MEF-2, GATA, and LIM proteins. LIM proteins contain one, two or multiple LIM domains and have been discovered to play important roles in a variety of fundamental biological processes including cytoskeleton organization, cell lineage specification and organ development. Recently, a new gene named as hhlim was cloned by three-element subtraction method; it is belong to one of the members of LIM family since it has the typical LIM domain. In recent years, accumulating results have continued to attribute essential functions to LIM proteins in a variety of different biological processes. But little is known about the new gene hhlim. On the basis of researches of hhlim gene regulation, we investigated the role of hhlim in cardiac hypertrophy at cellular and molecular level.1 Identification of hhlim gene regulatory region and study of hhlim gene expression regulationTo study the mechanisms of hhlim gene transcriptional regulation, a series of deleted fragments of hhlim gene 5' flanking region extending from -2537 to +16 bp were subcloned into the pGL3-Basic vector, respectively, to identify the specific functional regions by detecting the luciferase activities. The effect of ET-1 and bFGF on hhlim gene expression was observed at the same time.1.1 Compared with the full length of hhlim gene 5'flanking region, fragment of -253～+16 bp produced progressively increase in luciferase expression. The luciferase activity of -253～+16 bp region was 9.9-fold and 2.95-fold higher than that of -2537～+16 bp and -157～+16 bp region, respectively. Except for the region of pF2 and pF9, other region-drived luciferase expression in the differentiated C2C12 myotubes (MT) was higher than that of the undifferentiated C2C12 myoblasts. 1.2 Electrophoretic mobility shift assay showed that the patterns of shifted bands were different when the nuclear proteins from differentiated C2C12 (MT) and undifferentiated C2C12 cells (MB) bound to the region including the region of -253～-157 bp of hhlim gene. 1.3 To examine the transcriptional responsiveness of hhlim to hyperphictional agonists ET-1 and bFGF, C2C12 cells were transiently transfected with a luciferase reporter consisting of the hhlim promoter (pF5). Transfection of pF5, together with ET-1 stimulation, resulted in 2-fold activation of luciferase supporting the ability of ET-1 to augment hhlim promoter activity. Compared with ET-1, the effect of bFGF is weaker. As analyzed by RT-PCR, the treatment of differentiated C2C12 cells (MT) with ET-1 and bFGF for different times increased hhlim RNA levels. RT-PCR results showed that hhlim gene expression was upregulated by ET-1 and bFGF, respectively, and reached the peak at 24 h and 48 h after stimulation. Compared with the control, hhlim gene expression was enhanced by 4.5-fold and 3.38-fold, respectively, after being treated with ET-1 and bFGF for 24 h. These data identified that hhlim gene 5'flanking region from -253 to+16 bp was sufficient to drive hhlim transcription. The interaction between cis-element and transcriptional factor was complex. hhlim gene expression was regulated by ET-1 and bFGF.2 Cell localization of hhlim gene product and its effect on cel...
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