Liu X
Department of Internal Medicine, First Affiliated Hospital of West China University of Medical Sciences, Chengdu 610041, China.
Zhonghua Gan Zang Bing Za Zhi. 2000 Oct;8(5):279-81.
To elucidate the role of two transcription factors, c-myb and liver activator protein (LAP, a member of the C/EBP family) in the expression of alpha1(I) collagen gene in activated hepatic stellate cells (HSCs).
Rat HSCs were prepared from SD rats by in situ perfusion and single-step density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen alpha1(I) gene promoter fragments (-804 approximately +1452 or -804 approximately +222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene constructs and mammalian vectors expressing c-myb or/and LAP using the cationic-liposome mediated method.
Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL(3)-col and PGL(3)-col (intron) reporter gene vectors [(587 +/- 62)U/mg protein vs (315 +/- 45)U/mg protein and (326 +/- 52)U vs (220 +/- 70)U, t=10.4 and 3.6, respectively, both P<0.05]. C-myb showed no transactivation to both PGL(3)-col and PGL(3)-col (intron) reporter gene vectors. But co-expression of LAP and c-myb increased basal transcription from PGL(3)-col reporter gene by approximate 3 fold (1261 +/- 130)U vs (315 +/- 45)U, t=20.6, P<0.01. Moreover, co-expression of a specific c-myb antisense vector with the LAP vector inhibited the transactivation of LAP to collagen alpha1(I) gene promoter activity (334 +/- 29)U vs (315 +/- 45)U, t=1.06, P>0.05), which was observed only in PGL(3)-col plasmids.
The transcription factor LAP transactivates collagen alpha1(I) gene in activated HSCs. C-myb plays an important role in transcriptional regulation of alpha1(I) collagen gene in HSCs and this effect is mediated by the transcription factor LAP and the cis-acting element in the first intron of alpha1(I) collagen gene.
阐明两种转录因子，即c-myb和肝激活蛋白（LAP，C/EBP家族成员）在活化肝星状细胞（HSC）中α1（I）胶原基因表达中的作用。
通过原位灌注和单步密度Nycodenz梯度从SD大鼠制备大鼠HSC。构建了两种包含人胶原α1（I）基因启动子片段（-804至+1452或-804至+222）的嵌合荧光素酶报告基因质粒。使用阳离子脂质体介导的方法，将培养活化的HSC与报告基因构建体以及表达c-myb或/和LAP的哺乳动物载体共转染。
表达LAP的载体瞬时转染显著增加了PGL(3)-col和PGL(3)-col（内含子）报告基因载体的基础转录[（587±62）U/mg蛋白对（315±45）U/mg蛋白以及（326±52）U对（220±70）U，t分别为10.4和3.6，均P<0.05]。c-myb对PGL(3)-col和PGL(3)-col（内含子）报告基因载体均无反式激活作用。但LAP和c-myb共表达使PGL(3)-col报告基因的基础转录增加了约3倍[（1261±130）U对（315±45）U，t=20.6，P<0.01]。此外，特定的c-myb反义载体与LAP载体共表达抑制了LAP对胶原α1（I）基因启动子活性的反式激活作用[（334±29）U对（315±45）U，t=1.06，P>0.05]，这仅在PGL(3)-col质粒中观察到。
转录因子LAP在活化的HSC中转录激活胶原α1（I）基因。c-myb在HSC中α1（I）胶原基因的转录调控中起重要作用，且这种作用由转录因子LAP和α1（I）胶原基因第一内含子中的顺式作用元件介导。
