Synergistic activation of extracellular signal-regulated kinase in human dermal fibroblasts by human telomerase reverse transcriptase and transforming growth factor-beta1.
Synergistic activation of extracellular signal-regulated kinase in human dermal fibroblasts by human telomerase reverse transcriptase and transforming growth factor-beta1.
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人端粒酶逆转录酶和转化生长因子-β1协同激活人真皮成纤维细胞中的细胞外信号调节激酶。
DOI：
10.1016/j.jss.2007.02.030
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Mustoe,ThomasA
中科院分区：
文献类型：
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作者：
Liu,WRobert;Lu,Leonard;Rosenberg,DavidS;Procaccini,PieroSA;Mustoe,ThomasA
背景人类端粒酶逆转录酶（hTERT）主要以其延长端粒以维持染色体完整性和延缓细胞衰老的能力而闻名。最近，hTERT已经显示出在促进细胞增殖中具有独立于端粒延长的作用。hTERT如何发挥这种新的功能是细胞生物学中的一个基本问题。了解这个问题可能对器官或组织受损、心血管疾病、中风、缺血性慢性伤口和其他缺血再灌注损伤患者的再生医学具有治疗意义。为此，我们用转化生长因子（TGF）-β1处理hTERT转染的人真皮成纤维细胞（HDF），并研究细胞外信号调节激酶（ERK）1/2的活化，材料和方法用表达hTERT的重组腺病毒（Ad-hTERT）或对照腺病毒（Ad-NULL）转染原代HDF，随后用TGF-β1（2 μ g/mL）处理。使用仅识别在Thr 202和Tyr 204处双重磷酸化的活化ERK 1/2的抗体，通过Western印迹法测定ERK 1/2活化。实时荧光定量PCR检测TGF-β1、TGF β RI、TGFβ RII和Col 1 A1 mRNA表达水平，结果显示转染Ad-NULL的HDFs在TGF-β1作用下磷酸化ERK 1/2表达上调7倍以上。Ad-hTERT转染的HDFs中ERK 1/2的协同激活早在TGF-β1处理后10 min就出现，并持续至少30 min。TGF-β1处理后，转染Ad-hTERT和转染Ad-NULL的HDFs在TGF-β1、TGFβ-RI、TGFβ-RII和Col 1 A1 mRNA水平上无统计学显著差异。结论ShTERT和极低浓度的TGF-β1（2 pg/mL）通过一种独立于自分泌TGF-β1环的机制协同激活HDFs中的ERK 1/2。
BACKGROUNDHuman telomerase reverse transcriptase (hTERT) is primarily known for its ability to elongate telomeres for maintaining chromosomal integrity and delaying cellular senescence. Recently, hTERT has emerged as having a role in promoting cellular proliferation that is independent of telomere elongation. How hTERT elicits this novel function is a fundamental question in cell biology. Understanding this question may have therapeutic implications in regenerative medicine for patients with damaged organs or tissues, cardiovascular disorders, stroke, ischemic chronic wounds, and other ischemia-reperfusion injuries. Toward this end, we treated hTERT-transfected human dermal fibroblasts (HDFs) with transforming growth factor (TGF)-β1 and investigated the activation of extracellular signal-regulated kinase (ERK) 1/2, vital mediators of cell proliferation.MATERIALS AND METHODSPrimary HDFs were transfected with either recombinant adenovirus expressing hTERT (Ad-hTERT) or control adenovirus (Ad-NULL) and subsequently treated with TGF-β1 (2 pg/mL). ERK 1/2 activation was determined by Western blotting using an antibody recognizing only activated ERK 1/2 that is dually phosphorylated at Thr202and Tyr204. TGF-β1, TGFβ-RI, TGFβ-RII, and Col1 A1 mRNA levels were analyzed by real-time PCR.RESULTSAd-hTERT-transfected HDFs showed more than 7-fold up-regulation of phospho-ERK 1/2 over Ad-NULL-transfected HDFs upon TGF-β1 treatment. The synergistic ERK 1/2 activation in Ad-hTERT-transfected HDFs occurred as early as 10 min and was sustained for at least 30 min after TGF-β1 treatment. There were no statistically significant differences in TGF-β1, TGFβ-RI, TGFβ-RII, and Col1 A1 mRNA levels between HDFs that were transfected with Ad-hTERT and those that were transfected with Ad-NULL after TGF-β1 treatment.CONCLUSIONShTERT and extremely low concentrations of TGF-β1 (2 pg/mL) synergistically activate ERK 1/2 in HDFs by a mechanism that is independent of the autocrine TGF-β1 loop.